what is the phd domain

• Open access
• Published: 24 January 2020

Characterization of the plant homeodomain (PHD) reader family for their histone tail interactions

• Kanishk Jain 1 , 2 ,
• Caroline S. Fraser 2 , 3 ,
• Matthew R. Marunde 4 ,
• Madison M. Parker 1 , 2 ,
• Cari Sagum 5 ,
• Jonathan M. Burg 4 ,
• Nathan Hall 4 ,
• Irina K. Popova 4 ,
• Keli L. Rodriguez 4 ,
• Anup Vaidya 4 ,
• Krzysztof Krajewski 1 ,
• Michael-Christopher Keogh 4 ,
• Mark T. Bedford 5 &
• Brian D. Strahl   ORCID: orcid.org/0000-0002-4947-6259 1 , 2 , 3  

Epigenetics & Chromatin volume  13 , Article number:  3 ( 2020 ) Cite this article

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Plant homeodomain (PHD) fingers are central “readers” of histone post-translational modifications (PTMs) with > 100 PHD finger-containing proteins encoded by the human genome. Many of the PHDs studied to date bind to unmodified or methylated states of histone H3 lysine 4 (H3K4). Additionally, many of these domains, and the proteins they are contained in, have crucial roles in the regulation of gene expression and cancer development. Despite this, the majority of PHD fingers have gone uncharacterized; thus, our understanding of how these domains contribute to chromatin biology remains incomplete.

We expressed and screened 123 of the annotated human PHD fingers for their histone binding preferences using reader domain microarrays. A subset (31) of these domains showed strong preference for the H3 N-terminal tail either unmodified or methylated at H3K4. These H3 readers were further characterized by histone peptide microarrays and/or AlphaScreen to comprehensively define their H3 preferences and PTM cross-talk.

Conclusions

The high-throughput approaches utilized in this study establish a compendium of binding information for the PHD reader family with regard to how they engage histone PTMs and uncover several novel reader domain–histone PTM interactions (i.e., PHRF1 and TRIM66). This study highlights the usefulness of high-throughput analyses of histone reader proteins as a means of understanding how chromatin engagement occurs biochemically.

Histone proteins are fundamental to genome organization and packaging, and are chemically modified by a wide range of “writer” or “eraser” enzymes that, respectively, install or remove histone post-translational modifications (PTMs) [ 1 , 2 ]. These PTMs play a central role in chromatin function: some are believed to directly impact chromatin organization through biophysical means, but the vast number likely function through their ability to recruit effector or “reader” domain-containing proteins to chromatin. These reader proteins, which are often found in large multi-subunit complexes and in additional chromatin-modifying machines, interact with histone tails and chromatin in various ways that regulate gene transcription and other chromatin functions [ 2 , 3 ]. The varied and diverse patterns of histone PTMs that exist in vivo are referred to as the ‘histone code’, which is still poorly understood [ 2 , 3 ].

Histone PTMs often have either activating or repressive effects on gene transcription depending on the type of PTM (acetylation, methylation, etc.) and the position being modified (H3K4, H3S10, etc.). In general, distinct classes of reader domains bind to specific types of PTMs; for example, bromodomains recognize lysine acetylation [ 4 ], chromodomains recognize methyl-lysine [ 5 ], and the PHD fingers characterized to date generally recognize unmodified or methylated lysine residues [ 6 ]. Furthermore, many chromatin-associated proteins contain multiple reader domains, either multiples of the same type [ 7 ] or a variety of different domains [ 8 ], potentially meaning that the in vivo engagement with chromatin is multivalent. Significantly, increasing evidence shows that dysregulation of the epigenetic machinery, most notably the readers, writers, and erasers of the histone code, is causal for a wide range of human disease, including cancer [ 9 ].

Plant homeodomain fingers comprise one of the largest families of reader domains, with over 100 human proteins containing this module [ 6 ]. PHD fingers are Zn-coordinating domains that generally recognize unmodified or methylated lysines. To date, the majority of those characterized bind to histone H3 tails either methylated at K4 [ 7 ], or unmodified in that position (i.e., KDM5B PHD3 versus KDM5B PHD1 [ 10 , 11 ] or PHF21A, also known as BHC80 [ 12 ]). A smaller number of PHD fingers are reported as readers of H3K9 trimethylation (H3K9me3; e.g., CHD4) [ 13 , 14 ] and H3K36me3 (e.g., budding yeast Nto1) [ 15 ]. Intriguingly, the dual PHD finger region of DPF3b has been reported as a reader of H3K14ac [ 16 ], while PHD6 of MLL4 has been reported to recognize H4K16ac [ 17 ]. Additionally, a number of these PHD fingers occur in tandem (e.g., MLL1-4 [ 7 ] and PZP-containing proteins [ 18 , 19 ]) or next to additional reader domain types (e.g., bromodomains and chromodomains) [ 20 , 21 , 22 ], suggesting combinatorial interaction capabilities.

Despite great progress in uncovering the role of a subset of PHD fingers, many (over 100) of the annotated domain family remain uncharacterized. In this report, we set out to close the gap in our understanding of this reader domain class. Using a combination of complementary approaches (reader domain microarrays, peptide microarrays, pulldowns, and AlphaScreen peptide assays), we show (31/123) of the PHD-containing query proteins to bind histone H3 N-terminal peptides, with the majority of these preferring H3K4me3 over unmodified H3K4. Furthermore, a number of unreported histone PTM–PHD protein interactions were uncovered, with the PHD regions of PHRF1 and TRIM66 binding preferentially to an unmodified H3 N-terminal tail peptide. Given that many of these PHD fingers are mutated in diseases such as breast cancer and leukemia [ 7 , 20 , 21 , 22 , 23 , 24 ], these findings enhance our overall understanding of PHD reader–histone interactions and should serve as a resource and platform for future studies.

Analysis of the PHD finger proteome via protein domain microarrays

To define the histone binding preferences of the PHD finger proteome, we expressed and purified 123 annotated human PHD-containing domains as GST-tagged recombinant fusions from E. coli . The recombinant proteins consisted of either PHD fingers in isolation, or as tandem domains if a given PHD finger was located adjacent to another reader domain (e.g., one or more PHD fingers, Tudor, chromo and/or bromodomains) (Additional file 1 : Table S1). These GST fusions were printed in duplicate on nitrocellulose-coated microarray slides and probed with biotinylated peptides that represented the N-termini of H3, H4, H2A or H2B (Fig.  1 a and Additional file 2 : Figure S1). As the majority of PHD readers thus far characterized are H3K4me0/3 readers [ 6 ], we included additional peptides (H3K4 as either mono-, di-, or trimethylated) to further determine any H3K4 methyl preference (Additional file 2 : Figure S2 and Fig.  1 b). As a control, we also probed these microarrays with an α-Tubulin peptide (a.a. 30–50) that would not be predicted to interact with PHD fingers (Additional file 2 : Figure S1). As in Fig.  1 a, b, 31 of the 123 PHD-containing fusions showed positive binding to the H3 N-terminus, with the majority of these interactions showing preference for trimethylated H3K4. In contrast, the H2A, H2B, H4, and tubulin peptides showed little to no positive interactions, suggesting that the PHD finger family broadly prefers the histone H3 tail (Additional file 2 : Figure S1). We note that the absence of binding in these experiments does not rule out the possibility of PHD-finger:histone PTM recognition under different hybridization conditions. We also cannot exclude the possibility that some PHD fingers might not be functionally active on the microarrays (perhaps due to misfolding or the lack of an important adjacent region).

PHD domain array identifies 31 H3-interacting proteins. a PHD finger domain microarray probed with an unmodified H3 N-terminal peptide (1–20) (see “ Methods ”). Each positive binding interaction appears as a green circle, with each PHD protein in the array spotted in technical duplicate (indicated by connecting white lines). a PHD finger domain array probed with an H3 (1–20) peptide trimethylated at residue K4 (K4me3). c The 31 H3-interacting proteins are listed by their preference for binding H3 (1–20) K4me3 or K4me0. Each protein listed corresponds to the numbers in a , b . TTP Tandem Tudor domain + PHD, PPCC Dual PHD + Dual Chromodomain, PCC PHD + Dual Chromodomain, CW CW-type Zn-finger, PB PHD + Bromodomain, PPC2W2 Dual PHD + C2W2-type Zn-finger, SPB SAND + PHD + Bromodomain; domains not indicated, one PHD finger. For the entire list of proteins used and the microarray map, see Additional file 1 : Table S1

Based on the above, we were able to classify the [PHD–H3 tail] interactions into three groups, namely PHD fingers that: (1) bound specifically with methylated H3K4; (2) interacted only with unmethylated H3K4; or (3) bound without preference to the H3K4 methylated state. Many of the PHD fingers found to only bind H3K4 methylation have previously been described and include the well characterized domains from the ING and PHF protein families [ 6 , 24 ]. The PHD finger of MLL5, a member of the MLL/KMT2 family [ 25 , 26 , 27 , 28 ], showed strong preference for H3K4me2 and H3K4me3. This finding adds to the relatively small number of MLL5-histone PTM observations reported to date [ 25 ]. Of the PHD fingers that bound to H3K4 methylation specifically, we observed that H3K4me3 or H3K4me2 were largely recognized equivalently and these domains did not detect H3K4me1 to the same degree (Additional file 2 : Figure S2)—a result in agreement with other reports showing H3K4me binding occurs largely on higher methylated states [ 6 ]. Again, as with the H3K4me3 interacting PHDs, our findings for proteins such as KDM5A [third PHD finger (PHD3)] and KDM5B [third PHD finger (PHD3)] are consistent with their current classification as H3K4me3 binders [ 10 , 11 ]. In contrast to H3K4me2/3 binding, a smaller number of PHD fingers [e.g., PHD1 from KDM5A and KDM5B, PHF21A, AIRE (PP), and TRIM66 (PB)] showed preference for the unmethylated H3K4 state (Fig.  1 a, c). Furthermore, three PHD fingers we tested showed no preference between the H3K4me0 and H3K4me3 peptides: PHRF1 (RP), CHD5 (PCC), and KDM5B (PHD3) (Fig.  1 ). Collectively, these experiments identified 31 PHD-containing reader domains that showed positive interaction with the H3 N-terminus. While a majority of these reader domains preferentially interacted with H3K4me3 (18 out of 31) or H3K4me0 (10 out of 31), three showed no preference for the state of modification at K4. Importantly, these analyses uncovered several reader:histone interactions for poorly characterized PHDs (i.e., TRIM66, PHRF1, and SP140L): such insight could provide new avenues of investigation to these disease-relevant proteins [ 29 , 30 , 31 , 32 ].

Further characterization of H3-reading PHD fingers by peptide microarrays

To more comprehensively define the histone interactions of the 31 PHD readers identified from the domain microarray analyses, we probed each on an alternate microarray platform containing a library of 293 synthetic histone peptides with single or combinatorial PTMs [ 33 ] (Additional file 2 : Figure S4 and Additional file 3 : Table S2). All screening results can be found in Additional file 3 : Table S2, but for brevity, findings pertaining to peptides that contain K4 and K9 modifications as well as neighboring phosphorylation sites that impinge on the observed binding by reader domains are displayed in the form of a normalized heatmap (Fig.  2 ). In general, the 31 PHD fingers were confirmed to associate with the H3 tail with the same H3K4 methyl preferences as in the domain microarrays (Fig.  2 ; Additional file 3 : Table S2). Notably, the MLL5 PHD finger displays a strong preference for H3K4me3 over the un-, mono-, or di-methylated H3K4 peptides (Fig.  2 ), and further, over all other histone peptides on the array (Additional file 3 : Table S2), consistent with results from the domain array (Fig.  1 ). Since CHD4, a protein annotated to recognize H3K9me3 [ 13 , 14 ], was a positive binder in this assay, we compared its binding to H3K9me3 or H3K4 methyl peptides along with their unmodified counterparts at each position (K4me0/K9me0). The CHD4 (PPCC) fusion bound H3 N-terminal peptides more strongly when H3K4 was unmodified and dually acetylated at K9 and K18 versus when H3K4 is methylated in an identically acetylated context (Fig.  2 ); additionally, there was no difference in binding to the H3K4me0 peptide versus the H3K9me3 peptide. Interestingly, there also seems to be increased binding with CHD4 (PPCC) to the H3 K9ac peptide, potentially due to the “surface effect” (described in detail below). In addition, we confirmed the newly identified interactions observed with the domain microarrays for PHRF1 and TRIM66 (Fig.  2 ).

A majority of PHD-containing proteins identified in the domain array are H3 K4me3 readers. The heatmap represents relative binding of the indicated H3 N-terminal peptides (left side) to the PHD-containing GST-tagged proteins (top). Binding strength is shown as a color gradient from red to blue (stronger to weaker). Most of the 31 PHD proteins preferentially recognize H3K4me3 when residues K9 and K18 are acetylated. Array signals ( n  = 4) were normalized individually for each protein to the highest signal for each respective array; thus, comparisons should only be made between binding strengths of different peptides for the same protein. TTP Tandem Tudor domain + PHD, PPCC Dual PHD + Dual Chromodomain, PCC PHD + Dual Chromodomain, CW CW-type Zn-finger, PB PHD + Bromodomain, PPC2W2 Dual PHD + C2W2-type Zn-finger, SPB SAND + PHD + Bromodomain; domains not indicated, one PHD finger. For full construct information, see Additional file 1 : Table S1 and Additional file 2 : Figure S3. For full peptide microarray data, see Additional file 3 : Table S2

While findings between the domain microarrays and peptide microarrays largely agreed, there were some interesting differences. For example, PHRF1 (RP) showed no preference for the H3K4 methyl state on the domain array but strong preference for H3K4me0 on peptide microarray. Furthermore, KDM5B (PHD3), is reported to bind H3K4me3 [ 11 ], and showed such a preference on peptide microarrays but not on domain microarrays (Figs.  1 and 2 ). It should be noted that the comparison made here is between the H3K4me3 + K9ac + K18ac and the H3K4me0 + K9ac + K18ac peptides. Due to the limited binding, if any, observed by the non-acetylated versions of these peptides, it is difficult to assess the binding preference displayed by KDM5B (PHD3) with this comparison. Of note, certain PHD readers [i.e., DIDO1 and DPF2 (PPC2W2)] also showed some interaction with a number of H4 N-terminal peptides (Additional file 3 : Table S2), consistent with published reports [ 33 , 34 ].

During the course of this study, we observed that domain binding to H3 peptides tended to be enhanced when neighboring lysine residues were additionally acetylated (e.g., [K9ac + K18ac] for H3K4me0 or H3K4me readers) (Fig.  2 ). While at first approximation it might appear that these readers have an enhanced affinity for poly-acetylated states that neighbor H3K4, we note that solution-based peptide pulldown or AlphaScreen (see below) assays with several of these readers (i.e., KDM7A that binds H3K4me3 and KDM5B (PHD1) that reads H3K4me0) did not support this idea (Additional file 2 : Figure S5 and Fig.  3 i). We surmise that the enhanced binding caused by poly-acetylation is a property of the charged surface of the streptavidin-coated glass slides: when modified with bulky and neutral acetyl groups the highly charged histone N-terminal tail peptides become more accessible to reader domains.

dCypher histone peptide-binding assays define the PTM recognition preference of PHD proteins with high sensitivity. a – h Binding curves to determine optimal reader protein concentration for full peptide library screening on the dCypher ® AlphaScreen ® platform (see “ Methods ”). X -axes are log(protein concentration ( M )) at constant peptide concentration (100 nM); Y -axes are AlphaScreen counts, representing relative strength of binding ( n  = 2; error bars are S.D.). i Heat map represents relative binding to H3 N-terminal peptides (left) by PHD-containing GST-tagged proteins (top) using the dCypher AlphaScreen platform. Protein concentrations can be found in Additional file 5 : Table S4. Binding strength is indicated by color gradient from green to yellow (stronger to weaker). The asterisk (*) by MLL5 signifies its general preference for H3K4 methylation. Alpha counts ( n  = 2) were normalized individually for each protein to the highest signal for each respective assay. For full dCypher peptide screen data, see Additional file 4 : Table S3

Quantitative assessment of poorly defined PHD readers by the AlphaScreen dCypher assay

We next employed a highly sensitive proximity-based AlphaScreen histone peptide assay ( dCypher ® ) to provide a third and orthogonal approach to analyzing the histone binding preferences for a subset of the 31 PHD proteins with respect to various histone tail PTMs. In this assay, biotinylated peptides are bound to streptavidin “donor” beads and the GST-tagged reader domains bound to Glutathione “acceptor” beads. The donor beads are excited by 680 nm light, releasing a singlet oxygen which causes light emission (520–570 nm) in proximal acceptor beads (within 200 nm); emission intensity is then correlated to binding strength [ 35 ]. For further examination with this more sensitive approach we chose the PHD fingers with positive binding data from the domain and peptide microarrays that were less characterized in the literature [i.e., MLL5, PHRF1 (RP), and TRIM66 (PB)], or those that displayed weak interactions on the domain and/or peptide microarrays [i.e., CHD4 (PPCC) and CHD5 (PPCC)]. Additionally, we examined several well characterized PHD–PTM interactors [DIDO1, KDM7A, and DPF2 (PPC2W2)] for positive controls and to provide a benchmark. Initial binding assays were conducted for each fusion protein using three peptides [H3 (1–20) with K4me0, H3K4me3 or H3K9me3] to determine the optimal reader domain concentration for full peptide library studies (Fig.  3 a–h; Additional file 4 : Table S3 and Additional file 5 : Table S4). This is an important first step as signal often declined after query protein saturation (the ‘hook point’, caused by excess free query competing with bead bound).

Once the optimal protein concentration ranges for each of the eight readers were determined, we conducted the full dCypher peptide screen (293 histone peptides) (Fig.  3 i; Additional file 4 : Table S3). In agreement with our previous findings, the dCypher peptide assay demonstrated KDM7A to be a reader of H3K4me3. Furthermore, TRIM66 (PB) showed a preference for H3K4me0 and me1, consistent with findings from the peptide microarrays. For CHD4 (PPCC), the dCypher approach showed a clearer specificity for the H3K4me0 peptide over the methylated species in comparison to the peptide microarray results (Fig.  3 i versus Fig.  2 ). In the case of CHD5 (PPCC), the peptide microarray indicated this protein to be insensitive to the methylation status at H3K4 (Fig.  2 ), but the dCypher assay identifies a preference for H3K4me0/1 (Fig.  3 i), consistent with the domain microarray (Fig.  1 a, c).

Consistent with the results from the domain and peptide microarrays, dCypher assays confirmed that the PHD finger of DIDO1 and MLL5 recognized the higher methyl states of K4 (me3/2), but also identified interaction of these domains with the H3K4me1 peptide. Interestingly, the four H3K4me0 readers analyzed—CHD4 (PPCC), DPF2 (PPC2W2), TRIM66 (PB), CHD5 (PPCC)—also showed the ability to bind to the peptides containing H3K9me3; this may be due to H3K4me0 in the H3K9me3 peptide. However, CHD4 (PPCC) and TRIM66 (PB) showed stronger interaction with H3K9me3 compared with the unmodified peptide over a range of protein concentration (Fig.  3 d, f). We note that while the initial protein concentration optimizations in Fig.  3 a–h were performed over a range of protein concentrations, the full peptide screen (Additional file 5 : Table S4; summarized in panel Fig.  3 i) was performed at a single protein concentration. When presented with the [H3K9me3 + S10p] peptide, four out of five of the H3K4me0 readers lose binding capacity, suggesting that these readers are sensitive towards the bulky negative phosphate group at S10; this phenomenon is also observed with the H3S10p peptide alone (Additional file 4 : Table S3). To our knowledge, this would be the first report of a H3 tail binder outside the H3K9 position to be impacted by S10 phosphorylation, suggesting the phospho-methyl switch may function more broadly than previously thought. Intriguingly, PHRF1 (RP) binding specificity at 15 nM showed more limited interactions to H3K4me0 and H3K9me3 peptides (Fig.  3 i), which will be discussed further below. Finally we note that the shift for poly-acetyl peptides seen in the peptide microarrays (reflecting a possible “surface effect”; Fig.  2 ) is not observed in the dCypher screen (Fig.  3 i) which more closely resembles the peptide pulldown assays (Additional file 2 : Figure S5).

In the epigenetic landscape, histone PTMs can impact chromatin organization through their ability to recruit effector or “reader” domain-containing proteins. These reader proteins, which are also found in large multi-subunit chromatin-modifying machines, interact with histones and chromatin in various ways that regulate processes from gene transcription to chromosome segregation at mitosis [ 2 ]. Given that many of these reader proteins are widely dysregulated in human disease, understanding their histone binding preferences and modes of multivalent interactions is vital [ 36 ]. In this study, we screened 123 PHDs (singly and in tandem when next to another reader domain) against the core histone N-terminal tails to dissect the binding preferences for this poorly understood reader domain family. With over 100 PDHs represented on our domain microarrays, we determined that the family strongly prefers the histone H3 tail. Furthermore, the majority of the domains that displayed binding preferred the higher orders of H3K4 methylation, with two subsets showing either a preference for H3K4me0, or no preference to the H3K4 methyl state.

Our findings from domain and peptide microarray confirm the reported binding preferences of many PHD proteins such as those of the ING and PHF families [ 6 , 24 ]. Additionally, the PHD finger from MLL5 was shown to robustly bind peptides containing each methyl state at H3K4 (me1-2-3) on the domain microarray and dCypher screen, while the peptide microarrays suggest MLL5 is a specific reader for H3K4me3. Intriguingly, we note that previous studies have found discrepancies in whether the PHD finger of MLL5 is a H3K4me3 or H3K4me2 reader [ 25 , 26 ]. We surmise that the basis of this difference may be due to the overall sensitivity of the various assays employed, which also may account for different observations in the literature. Nonetheless, our analyses provide strong support for MLL5 as a binder of H3K4 methylation on peptides. While recent work has suggested the disease relevance of MLL5 [ 26 ], few studies have characterized its histone PTM binding preferences and whether such interaction contributes to its normal or disease functions [ 25 ]. The domain microarrays also identified two poorly characterized proteins—TRIM66 and PHRF1—as readers of the unmodified H3 tail. Both proteins are E3 ligases that contain a PHD finger, but whose histone binding capabilities have not been well documented [ 29 , 30 , 31 ]. How these histone interactions contribute to the function of these ligases is currently unknown but will be interesting to determine in future studies.

While our domain microarrays revealed 31 out of 123 tested PHD proteins to be binders of the H3 N-terminus (Fig.  1 and Additional file 1 : Figures S1, S2), this does not preclude the potential for other PHD fingers to bind under alternate hybridization conditions or to unrepresented targets. Reader domain–histone PTM interactions are multifaceted, and while the results of this study’s domain array do confirm published observations as well as revealing new and interesting binding preferences, we point out that they are not meant to represent an exhaustive list of PHD-mediated interactions but rather to serve as a community resource.

Although domain microarrays are useful in probing many domains in high-throughput, they are limited by the ability to probe with one peptide of interest at a time. To further define the histone PTM landscape to which the subset of 31 PHD proteins identified in the domain microarray might bind, we employed the opposite approach of analyzing each individual domain against a microarray containing ~ 300 singly or combinatorially modified histone peptides (Fig.  2 ; Additional file 3 : Table S2). Through this approach, we were able to confirm many of the interactions observed on the domain microarray with respect to the H3K4me0/1/2/3 peptides. Significantly, the peptide microarray showed that PHRF1 (RP) specifically bound H3K4me0 over K4me, whereas it had no preference on the domain array—which may be explained by the fact that proteins and peptide concentrations on the domain microarrays are high, and thus may capture weak binding events that may not be observed on other platforms.

Despite the obvious potential of peptide microarrays, it would be remiss not to note possible limitations of the platform. The dynamic range of detected interactions is narrow, and from extensive experience, we are only able to characterize domain–peptide interactions on a four-point scale (very strong, strong, weak, or not detected). In addition, these interactions do not represent values that can be translated into binding affinities. Furthermore, comparing values between different probed arrays is also challenging given the lack of a platform control that can be used to normalize signals between arrays. We have also identified potential biophysical artifacts of the platform: we confirmed with these arrays that domains interacting with the H3 N-terminus are influenced by the neighboring acetylation status—a result observed in past publications with PHD readers using these or similar microarrays [ 37 , 38 ]. However, the impact of H3 acetylation on reader domain binding in the platform context appear to be indirect, as the solution-based binding reactions conclusively show that PHD fingers do not prefer H3K4me0-3 in the context of neighboring acetylation. Rather, it appears that streptavidin-coated slides may carry some amount of negative charge that binds the positively charged histone tails except when this is neutralized (e.g., by acetylation) and thus released from the surface. This “surface effect” shifts the H3 N-terminal binding preferences for many reader proteins towards acetylated peptides, but it is clear that the binding preferences for PHD fingers are primarily driven by direct interactions towards H3K4 ( ∓ methylation). Although this is a technical challenge, it does not preclude the use of peptide microarrays as the end user can be aware of the role of neighboring acetylation and how to put such results in context.

In contrast to the histone peptide microarrays, the dCypher AlphaScreen histone peptide assay has recently emerged as a highly sensitive and robust technique in gauging the binding interactions between reader domains and histone PTMs [ 35 ]. Furthermore, this method allows for the thorough optimization of reaction conditions in terms of buffers, protein/peptide/salt concentration, and cofactor/competitor additives to enable the study of otherwise poorly behaved proteins of interest. Given the advantages of this platform, we used the dCypher assay to first optimize the binding conditions for PHD fingers, and then proceeded to a variety of the PHD fusions that showed low/weak binding or novel histone PTM interactions on the microarrays. The dCypher approach is sensitive and benefits from an initial optimization step for each protein (see Fig.  3 a–h) to find the optimal concentration needed in the assay (see Fig.  3 i). Using this approach, we were able to confirm that several poorly characterized proteins including TRIM66 are indeed robust readers of H3K4me0 peptides. Intriguingly, the highly sensitive nature of the dCypher assay allowed comparison of peptide-binding signal at low versus high protein concentrations, which revealed that PHRF1 had a distinct binding preference for the H3K9me3 peptide over the H3K4me0 peptide. Importantly, the domain and peptide microarrays rely on micromolar reader domain concentrations, while the dCypher assay can reliably measure binding signal with proteins in the picomolar range. Thus, the dCypher screen revealed the ability of some domains to have distinct preferences at different concentrations that could not be determined from the other approaches. Whether such distinct histone binding preferences in the context of N-terminal peptides are physiologically relevant and could effectively represent the local concentration of particular reader domain on chromatin is currently unknown but is interesting to consider.

In this report, we have employed multiple high-throughput methods such as domain and peptide microarrays, as well as the proximity-based dCypher peptide screen to assemble a large dataset describing histone PTM binding preferences for PHDs, starting from a broad analysis of the entire family narrowing down to 31 histone H3-interacting readers. While we used the domain microarrays as an initial guide for which proteins to employ in further characterizations, we expect that further exploration of the remaining readers on this microarray platform will uncover additional interactions when binding conditions are further explored (e.g., the PHD domains of UHRF1/2 that were negative in the assays but reported to also bind H3 [ 39 , 40 ]). Assay development for studying chromatin-interacting proteins has been on the rise in the last decade and we believe that it will be necessary to understand how PHD readers interact with histone PTMs in a nucleosomal context alongside peptides to better replicate physiological conditions. Further, while the bulk of literature and indeed the focus of this study concerning PHD proteins has focused on their interactions with histones, the possibility of these readers binding non-histone biomolecules is intriguing and merits further study. Taken together, we expect our findings to serve as a resource for the chromatin community and to provide a framework for future studies regarding plant homeodomain proteins.

Protein domain array

The protein domain microarray was designed to include 123 GST-tagged PHD-domain containing recombinant proteins. Protein domain microarray development and probing was as previously [ 41 , 42 , 43 ]. Briefly, recombinant proteins were synthesized and cloned into pGEX-4T-1 vector by Biomatik Corporation. These GST-PHD readers were subsequently expressed, purified, and spotted in duplicate onto nitrocellulose-coated glass slides (Oncyte Avid slides, Grace Bio-Labs) using a pin arrayer (Aushon 2470, Aushon). For probing, microarray slides were blocked with 3% milk, 3% bovine serum albumin, 0.1% Tween 20 in PBS. Biotinylated peptides were pre-labeled with streptavidin-Cy3 fluorophore (GE Healthcare) and incubated with the blocked array slides. Slides were then washed with PBST and allowed to air dry. Fluorescent interactions were visualized using a GenePix 4200A Microarray Scanner (Molecular Devices).

Protein purification, histone peptide microarrays, and peptide pulldown assays

The 31 GST-tagged PHD readers identified in the PHD finger domain array were expressed and purified as previously [ 33 ]. Histone peptide arrays and peptide pulldown assays were conducted as recently described (specifically, the optimized protocol from Petell et al. for the former) [ 33 ].

dCypher Alphascreen peptide screen assay

The dCypher peptide screen assay was performed as previously described [ 35 ]. Briefly, 5 μL of GST-tagged reader domains (optimal protein concentration for library screening determined by initial binding curves to candidate peptides) were incubated with 5 μL of 400 nM (100 nM Final) biotinylated histone peptides ( EpiCypher ) for 30 min at 23 °C in 1× AlphaLISA Epigenetics buffer + epigenetics buffer supplement ( PerkinElmer , AL1008) in a 384-well plate. A 10 μL mix of 5 µg/mL (2.5 μg/mL final) glutathione Acceptor beads ( PerkinElmer , AL109M) and 10 μg/mL (5 μg/mL final) streptavidin Donor beads ( PerkinElmer , 6760002) was prepared in 1× [Epigenetics buffer + supplement] and added to each well. Plates were incubated at 23 °C in subdued lighting for 60 min and AlphaLISA signal measured on a PerkinElmer 2104 EnVision (680 nm laser excitation, 570 nm emission filter ± 50 nm bandwidth).

Availability of data and materials

The datasets used and/or analyzed during this study are included as additional files. All plasmids are available from the corresponding authors on request.

Abbreviations

plant homeodomain

post-translational modifications

Tandem Tudor domain + PHD

Dual PHD + Dual Chromodomain

PHD + Dual Chromodomain

CW-type Zn-finger

PHD + Bromodomain

Dual PHD + C2W2-type Zn-finger

SAND + PHD + Bromodomain

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Acknowledgements

We thank the Strahl and Bedford labs for helpful comments and suggestions.

This work was supported by NIH Grant GM126900 to BDS. The UT MDACC Protein Array & Analysis Core (PAAC) is supported by CPRIT Grant RP180804 (MTB). EpiCypher and MTB are supported by NIH Grant R44GM116584, with EpiCypher further supported by R44GM117683 and R44CA214076. KJ is supported by Postdoctoral Training Fellowship T32CA217824 from the NCI and UNC Lineberger Comprehensive Cancer Center.

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Kanishk Jain, Caroline S. Fraser, Madison M. Parker & Brian D. Strahl

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Caroline S. Fraser & Brian D. Strahl

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Matthew R. Marunde, Jonathan M. Burg, Nathan Hall, Irina K. Popova, Keli L. Rodriguez, Anup Vaidya & Michael-Christopher Keogh

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Contributions

KJ, BDS, and MTB conceived the project, with input from all authors. KJ, CSF, and MMP purified proteins and performed peptide microarrays and peptide pulldowns. CSF and CS performed domain microarrays. MRM and JMB were responsible for dCypher assay design, with NH, IKP, KLR, and AV performing dCypher analyses. All authors analyzed the data and discussed the results. KJ and BDS wrote the manuscript with input from all authors. All authors read and approved the final manuscript.

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Correspondence to Mark T. Bedford or Brian D. Strahl .

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BDS and MTB are co-founders, shareholders, and scientific advisory board members of EpiCypher, Inc. EpiCypher is a commercial developer and supplier of reagents (e.g., synthetic histone peptides) and the dCypher ® peptide-binding platform.

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Supplementary information

Additional file 1: table s1..

PHD finger Domain Array Constructs and Map. Excel Tab PHD Proteins is a compiled list of the 123 GST-tagged proteins used for the domain array. The list contains information on the domains, UniProt accession numbers, sequence coverage, and amino acid sequence for each protein. Excel Tab Array Map represents the pattern in which each of the GST-tagged proteins has been printed for the domain microarray.

Additional file 2: Figure S1.

Positive and negative controls of PHD finger domain arrays. Figure S2. PHD finger domain array with H3 (1-20) K4me1 and K4me2. Figure S3. Domain architecture of the 31 human PHD-containing proteins identified as hits for H3K4me0 or H3K4me3 via protein domain array (Fig.  1 ). Figure S4. Peptide arrays for 31 PHD-containing proteins. Figure S5. Peptide Pulldowns with KDM7A and KDM5B (PPC2W2).

Additional file 3: Table S2.

Histone Peptide Microarray Results. Excel Tab Peptide List is a compiled list of the entire 293 histone peptide library with amino acid sequence ranges. Excel Tab Peptide Array Grid Map is a table representing the pattern in which peptides have been printed for the peptide microarray. Each number corresponds to the peptide number designated in Peptide List. Excel Tab Average Signals is a compiled set of binding data: chromatin reader at 0.5 μM vs. entire 293 peptide library. Data is presented as the average and standard deviation of 4 replicates. The average signals are colored to indicate signal intensity with green being strong and white being weak/low. Excel Tab Array Heatmap contains a condensed form of Average Signals data displaying an average of 4 replicates for each chromatin reader tested and individually formatted to demonstrate binding specificity. Key: red= stronger relative binding; blue = weaker relative binding. Excel Tab Array info summary is a table summarizing the domains, sequence coverage, array signal range, and noise for each of the GST-tagged readers assayed on the peptide microarrays.

Additional file 4: Table S3.

dCypher Results. Excel Tab Peptide Phase A is a compiled set of binding data: chromatin reader titration vs. several peptide targets. Data is presented as raw Alpha counts in duplicates at each concentration tested. Excel Tab Peptide Phase B is a compiled set of binding data: chromatin reader at optimized concentrations vs. entire 293 peptide library. Data is presented as the average and standard deviation of duplicates. Excel Tab Peptide Phase B Heatmap contains a condensed form of Peptide Phase B data displaying an average of duplicates for each chromatin reader tested and individually formatted to demonstrate binding specificity. Key: red = strong binding; blue = weak/no binding.

Additional file 5: Table S4.

dCypher screen reader domain concentrations.

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Jain, K., Fraser, C.S., Marunde, M.R. et al. Characterization of the plant homeodomain (PHD) reader family for their histone tail interactions. Epigenetics & Chromatin 13 , 3 (2020). https://doi.org/10.1186/s13072-020-0328-z

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• Histone methylation
• PHD fingers
• Histone peptide microarray
• Protein domain microarray

Epigenetics & Chromatin

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Genome wide survey, evolution and expression analysis of PHD finger genes reveal their diverse roles during the development and abiotic stress responses in Brassica rapa L.

• Intikhab Alam 1 ,
• Cui-Cui Liu 1 ,
• Hong-Liu Ge 1 ,
• Khadija Batool 1 ,
• Yan-Qing Yang 1 &
• Yun-Hai Lu   ORCID: orcid.org/0000-0003-2044-2511 1 , 2  

BMC Genomics volume  20 , Article number:  773 ( 2019 ) Cite this article

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Plant homeodomain (PHD) finger proteins are widely present in all eukaryotes and play important roles in chromatin remodeling and transcriptional regulation. The PHD finger can specifically bind a number of histone modifications as an “epigenome reader”, and mediate the activation or repression of underlying genes. Many PHD finger genes have been characterized in animals, but only few studies were conducted on plant PHD finger genes to this day. Brassica rapa (AA, 2n = 20) is an economically important vegetal, oilseed and fodder crop, and also a good model crop for functional and evolutionary studies of important gene families among Brassica species due to its close relationship to Arabidopsis thaliana.

We identified a total of 145 putative PHD finger proteins containing 233 PHD domains from the current version of B. rapa genome database. Gene ontology analysis showed that 67.7% of them were predicted to be located in nucleus, and 91.3% were predicted to be involved in protein binding activity. Phylogenetic, gene structure, and additional domain analyses clustered them into different groups and subgroups, reflecting their diverse functional roles during plant growth and development. Chromosomal location analysis showed that they were unevenly distributed on the 10 B. rapa chromosomes. Expression analysis from RNA-Seq data showed that 55.7% of them were constitutively expressed in all the tested tissues or organs with relatively higher expression levels reflecting their important housekeeping roles in plant growth and development, while several other members were identified as preferentially expressed in specific tissues or organs. Expression analysis of a subset of 18 B. rapa PHD finger genes under drought and salt stresses showed that all these tested members were responsive to the two abiotic stress treatments.

Conclusions

Our results reveal that the PHD finger genes play diverse roles in plant growth and development, and can serve as a source of candidate genes for genetic engineering and improvement of Brassica crops against abiotic stresses. This study provides valuable information and lays the foundation for further functional determination of PHD finger genes across the Brassica species.

Zinc finger proteins are abundantly present in both prokaryotic and eukaryotic genomes, including the plant kingdom [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. They are characterized by the presence of one or more sequence motifs in which cysteines and/or histidines coordinate one or more zinc atoms to form stable local peptide structures (zinc fingers, ZFs) that are required for their specific functions [ 2 , 4 ]. The zinc finger was first identified in Xenopus laevis transcription factor IIIA (TFIIIA) in 1985 [ 9 ], and the three dimensional solution structure of a single zinc finger was first reported in 1989 [ 10 ]. Since then, various other zinc binding motifs have been identified and characterized, and as high as 30 types of Zinc finger proteins were currently identified in human genome based on the zinc-finger domain structure [ 11 , 12 ]. The most common types of zinc finger proteins include C2H2, RING ( really interesting new gene ), PHD ( plant homeodomain ), and LIM ( Lin-ll, Isl-1 and Mec-3 ) families [ 2 , 12 , 13 ]. These varied zinc finger domains enable different proteins to interact specifically with cognate DNA, RNA, proteins, lipids (or membrane), and small molecules through hydrogen bonds and hydrophobic interactions [ 14 , 15 , 16 ]. Proteins containing zinc finger domain (s) were found to play important roles in various molecular, physiological and cellular processes in cells or tissues, and some of them may function as part of a large regulatory network that senses and responds to different environmental stimuli, and regulate different signal transduction pathways and controlling processes, such as development and programmed cell death [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 12 , 17 , 18 , 19 , 20 , 21 ].

The PHD finger was first identified in Arabidopsis thaliana transcription factor HAT3.1 (a homeodomain-containing protein) and its maize homolog Zmhox1a in 1993 [ 22 ]. Since then, many other PHD-finger proteins have been identified in various eukaryotes, including the yeast [ 23 , 24 ], Drosophila [ 25 , 26 ] and human [ 12 , 27 , 28 ]. The PHD finger can be defined as a Cys-rich domain of approximately 50~80 amino acids with spatially conserved 8 metal ligands arranged as unique Cys4-His-Cys3 pattern in 4 pairs which can chelate two Zn 2+ atoms and form a cross-brace structure [ 13 , 29 , 30 ]. The PHD finger can specifically bind a number of histone modifications as an “epigenome reader”, and mediate the activation or repression of underlying genes [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. In human, mutations in PHD fingers or deletions of these domains are linked to a number of diseases such as cancer, mental retardation, and immunodeficiency [ 32 , 33 ]. In plant, the PHD domains were found to be involved in the transcriptional regulation of developmental processes such as meiosis and postmeiotic events during pollen maturation, embryo meristem initiation and root development, germination, flowering time, etc. [ 36 ].

The Brassicaceae or Cruciferae is one of the most important families of flowering plants, containing some 338 genera and approximately 3709 species, with an extreme high level of morphological diversity [ 37 , 38 ]. The family includes a number of economically important species of the genus Brassica cultivated worldwide as vegetables, oil seed crops, condiments and fodder crops, as well as the extensively studied model plant Arabidopsis thaliana [ 39 ]. The genomic relationships among the six cultivated Brassica species, including B. rapa (2n = 20, AA genome, 529 Mb genome size), B. nigra (2n = 16, BB, 632 Mb), B. oleracea (2n = 18, CC, 696 Mb), B. juncea (2n = 36, AABB, 1068 Mb), B. napus (2n = 38, AACC, 1132 Mb) and B. carinata (2n = 34, BBCC, 1284 Mb), has long been established as the Triangle of U [ 40 , 41 ]. Previous studies revealed that all the species of the tribe Brassiceae shared a common whole-genome triplication (WGT) event occurred ~ 15.9 million years ago (MYA) just after the divergence of their ancestor from that of A. thaliana (tribe Arabideae) [ 42 , 43 , 44 , 45 ] . This whole genome triplication event was followed by genome diploidization involving substantial genome reshuffling and gene losses in duplicated genomic blocks, and resulted in three subgenomes with different degree of gene losing, e.g. least fractionized (LF), moderately fractionized (MF1) and most fractionized (MF2) subgenomes [ 46 , 47 ]. B. rapa is an important, worldwide cultivated crop with various morphotypes, such as leafy vegetables, turnips and oilseed rape [ 38 , 48 ]. Because of its smallest genome size of the genus Brassica , rapid life cycle, high morphological diversity, and origin from a common hexaploid ancestor as all other members of the tribe Brassiceae, B. rapa became a model plant for genetic, genomic and evolutionary studies in Brassica species [ 47 , 49 ]. The complete sequencing of the B. rapa genome makes it possible to analyze some important gene families at a whole genome level [ 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ].

Abiotic stresses, especially salt and drought stresses, affect many aspects of plant physiology and metabolism, and cause severe crop yield losses around the world [ 57 ]. Brassica crops are mainly grown in arid and semiarid areas, and they are the most affected by drought and salinity among the major food crops [ 58 ]. Several drought or salt-tolerant genes isolated in A. thaliana as well as in Brassica crops showed great potential for genetic improvement of plant tolerance [ 58 ]. In several previous studies [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 ], some PHD finger genes were found to be highly responsive to abiotic stresses, including salt and drought stresses, suggesting that they might play important roles for the response and adaptation to abiotic stresses in plants. In the current paper, we reported the identification and comprehensive analysis of the PHD finger genes in B. rapa genome. Our results lay a foundation for further functional characterization of PHD finger genes among Brassica species.

Identification and characterization of PHD finger genes in B. rapa

A total of 145 non-redundant predicted PHD finger proteins containing 233 PHD finger domains were identified from the Brassica Database (BRAD), of which 92 (65.7%), 27 (18.6%), 17 (11.7%) and 9 (6.2%) contain one, two, three and four putative PHD domain (s), respectively (Additional file  2 : Table S1). The details of these B. rapa PHD protein genes, such as locus name, chromosome location, CDS and amino acid lengths, protein masse, and isoelectric points (pIs), were summarized in Additional file 2 : Table S1. We also identified 16 PHD-suspected domain-containing proteins that contain each an imperfect PHD motif (Additional file 2 : Table S2).

The identified 233 B. rapa PHD domains were extracted from their corresponding protein sequences, and archived in Additional file 2 : Table S1. Based on these domain sequences, a multiple sequence alignment, a sequence logo of the over-represented residues and a phylogenetic tree were generated and illustrated in Additional file  1 : Figure S1, S2 and S3, respectively. Additional file 1 : Figures S1 and S2 illustrated the conservation of eight metal ligands as well as the spacing between them, while Additional file 1 : Figure S3 showed the close evolutionary inter-relationships among some of these PHD domains and their multiplication during the B. rapa genome evolution.

In order to gain a global idea about the function of the B. rapa PHD finger genes, we retrieved their associated Gene Ontology (GO) terms from Phytozome database, and performed the prediction of subcellular localization by CELLO software (Additional file 2 : Table S1). The GO Molecular Function term is available for 138 out of 145 (95%) identified PHD finger proteins, which can be classified into protein-binding activity (126/138 = 91.3%), proteins-disulfide reductase activity (20/138 = 14.4%), and transcription cofactor activity (10/138 = 7.2%) (Fig.  1 a). Ninety-eight out of 145 (67.6%) B. rapa PHD finger proteins were predicted to be localized in nucleus, 25 (17.2%) in extracellular, 10 (6.9%) in plasma membrane, 7 (4.8%) in cytoplasm, and 5 (3.5%) in chloroplast (Fig.  1 b). The distribution of 145 B. rapa PHD proteins containing one to four PHD domain (s) in different cellular compartments was summarized in Table  1 . We observed that near 80% of 1-PHD domain-containing B. rapa PHD finger proteins were localized in nucleus, while only 12.0% in extracellular, 7.6% in cytoplasm, 2.2% in chloroplast, and 0% in plasma membrane. Among both the 2- and 3-PHD domain-containing proteins, a little more than 50% were localized in nucleus, about 20–30% in extracellular, about 20% in plasma membrane, 11 or 0% in chloroplast, and 0% in cytoplasm. Among the nine 4-PHD domain-containing proteins, three (33%) were localized in nucleus, four (44%) in extracellular, and two (22%) in plasma membrane.

Distribution of GO molecular function terms ( a ) and sub-cellular localization ( b ) of 145 Brassica rapa PHD finger proteins

Phylogenetic and gene structure analyses of B. rapa PHD finger proteins

To gain insights into the evolutionary relationships among B. rapa PHD finger proteins, a phylogenetic tree was generated based on the sequences of 145 B. rapa as well as 97 A. thaliana PHD finger proteins (Fig.  2 ). The result showed that these PHD finger proteins could be divided into six major groups, named A, B, C, D, E and F, within which the orthologous or homologous proteins from B. rapa and A. thaliana were closely clustered together (Fig.  2 ). The largest group A contains 51 B. rapa and 30 A. thaliana PHD finger genes. The group B contains 9 B. rapa and 7 A. thaliana PHD finger genes. The group C contains 27 B. rapa and 26 A. thaliana PHD finger genes. The group D contains 9 B. rapa and 7 A. thaliana PHD finger genes. The group E contains 30 B. rapa and 15 A. thaliana PHD finger genes. The group F contains 19 B. rapa and 12 A. thaliana PHD finger genes. Obviously, groups A, C, E and F can be further divided into several subgroups. An enlarged phylogenetic tree including PHD finger proteins from A. thaliana , B. rapa , Oryza sativa , Populus trichocarpa and Zea mays was also generated and presented in Additional file 1 : Figure S4, showing the orthologous relationships and high evolutionary conservation between PHD finger proteins of different species. The Gene Ontology terms associated with these 97 A. thaliana PHD finger genes were summarized in Additional file 2 : Table S3, showing the rich information concerning their functions in A. thaliana that can be used to explore the functions of their corresponding orthologs in Brassica crops.

Phylogenetic tree analysis of the PHD finger proteins from Arabidopsis and Brassica rapa . The tree was determined by using MEGA6.06 software with the Neighbor–Joining (NJ) algorithm and a bootstrap analysis of 1000 replicates. The PHD finger proteins were clustered into six major groups (A-F). Proteins from Arabidopsis and B. rapa are indicated by red and green colors, respectively

Intron loss or gain is another important evolutionary mechanism that generates gene structural diversity and complexity, and contributes to the functional diversity and divergence during the evolution of multi-gene families in plants [ 68 , 69 ]. To obtain insights into the structural variation of B. rapa PHD-finger protein genes, we analyzed their exon/intron organization from the genomic sequences of individual B. rapa PHD-finger protein genes, in relation to their phylogenic tree by groups extracted from the Fig.  2 (Fig.  3 ). The result showed that the most closely related members tended to be clustered together and shared similar exon/intron structures but with exceptions, such as between Bra036726 and Bra026189 in group A, between Bra02633 and Bra07814 in group B, between Bra030481 and Bra035110 in group C, between Bra020445 and Bra035572 in group D, between Bra026708 and Bra016634 in group E, and between Bra029401 and Bra009752 in group F. Among the identified 145 B. rapa PHD finger proteins, 26 (17.9%) (including 19 in group A, one in group C, four in group E, and six in group F) possess each 0 intron, 15 (10.3%) (five in group A, one in group B, one in group C, five in group E, three in group F) contain each 1 intron, while the remaining 104 contain each 2–30 introns (Bra035110 in group C contains 30 introns, Bra007814 in group B contains 28 introns). Bra036288 in group A is the longest gene covering a genomic sequence as long as ~ 21 kb, contrasting to the shortest member Bra006562 covering a genomic sequence of ~ 0.2 kb (Fig. 3 ).

Phylogenetic relationships and gene structure of PHD finger genes in Brassica rapa . The tree was generated by using Neighbor-Joining method with 1000 bootstrap replicates. The tree shows six major phylogenetic groups (group A to F) indicated with six differently colored backgrounds. Green color boxes represent exons and grey color lines indicate introns, and the untranslated regions (UTRs) are represented by blue color boxes. The sizes of exons and introns can be estimated using the scale at the bottom. The tightly clustered genes with remarkable differences in their gene structures are indicated by red stars

Previous studies have shown that tandem PHD fingers can fold as one functionally cooperative unit and be used to read more complex combinations of histone modifications, thus reinforcing the notion that the unequal numbers of PHD-finger domains detected in each protein sequence may contribute to their functional diversity and complexity [ 35 , 70 ]. The distribution of 145 B. rapa PHD proteins containing one to four PHD domain (s) in different phylogenetic groups of Fig.  2 was summarized in Table  2 . We observed that the proportion of 1-PHD domain-containing proteins was very high in group D (88.9%) and C (85.2%), followed by F (63.2%), E (60.0%), A (54.9%) and B (33.3%). The twenty-seven 2-PHD domain-containing proteins were distributed into group A (15.7%), B (44.4%), C (14.8%), E (13.3%) and F (36.8%), while the 17 3-PHD domain-containing proteins into group A (16.7%), D (11.1%) and E (23.3%), and the nine 4-PHD domain-containing proteins into A (11.7%), B (22.2%) and E (3.3%).

Additional domain analysis outside of PHD finger domain

For each of the identified 145 PHD finger protein, the presence or not of any additional known domain outside of the PHD finger domain (s) was inspected through the Smart analysis. A total of 56 additional known domains were identified, allowing classifying the 145 PHD finger proteins into 28 groups and subgroups (Additional file 2 : Table S4). The largest group (group 1) includes 42 members (42/145 = 29.0%) which all contain no other additional domain besides the 1–4 PHD domain (s). The second group includes 15 members (15/145 = 10.3%) which contain each a DUF3594 domain besides a single PHD domain. The third groups includes 9 members (9/145 = 6.2%) which contain each a KAT11, a ZnF_ZZ and a ZnF_TAZ domain besides a single PHD domain. The other 25 groups include each 1–8 members with 1–5 additional known domains. These additional known domains may be involved in protein-protein interaction (Ald_Xan_dh_C2, Coiled-coil, JmJC, PWWP), protein binding (GYF, SWIB), histone binding/acetylation/methylation (BAH, Cohisin Heat, DUF295, DUF1086, DUF3594, DUF1087, ING, KAT11, NIPPED-B_C, Post-SET, SET, SRA), nucleic acid binding (AAA, Ald_Xan_Dh_C2, ARID, AT hook, DDT, DEXDc, DNMT1, HELICc, Helicase_C_4, MBD, PLUS3, PPR, Res III, SANT, SAP, WHIIM, Znf-C2H2, Znf-C5HC2), and Zinc ion binding (Znf-C2H2, Znf-C5HC2, Znf-CCCH, Znf-TAZ, Znf-UBA, Znf-ZZ). Other known domains such as AMP-Binding (catalytic activity), C1 (intracellular signal transduction), DYW_deaminase, ELM2, EloA-BP1, FLU-1, FYRC, FYRN, JAS (jasmonate signaling), MBOAT_2, Oberon_cc and Transmembrane, were also detected.

Table  3 summarized the distribution of 56 additional known domains in the different phylogenetic groups of Fig.  2 . We observed that 46 out of 56 (82.1%) additional domains were specific to a single group, i. e., 7 were specific to group A, 3 to group B, 19 to group C, 6 to group D, 5 to group E, and 6 to F; five out of 56 (8.9%) were simultaneously present in two groups; four out of 56 (7.1%) were simultaneously present in three groups; and one (Coiled-coil domain) out of 56 (1.8%) was simultaneously present in five (A, B, C, E and F) of six groups. The group C contains as high as 26 types of additional domains, compared to a value of 11, 7, 8, 11 and 10 for group A, B, D, E and F, respectively. It is worth to remark that, among the 51 members of group A, 15 (29.4%) shared DUF3594 domain known for involving in histone binding and regulation of transcription activities, and eight (15.7%) shared the C1 domain known for intracellular signal transduction activity. Among the nine members of group B, five (55.6%) shared JAS domain known for jasmonate signaling activity. Group C (27 members) contains very diverse additional domains, including Coiled-coil (14.8%) and PWWP (14.8%) known for protein-protein interaction activity, Post-SET (14.8%) and SET (14.8%) for histone methyltransferase activity, and SAP (11.1%) for DNA-binding involved in chromosomal organization. Group D (9 members) contains additional domains such as GYF (44.4%) and SWIB (66.7%) known for protein binding activity, and PLUS3 (44.4%) for DNA binding activity. Group E (30 members) contains additional domains such as DDT (20.0%) known for DNA binding activity, KAT11 (30.0%) for histone acetylation activity, and Znf-TAZ (30.0%) and Znf-ZZ (30.0%) for zinc ion binding activity. Group F (19 members) contains additional domains such as HOX (21.1%) and Znf-C5HC2 (5.3%) known for DNA binding activity, and JmjC (5.3%) for demethylase activity.

Chromosomal distribution, gene duplication and syntenic relationships

Based on the chromosome location data of each identified PHD finger gene retrieved from BRAD database (Additional file 2 : Table S1), 140 out of 145 (96.6%) B. rapa PHD finger genes were mapped into the 10 chromosomes of B. rapa (Fig.  4 ) , while the remaining 5 PHD genes were not mapped to a specific chromosome because they were currently assigned to isolated scaffolds. Our results showed that these PHD finger genes were unevenly distributed across the 10 B. rapa chromosomes. The number of mapped PHD finger genes is 21 on A09, 19 on A02, 16 on A07, 15 on A01, 15 on A03, 15 on A05, 14 on A06, 10 on A08, 9 on A10, and 6 on A04. B. rapa PHD finger genes tend to be clustered in some chromosomal regions. Our mapping analysis showed also that 58 out of 140 (41.4%) B. rapa PHD finger genes were involved in segmental duplication and only two genes (1.4%) were involved in tandem duplication (Fig.  4 ).

Distribution of 140 PHD finger genes on 10 chromosomes of Brassica rapa . The 140 BrPHD genes unevenly located on each conserved collinear blocks of the chromosomes. Chromosome number (A01-A10) is indicated at the top of each chromosome. Gene name is indicated on the right side of each chromosome. The physical position (Mb) of each mapped gene is indicated on the left side of each chromosome. The genes located on duplicated chromosomal segments are framed by same colors and connected by same color lines between the two relevant chromosomes. The tandem repeated genes are marked by red color on the chromosomes

Brassica species have all undergone a whole genome triplication (WGT) event ~ 15.9 MYA following their divergence from the Arabidopsis lineage ~ 20 MYA [ 42 , 47 , 71 , 72 , 73 ]. B. rapa is considered as a paleohexaploid, and contains three subgenomes commonly called as least fractionized (LF), moderately fractionized (MF1) and most fractionized (MF2) [ 47 , 71 , 72 , 73 ]. The syntenic relationships between the PHD finger genes of B. rapa and A. thaliana were determined from BRAD database, and summarized in Additional file 2 : Table S5. Among the 145 B. rapa PHD finger genes, 59 (40.7%) were assigned on LF, 46 (31.7%) on MF1, and 40 (27.6%) on MF2. In seven cases, the three paralogous copies were simultaneously conserved on the three subgenomes LF, MF1 and MF2, while in 21 cases, only two of the three expected paralogous copies were conserved, and in 68 cases, only one of the three expected paralogous copies was conserved. One hundred eighteen out of 145 (81.4%) B. rapa PHD finger genes had their syntenic orthologs in A. thaliana, covering 23 blocks of seven chromosomes of ancestral translocation Proto-Calepineae Karyotype (tPCK) [ 43 , 44 , 71 , 72 , 73 ]. Twenty seven out of 145 (18.6%) B. rapa PHD finger genes didn’t have their syntenic orthologs in A. thaliana, while 18 out of 97 (18.6%) A. thaliana PHD finger genes didn’t have their syntenic orthologs in B. rapa (Additional file 2 : Table S5).

Expression analysis of B. rapa PHD finger genes in different tissues

The expression patterns of individual B. rapa PHD finger genes in different tissues (callus, root, stem, leaf, flower and silique) were analyzed based on a publicly available B. rapa RNA-Seq transcriptomic dataset [ 74 ]. Except for Bra002401, Bra004233, Bra010170 and Bra021575, the expression data of 141 other B. rapa PHD finger genes were available from the dataset, of which one (Bra013261) showed an expression value of zero for all the six tissues, while the remaining 140 genes were expressed in at least one of the six tissues., A clustered heat map displaying the expression patterns of the 140 B. rapa PHD finger genes in callus, root, stem, leaf, flower and silique was generated based on their log2-transformed fragments per kilobase of transcript per million fragments mapped (FPKM) values (Fig.  5 ). The result showed that these 140 B. rapa PHD finger genes were clustered into three major groups with subgroups. The group I (biggest) includes 78 genes, which were almost all constitutively expressed in all the tested tissues with relatively higher expression levels. The group II includes 36 genes, preferentially (> 2-folds higher) expressed in one or more tissues with relatively higher expression levels. For example, Bra028465 (corresponding to Arabidopsis gene At5g40590, a cysteine/histidine-rich C1 domain family protein gene) was preferentially expressed in root, but very lowly (or not) expressed in other tested tissues; Bra025864 (corresponding to At1g20990, another cysteine/histidine-rich C1 domain family protein gene) was preferentially expressed in root, and only very lowly expressed in other tissues; Bra029401 (corresponding to At5g24330 or Arabidopsis TRITHORAX-RELATED protein 6, ATXR6) was preferentially expressed in stem than in other tissues; Bra020445 (corresponding to At5g57380 or VERNALIZATION INSENSITIVE 3, VIN3) was preferentially expressed in leaf than in other tissues. The group III includes 26 genes which were almost all very lowly (or not) expressed in the tested tissues, except for Bra028463 (corresponding to At5g40320, a cysteine/histidine-rich C1 domain family protein gene) preferentially expressed in callus but very lowly (or not) expressed in other tissues; Bra012982 (corresponding to At5g61090, a polynucleotidyl transferase gene) was preferentially expressed in silique, moderately expressed in flower, but very lowly (or not) expressed in other tissues; and Bra033990 (homologous to At2g21840, At2g21850 and At2g21830, cysteine/histidine-rich C1 domain family protein genes) was moderately but preferentially expressed in root.

Expression profile of 140 Brassica rapa PHD finger genes in different tissues revealed by clustering analysis of RNA-Seq data. The 140 genes were divided into three major groups (I-III) based on the log2-transformed fragments per kilobase of transcript per million fragments mapped (FPKM) values. The scale representing the relative signal values is shown above. The tissue types are indicated on the top. The individual gene names are indicated on the right side

To obtain information about the variation in expression pattern among triplicated PHD finger gene members caused by WGT [ 42 , 71 ], we compared the expression levels (FPKM values) of six sets of three triplicated members that were well conserved across the three subgenomes (LF, MF1 and MF2) of B. rapa in different tissues (Fig.  6 , Additional file 2 : Table S5). The results showed that these triplicated members display different expression patterns between them. For four of six triplet sets, two members maintained relatively higher expression levels while the third one was significantly lowly expressed in the tested tissues. For one triplet set, one member showed a dominant high expression level over two other members in all tested tissues, while for another triplet set, one member was dominantly expressed over two other members in some tissues but not in others (Fig.  6 ).

Comparison of the expression levels (by FPKM values) in different tissues between the triplicated Brassica rapa PHD finger gene members conserved across the three subgenomes LF, MF1 and MF2

Table  4 summarized the distribution of 140 PHD finger genes in different expression groups of Fig.  5 in relation to the phylogenetic classification of their encoded proteins in Fig.  2 . We observed that 42.9% of genes in phylogenetic group A, 88.9% in group B, 70.4% in group C, 55.6% in group D, 56.7% in group E and 50.0% in group F were clustered into the expression group I (constitutively expressed in almost all the tested tissues). About 20% of genes in phylogenetic group A, 10% in group B, and 30% in group C, D, E and F were clustered into the expression group II (preferentially expressed in some tissues). About 40% of genes in phylogenetic group A, 0% in group B and C, 10% in group D and E, and 20% in group F were clustered into the expression group III (very lowly or not expressed in almost all the tested tissues).

Expression analysis of B. rapa PHD finger genes under salt and drought stresses

In order to relate our results with the existing data from other species, we generated a phylogenetic tree by using the protein sequences of 145 B. rapa PHD finger genes together with those of a few previously reported as stress- or development-related in maize [ 64 ], poplar [ 65 ], soybean [ 61 , 62 ], alfalfa [ 60 ], Arabidopsis [ 75 , 76 , 77 , 78 , 79 , 80 ] and rice [ 81 , 82 , 83 ] (Additional file 1 : Figure S5). We found that 18 B. rapa PHD finger genes were clustered together with those previously characterized as stress-related, while 63 others were clustered together with those previously reported as development-related. Genes closely clustered together in a phylogenetic tree may share common ancestors, and their functions may be conserved across species. Based on this phylogenetic tree (Additional file 1 : Figure S5), we selected nine genes (Bra001393, Bra016698, Bra017415, Bra026210, Bra026825, Bra034169, Bra034860, Bra034950 and Bra036568) representing the “stress-related”, and nine other genes (Bra007814, Bra015682, Bra020249, Bra020856, Bra026192, Bra027574, Bra037238, Bra037299, Bra040028) representing the “development-related” or non-characterized genes for qRT-PCR analysis to examine their expression response to salt (200 mM NaCl) (Fig.  7 ) and drought (20% (w/v) PEG 6000 ) (Fig.  8 ) stresses in the leaves of three-week-old seedlings. Our results showed that all the selected 18 B. rapa PHD finger genes were responsive to the two abiotic stress treatments.

qRT-PCR expression patterns of 18 Brassica rap PHD finger genes under salt treatment. The time points represent by x-axis and the scale of relative expression showed by y-axis. Statistical significance of deference’s between control and treated groups was analyzed using Student’s t-test (*indicates P  < 0.05, ** indicates P  < 0.01). The “stress-related” genes (see the text) are framed by red box

qRT-PCR expression patterns of 18 Brassica rap PHD finger genes under drought treatment. The time points represent by x-axis and the scale of relative expression showed by y-axis. Statistical significance of deference’s between control and treated groups was analyzed using Student’s t-test (*indicates P  < 0.05, ** indicates P  < 0.01). The “stress-related” genes (see the text) are framed by red box

For salt stress analysis, all the 18 tested genes were responsive to the treatment with 11 PHD finger genes up-regulated and seven genes down-regulated compared to control (CK) after 1 h, 3 h or 24 h of treatment, respectively (Fig.  7 ). The most spectacular case is the gene Bra026210 (corresponding to At1g14510 or ALFIN-LIKE 7, AL7, involved in covalent chromatin modification or regulation of transcription) which was induced by more than 18 fold under salt treatment at 1 h. Interestingly, Bra016698, a paralogous copy of Bra026210 produced by WGT (Additional file 2 : Table S5), was progressively induced along with the time under salt treatment and reached as high as nine fold of the control at 24 h, while another paralogous copy Bra026825 was down-regulated by more than two fold. Another gene, named Bra007814 (corresponding to At2g25170 or CYTOKININ-HYPERSENSITIVE 2, CKH2, involved in covalent chromatin modification and negative regulation of transcription) was up-regulated by four fold at 1 h of treatment but significantly down-regulated at 3 and 24 h.

For drought stress analysis, all the 18 tested genes were responsive to treatment with 16 genes up-regulated and two down regulated compared to control (CK) after 1 h, 3 h or 24 h of treatment, respectively (Fig.  8 ). Globally, the variation extents induced by drought stress were more spectacular than salt stress. Interestingly, Bra026210 was also highly induced by drought stress as it was the case for salt stress (Fig.  7 ), and reached an expression level of as high as 55 times compared to the control at 1 h, followed by an expression level of about 15 times of the control at 3 or 24 h. Bra037238 (corresponding to At2g18090, involved in regulation of transcription), Bra015682 (corresponding to At1g77250, involved in regulation of transcription) and Bra034860 (corresponding to At3g11200 or ALFIN-LIKE 2, AL2, involved in covalent chromatin modification or regulation of transcription) were induced by about 13, seven and five fold, respectively, compared to control at 1 h of treatment. Bra017415 (corresponding to At2g02470 or ALFIN-LIKE 6, AL6, involved in covalent chromatin modification or regulation of transcription) and Bra016698 (corresponding to At1g14510, or ALFIN-LIKE 7, AL7, involved in covalent chromatin modification or regulation of transcription) were induced by about eight and seven fold, respectively, compared to control at 3 h of treatment. It is worth to note that the three triplicated paralogous genes, Bra016698, Bra026210 and Bra026825, display different expression patterns along with the time of treatments of both salt and drought stress (Figs.  7 ) and ( 8 ).

PHD fingers can specifically recognize various histone marks or post-translational histone modifications (PTMs) such as trimethylated Lysine 4 in histone H3 (H3K4me3), trimethylated Lysine 9 in histone H3 (H3K9me3), trimethylated Lysine 36 in histone H3 (H3K36me3), acetylated Lysine 9 in histone H3 (H3K9ac), acetylated Lysine 14 in histone H3 (H3K14ac) , etc. , as well as unmodified histone tails such as H3K4, and other non-histone proteins [ 34 , 35 , 36 , 84 ] . For example, all the PHD domains of the seven Arabidopsis Alfin1-like proteins (AL1 to AL7) can bind to the histone H3K4me3 peptide with varying methylation state preference and binding affinities [ 85 ] ; rice CHD3 protein acts as a bifunctional chromatin regulator able to recognize and modulate H3K4 and H3K27 methylation over repressed or tissue-specific genes [ 86 ]; PHD finger of the SUMO ligase Siz/PIAS family in rice reveals specific binding for methylated histone H3 at lysine 4 and arginine 2 [ 87 ]. These features highlight the functional versatility of PHD fingers as epigenome readers that regulate gene expression (activation or repression) according to the status of the chromatin, and reinforce the hypothesis that evolutionary changes in amino acids surrounding the eight conserved metal ligand positions on a conserved structural fold would increase the functional diversity of these PHD finger proteins [ 35 ].

More and more plant PHD-finger protein genes have been identified as involved in various important biological processes. For examples, in the model plant A. thaliana , MMD1 (AT1G66170), SCC2 (AT5G15540), MS1 (AT5G22260) and ASHR3 (AT4G30860) are involved in the meiosis and post-meiotic processes, and their mutations can cause male sterility [ 36 ]; OBE1 (AT3G07780), OBE2 (AT5G48160) and PKL (AT2G25170) are involved in the embryonic meristem initiation and root development, and their mutations can result in an absence of root and defective development of the vasculature [ 36 ]; AL6 (AT2G02470) and AL7 (AT1G14510) are involved seed germination, and their double mutation can result in a germination delay under osmotic stress conditions [ 36 ]; VIL1 (AT3G24440), VRN2 (AT4G16845), VIN3 (AT5G57380), VRN5 (AT3G24440), ATX1 (AT2G31650), EBS (AT4G22140) and SHL (AT4G39100) are involved in the control of flowering time [ 36 ]; GSR1 (AT3G27490) is involved in auxin-mediated seed dormancy and germination [ 88 ]; EDM2 (AT5G55390) is involved the resistance to downy mildew [ 89 ]; AL5 (AT5G20510) is involved in abiotic stress tolerance [ 63 ]. In rice, Ehd3 acts as a promoter in the unique genetic pathway responsible for photoperiodic flowering [ 81 ]; PTC1 is involved in tapetal cell death and pollen development [ 83 ]; OsVIL2 is involved in the control of flowering time, and its insertion mutations cause late flowering under both long and short days [ 90 ]; OsTTA is a constitutively expressed regulator of multiple metal transporter genes responsible for essential metals delivery to shoots for their normal growth [ 91 ]; OsMS1 functions as a transcriptional activator to regulate programmed tapetum development and pollen exine formation [ 92 ]. In barley, HvMS1 silencing and overexpression can result in male sterility [ 93 ]. In maize, the mutation of ZmMs7 (ortholog of PTC1 ) can result in male sterility [ 94 ]. In soybean, all six Alfin1-type PHD finger genes were found to be responsive to various stress treatments, and overexpressing the GmPHD2 showed salt tolerance when compared with the wild type plants [ 61 ]; GmPHD5 encodes an important regulator for crosstalk between histone H3K4 di-methylation and H3K14 acetylation in response to salinity stress [ 62 ]. In alfalfa, Alfin1 is involved in salt tolerance [ 59 , 60 ]. In cassava, MePHD1 is involved in starch synthesis [ 95 ]. Although the number of identified PHD-finger genes is increasing in different species, most of putative PHD finger genes remain to be characterized, and no any research on this category of genes has been conducted in Brassica species to this day .

B. rapa (AA genome) is not only an economically important vegetal, oilseed and fodder crop widely grown around the world, but also one of the diploid progenitor parents of amphidiploid oilseed crops B. napus (AACC) and B. juncea (AABB), and can be used as a model plant for functional and evolutionary studies of important gene families among Brassica species [ 49 ]. In this study, a total of 145 PHD finger proteins containing 233 PHD domains were identified from the current version of the B. rapa proteome database (Additional file 2 : Table S1). This number is considerably higher than those previously identified in maize (67) [ 64 ], poplar (73) [ 65 ] and rice (59) [ 66 , 96 ], pear (31) [ 97 ] and moso bamboo (60) [ 67 ], although it might not yet be exhaustive as other PHD-suspected domain-containing proteins were also detected (Additional file 2 : Table S2). This is the consequence of the WGT event occurred ~ 15.9 MYA in Brassica ancestor followed by gene losing [ 42 , 47 , 71 ], while only one tandem duplication event was observed among these PHD finger genes (Fig.  4 ). Interestingly, these PHD finger genes were unevenly distributed on the 10 B. rapa chromosomes (Fig.  2 ), a phenomenon also observed in A. thaliana [ 8 ], maize [ 64 ] [ 65 ] and rice [ 66 ], implying a possible relationship between chromosomal location and their cellular functions.

Our gene ontology analysis showed that 67.7% of the identified B. rapa PHD finger proteins were predicted to be located in nucleus, and 91.3% members were putatively involved in protein binding activity (Fig.  1 ). These features support the previous findings about the main functions of these PHD finger genes as epigenomic effectors regulating gene expression in cells [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. Based on the presence or not of additional domains (Additional file 2 : Table S4), gene structure and phylogenic analysis (Figs.  2  and 3 ; Additional file 1 : Figure S2), these B. rapa PHD finger genes can be classified into several groups and subgroups, illustrating the evolution and functional diversification of these genes in B. rapa . Our phylogenetic (Fig.  2 ) and syntenic (Additional file 2 : Table S5) analyses showed that, for the majority of B. rapa PHD finger genes, their corresponding orthologs were also found in the model plant A. thaliana , meant that the functional study of B. rapa PHD finger genes can largely benefit from the rich information available in A. thaliana (Additional file 2 : Table S3). However, as shown in Fig.  2 , the duplicated gene members in B. rapa generally evolved at different rates in comparison with their orthologs in A. thaliana , furthermore, some B. rapa finger protein genes, such as Bra038151 and Bra029800 in phylogenetic group A, Bra026192 in group B, and Bra034957 in group C, etc ., cannot find their corresponding orthologs in A. thaliana , suggesting that these genes may provide some new or specific functions for the growth and development of Brassica crops or their responses to various stresses.

Our analysis on RNA-Seq data (Fig.  5 ) showed that 55.7% of the B. rapa PHD finger genes were constitutively expressed in all the tested tissues with relatively higher expression levels, suggesting their important housekeeping roles in plant growth and development. A few PHD finger genes were also identified as preferentially expressed in specific tissues, constituting then an interesting panel of candidates for future targeted studies on the function of PHD finger genes and genetic improvement of Brassica species. Comparison of expression levels between triplicated members (Fig.  6 ) showed that in the majority of cases the three triplicated members display varied expression levels and patterns across different tissues, indicating that their biological roles may be also varied in plant growth and development, a phenomenon of neo-functionalization or sub-functionalization of duplicated genes [ 98 ]. Existence of 1–2 triplicated members that display a very low (or not) expression level contrasting to the higher expression levels of other triplicated members of the same triplet set, such as the case of Bra006562 in Fig.  6 , indicates that they may be degenerated during the evolution, a phenomenon already observed previously for RING finger protein genes [ 99 ].

In this study, we also analyzed the expression patterns of 18 B. rapa PHD finger genes in response to drought and salt stresses, of which nine have been characterized previously as “stress-related” in other plant species [ 60 , 61 , 62 , 64 , 65 ], while nine other genes representing the “development-related” or non-characterized genes (Additional file 1 : Figure S5). Our results showed that all these genes were responsive to the two abiotic stress treatments with different amplitudes and varied expression patterns: some members were highly up-regulated while others were down-regulated along with the time of treatments (Fig.  7 , Fig.  8 ). This means that some PHD finger genes may play important roles in plant adaption to adverse environmental stresses, an idea that was also supported by other studies [ 61 , 64 , 65 , 66 ]. These identified PHD finger genes can then serve as a source of candidate genes for genetic engineering and improvement of Brassica crops against abiotic stresses. Further studies extended on other PHD finger genes with more types of abiotic stress treatments would allow us to obtain a global view on the involvement of these PHD finger genes in response to abiotic stresses, and identify the most prominent ones for use as targets in genetic improvement of stress resistance in plants.

We identified a total of 145 putative PHD finger proteins containing 233 PHD domains from the current release of B. rapa genome database. These PHD finger genes were further characterized and classified into different groups or categories by analyses of gene ontology, additional domain, gene structure, synteny and phylogeny. We also analyzed the RNA-Seq data of these PHD finger genes, and found that 55.7% of them were constitutively expressed in all the tested tissues with relatively higher expression levels. Expression analysis of a subset of 18 PHD finger genes under salt and drought treatments showed that all of them were responsive to the two abiotic stresses, indicating that PHD finger genes can be a source of candidate genes for genetic improvement of Brassica crops against abiotic stresses. Our results lay the foundation for further functional determination of each PHD finger gene across the Brassica species, and may help to select the most promising gene targets for further genetic engineering and improvement of Brassica crops.

Identification and characterization of PHD finger proteins in B. rapa

To identify all B. rapa PHD finger proteins, we followed two different strategies as have been described in a previously study [ 50 ]. First, all previously identified Arabidopsis PHD finger proteins [ 96 , 100 ] were used as query sequences for BLASTp searches against the B. rapa proteome database at BRAD ( http://brassicadb.org/brad/ ). Second, all Arabidopsis PHD finger domains as well as those of maize [ 64 ] and poplar [ 65 ] were used as query sequences for BLASTp searches against the same B. rapa proteome database at BRAD. The irredundant candidate sequences were then analyzed online by SMART ( http://smart.embl-heidelberg.de ) (option Pfam) and occasionally by InterPro ( http://www.ebi.ac.uk/interpro/ ) to confirm the presence or not of PHD domains. This was followed by visual inspections based on the conservation of eight metal ligands (Cys4-His-Cys3) and the residue number between two neighboring metal ligands, especially between the 4th and 5th metal ligands where the number of residues should be four or five for a PHD domain contrasting to two or three for RING and two for LIM [ 13 ]. Those proteins that were predicted as PHD domain-containing by SMART but lacked two or more metal ligands, or those containing a sequence motif visually resembled to a PHD domain but not validated by SMART were classified as PHD-suspected domain-containing. The protein size, molecular weight (MW), and theoretical isoelectric point (pI) of each PHD finger protein were computed by using Pepstats ( http://www.ebi.ac.uk/Tools/seqstats/emboss_pepstats/ ).

For each identified B. rapa PHD finger protein, their associated Gene Ontology (GO) terms were retrieved from the Phytozome database ( http://phytozome.jgi.doe.gov/pz/portal.html ), and their subcellular localization was predicted by CELLO v2.5 software ( http://cello.life.nctu.edu.tw ).

Multiple sequence alignment, gene structure and phylogenetic analysis

The PHD finger domains were aligned by Clustal W and manually edited by BioEdit software. The sequence logo of over-represented motif among the identified PHD domains was generated by using the Web Logo software (http: // weblogo.berkeley.edu /logo.cgi). Phylogenetic trees based on B. rapa PHD finger domain sequences and the PHD finger protein sequences of B. rapa and A. thaliana were generated by using MEGA6.06 software with the Neighbor–Joining (NJ) algorithm and a bootstrap analysis of 1000 replicates. The exon/intron structure of each B. rapa PHD finger gene was generated by using the Gene Structure Display Server 2.0 ( http://gsds.cbi.pku.edu.cn/ ).

Additional domain analysis

To identify additional known domains, each predicted B. rapa PHD finger protein was analyzed by Smart (http:// smart.embl-heidelberg.de) with option Pfam. According to the presence and organization of different known domains, these B. rapa PHD finger proteins were divided into different groups. These additional domains were then used as query sequences for BLASTp searches against the NCBI database to determine if they were also present in other plant species.

Chromosome location of PHD-finger protein genes in B. rapa

For chromosome mapping of the identified B. rapa PHD finger genes, we followed the same procedure that was described in our previous study [ 50 ]. For each putative PHD-finger protein gene, their physical chromosome location data were retrieved from the BRAD database. The Map Chart 2.3v software was used for mapping analysis.

Syntenic relationships between B. rapa and A. thaliana PHD finger genes

For establishing the syntenic relationships among the identified B. rapa PHD finger genes, we followed the same procedure that was described previously [ 50 ]. The Search Syntenic Gene function of the BRAD database was used to find out the syntenic paralogs in B. rapa and orthologs in A. thaliana . The information such as gene name (s), localization on ancestral chromosome blocks of the tPCK (Translocation Proto-Calepineae Karyotype), Arabidopsis chromosomes and B. rapa LF, MF1 and MF2 subgenomes [ 43 , 44 , 45 , 72 ], as well as the possible tandem repeats in the two species, were recorded and summarized in Additional file 2 : Table S5.

Expression pattern of PHD finger genes in B. rapa

The RNA-Seq data of six tissues (callus, root, stem, leaf, flower and silique) of the B. rapa accession Chiifu-401–42 was downloaded from NCBI ( http://www.ncbi.nlm.nih.gov/geo/ ) (GEO accession GSE43245) [ 74 ]. For each identified B. rapa PHD finger gene, their expression values (Fragments Per Kilobase of exon model per Million mapped, FPKM) of were extracted from the data set. The clustering analysis was then conducted by using Cluster software v3.0 ( http://bonsai.hgc.jp/~mdehoon/software/cluster/ ) with the options of log2-transformed, Euclidean distances and the average linkage clustering method. The Java Tree view software ( http://jtreeview.sourceforge . net/) was used to generate a clustering gene expression heatmap.

Plant materials and stress treatments

For preparation of plant materials and stress treatments, we followed the same procedures that were described in our previous paper [ 50 ]. B. rapa accession Chiifu-401–42 seeds were first germinated in a Petri dish at 25 ° C, then transferred into plastic pots in a greenhouse at 22  ° C with 16/8 h for light/dark. Stress treatments were conducted on 21-days-old seedlings., The plants were irrigated with 200 mM NaCl and 20% (w/v) polyethylene glycol (PEG 6000) for salt and drought stress treatments, respectively. For each treatment, three biological replicates were prepared. The leaves from control and stressed plants were harvested in liquid nitrogen after 0, 1, 3, and 24 h of treatments, and placed at − 80 °C before RNA extraction.

RNA isolation and quantitative real-time PCR (qRT-PCR) analysis

For RNA isolation and quantitative real-time PCR (qRT-PCR) analysis, we followed the same procedures that were described in a previous study [ 50 ]. Total RNA was isolated from approximately 100 mg of the frozen leaves of each sample using an OMEGA Plant RNA extraction Kit. RNA concentrations were estimated by using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Inc., USA). First-strand cDNA was synthesized by using a TaKaRa cDNA Synthesis Kit (Dalian, China). Gene-specific primers were designed using the online Primer3Plus software ( http://www.primer3plus.com/ ). The B. rapa Actin -2 gene (XM_018658258) was used as internal reference gene. The primers used in this study were presented in Additional file 2 : Table S6. The qRT-PCR analysis was conducted on an ABI 7500 Fast Real-time PCR amplification system (Applied Biosystems, USA) in a volume of 20 μL: 2 μL cDNA template, 0.8 μL forward primers (10 μM), 0.8 μL reverse primers (10 μM), 10 μL SYBR Green PCR Master (ROX) (Roche, China), and 6.4 μL sterile water. The amplification parameters were: 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 70 s. The 2 −ΔΔCt method [ 101 ] was usd for data analysis. The Student’s t-test was used to determine the significance of differences among relative expression levels of each tested gene at different time points of treatment (with P  < 0.05 considered as statistically significant).

Availability of data and materials

Not applicable.

Abbreviations

Fragments Per Kilobase of exon model per Million mapped

Gene Expression Omnibus

Least fractionized subgenome

Lin-ll, Isl-1 and Mec-3

Moderately fractionized subgenome

Most fractionized subgenome

Molecular weight

Million years ago

Polyethylene glycol

Plant homeodomain

Theoretical isoelectric point

quantitative real-time PCR

Really interesting new gene

RNA sequencing;

Transcription factor IIIA

translocation Proto-Calepineae Karyotype

Whole genome triplication

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Acknowledgements

We thank Dr. Xiao-Ming Wu of Oil Crop Research Institute, Chinese Academy of Agricultural Sciences, for kindly providing the plant seeds used in this study. We are grateful to the anonymous reviewers for their valuable and constructive suggestions about the manuscript, and to Dr. Anne-Marie Chèvre for handling the manuscript.

This work was supported by a startup fund for distinguished scholars of Fujian Agriculture and Forestry University, No. 114120019, awarded to YHL. The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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Intikhab Alam, Cui-Cui Liu, Hong-Liu Ge, Khadija Batool, Yan-Qing Yang & Yun-Hai Lu

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YHL conceived and designed the experiments; IA, CCL, HLG, KB, and YQY conducted the experiments; IA and YHL processed the data and wrote the manuscript; YHL revised the manuscript; all authors have read and approved the manuscript.

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Additional file 1: fig. s1..

Multiple sequence alignment of 233 PHD domains from 145 PHD finger proteins of Brassica rapa. Fig. S2. Sequence logo of the overrepresented motif found in 233 PHD domains of Brassica rapa . Fig. S3. Phylogenetic tree based on multiple sequence alignment of 233 PHD domains from 145 putative PHD finger proteins of Brassica rapa . Fig. S4. Phylogenetic tree based on multiple sequence alignment of PHD finger proteins from Arabidopsis thaliana , Brassica rapa , Oryza sativa , Populus trichocarpa  and Zea mays. Fig. S5. Phylogenetic tree analyses of all 145 Brassica rapa PHD finger proteins and a few PHD finger proteins from other species previously characterized as stress or plant development related.

Additional file 2: Table S1.

List of 145 B. rapa PHD finger protein genes, and their related informations . Table S2 . List of 16 suspected B. rapa PHD finger proteins. Table S3. Summary of gene ontology terms of 98 A. thaliana PHD finger protein genes (retrieved from TAIR database, https://www.arabidopsis.org/index.jsp) in relation to the phylogenetic classification of their encoded proteins along with the 145 B. rapa PHD finger proteins in Fig.  2 . Table S4. Classification of145 B. rapa PHD domain-containing proteins based on the presence or not and organization of additional domain (s). Table S5. Synteny relationships between Arabidopsis and B.rapa PHD finger protein genes. Table S6. The information of primers used in the quantitative real-time PCR (qRT-PCR) analysis.

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Alam, I., Liu, CC., Ge, HL. et al. Genome wide survey, evolution and expression analysis of PHD finger genes reveal their diverse roles during the development and abiotic stress responses in Brassica rapa L.. BMC Genomics 20 , 773 (2019). https://doi.org/10.1186/s12864-019-6080-8

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DOI : https://doi.org/10.1186/s12864-019-6080-8

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• Brassica rapa
• PHD finger genes
• Gene duplication
• Genes expression
• Abiotic stress

BMC Genomics

ISSN: 1471-2164

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• General enquiries: [email protected]

NORMAL GENOMIC

Letunic et al. (2017) Nucleic Acids Res doi: 10.1093/nar/gkx922 Letunic et al. (2020) Nucleic Acids Res doi: 10.1093/nar/gkaa937

There are 144042 PHD domains in 86307 proteins in SMART's nrdb database.

Click on the following links for more information.

• Evolution (species in which this domain is found)

Taxonomic distribution of proteins containing PHD domain.

This tree includes only several representative species. The complete taxonomic breakdown of all proteins with PHD domain is also avaliable .

Click on the protein counts, or double click on taxonomic names to display all proteins containing PHD domain in the selected taxonomic class.

• Cellular role (predicted cellular role)

Cellular role : transcription

• Literature (relevant references for this domain)

Primary literature is listed below; Automatically-derived, secondary literature is also avaliable.

• Ragvin A et al.
• Nucleosome binding by the bromodomain and PHD finger of the transcriptional cofactor p300.
• J Mol Biol. 2004; 337 : 773-88
• Display abstract

The PHD finger and the bromodomain are small protein domains that occur in many proteins associated with phenomena related to chromatin. The bromodomain has been shown to bind acetylated lysine residues on histone tails. Lysine acetylation is one of several histone modifications that have been proposed to form the basis for a mechanism for recording epigenetically stable marks in chromatin, known as the histone code. The bromodomain is therefore thought to read a part of the histone code. Since PHD fingers often occur in proteins next to bromodomains, we have tested the hypothesis that the PHD finger can also interact with nucleosomes. Using two different in vitro assays, we found that the bromodomain/PHD finger region of the transcriptional cofactor p300 can bind to nucleosomes that have a high degree of histone acetylation. In a nucleosome retention assay, both domains were required for binding. Replacement of the p300 PHD finger with other PHD fingers resulted in loss of nucleosome binding. In an electrophoretic mobility shift assay, each domain alone showed, however, nucleosome-binding activity. The binding of the isolated PHD finger to nucleosomes was independent of the histone acetylation levels. Our data are consistent with a model where the two domains cooperate in nucleosome binding. In this model, both the bromodomain and the PHD finger contact the nucleosome while simultaneously interacting with each other.

• Townsley FM, Thompson B, Bienz M
• Pygopus residues required for its binding to Legless are critical for transcription and development.
• J Biol Chem. 2004; 279 : 5177-83

Pygopus and Legless/Bcl-9 are recently discovered core components of the Wnt signaling pathway that are required for the transcriptional activity of Armadillo/beta-catenin and T cell factors. It has been proposed that they are part of a tri-partite adaptor chain (Armadillo>Legless>Pygopus) that recruits transcriptional co-activator complexes to DNA-bound T cell factor. Here, we identify four conserved residues at the putative PHD domain surface of Drosophila and mouse Pygopus that are required for their binding to Legless in vitro and in vivo. The same residues are also critical for the transactivation potential of DNA-tethered Pygopus in transfected mammalian cells and for rescue activity of pygopus mutant embryos. These residues at the Legless>Pygopus interface thus define a specific molecular target for blocking Wnt signaling during development and cancer.

• Aravind L, Iyer LM, Koonin EV
• Scores of RINGS but no PHDs in ubiquitin signaling.
• Cell Cycle. 2003; 2 : 123-6

Recently, it has been reported that PHD fingers of MEKK1 kinase and a family of viral and cellular membrane proteins have E3 ubiquitin ligase activity. Here we describe unique sequence and structural signatures that distinguish PHD fingers from RING fingers, which function primarily as E3 ubiquitin ligases, and demonstrate that the Zn-binding modules of the above proteins are distinct versions of the RING domain rather than PHD fingers. Thus, currently available data reveal extreme versatility of RINGs and their derivatives that function as E3 ubiquitin ligases but provide no evidence of this activity among PHD fingers whose principal function appears to involve specific protein-protein and possibly protein-DNA interactions in chromatin.

• Fair K, Anderson M, Bulanova E, Mi H, Tropschug M, Diaz MO
• Protein interactions of the MLL PHD fingers modulate MLL target gene regulation in human cells.
• Mol Cell Biol. 2001; 21 : 3589-97

The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservation but their function is unknown. We have determined that these fingers mediate homodimerization and binding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammalian cells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cells results in altered expression of HOX genes that are targets for regulation by MLL. These alterations are suppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHD fingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.

• Schultz DC, Friedman JR, Rauscher FJ 3rd
• Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD.
• Genes Dev. 2001; 15 : 428-43

Macromolecular complexes containing histone deacetylase and ATPase activities regulate chromatin dynamics and are vitally responsible for transcriptional gene silencing in eukaryotes. The mechanisms that target these assemblies to specific loci are not as well understood. We show that the corepressor KAP-1, via its PHD (plant homeodomain) and bromodomain, links the superfamily of Kruppel associated box (KRAB) zinc finger proteins (ZFP) to the NuRD complex. We demonstrate that the tandem PHD finger and bromodomain of KAP-1, an arrangement often found in cofactor proteins but functionally ill-defined, form a cooperative unit that is required for transcriptional repression. Substitution of highly related PHD fingers or bromodomains failed to restore repression activity, suggesting high specificity in their cooperative function. Moreover, single amino acid substitutions in either the bromodomain or PHD finger, including ones that mimic disease-causing mutations in the hATRX PHD finger, abolish repression. A search for effectors of this repression function yielded a novel isoform of the Mi-2alpha protein, an integral component of the NuRD complex. Endogenous KAP-1 is associated with Mi-2alpha and other components of NuRD, and KAP-1-mediated silencing requires association with NuRD and HDAC activity. These data suggest the KRAB-ZFP superfamily of repressors functions to target the histone deacetylase and chromatin remodeling activities of the NuRD complex to specific gene promoters in vivo.

• Aasland R, Gibson TJ, Stewart AF
• The PHD finger: implications for chromatin-mediated transcriptional regulation.
• Trends Biochem Sci. 1995; 20 : 56-9
• Schindler U, Beckmann H, Cashmore AR
• HAT3.1, a novel Arabidopsis homeodomain protein containing a conserved cysteine-rich region.
• Plant J. 1993; 4 : 137-50

Homeodomain proteins have been shown to play a major role in the development of various organisms. A novel Arabidopsis homeodomain protein has been isolated based on its capability to interact with a DNA motif derived from the light-induced cab-E promoter of Nicotiana plumbaginifolia. The homeodomain of this protein, designated HAT3.1, differs substantially from those in other plant homeobox proteins identified so far. Furthermore, HAT3.1 is unique among other Arabidopsis proteins in that it does not contain a leucine zipper motif following the homeodomain. HAT3.1 is further characterized by an N-terminal region that shares substantial sequence similarity with the maize homeodomain protein Zmhox1a. Within this conserved region, the presence of eight regularly spaced cysteine/histidine residues was observed reminiscent of other metal-binding domains. Based on the strong evolutionary conservation of this domain, it is proposed that this region represents a novel protein-motif which is denoted PHD-finger (plant homeodomain-finger). In vitro DNA binding studies demonstrated that HAT3.1 is capable of interacting with any DNA fragment larger than 100 bp. Interestingly, a deletion of the N-terminal PHD-finger domain completely abolished DNA binding, suggesting that this region may play an important functional role in protein-protein or protein-DNA interaction. HAT3.1 mRNA was primarily detected in root tissue, implying a regulatory function of this protein in root development.

• Metabolism (metabolic pathways involving proteins which contain this domain)

Click the image to view the interactive version of the map in

% proteins involved KEGG pathway ID Description 22.22 Tryptophan metabolism 22.22 Tight junction 11.11 Lysine degradation 11.11 Purine metabolism 7.41 Cell cycle 7.41 Cell cycle - yeast 3.70 RNA polymerase 3.70 Glycerophospholipid metabolism 3.70 Oxidative phosphorylation 3.70 Phosphatidylinositol signaling system 3.70 Glycerolipid metabolism This information is based on mapping of SMART genomic protein database to KEGG orthologous groups. Percentage points are related to the number of proteins with PHD domain which could be assigned to a KEGG orthologous group, and not all proteins containing PHD domain. Please note that proteins can be included in multiple pathways, ie. the numbers above will not always add up to 100%. Structure (3D structures containing this domain) 3D Structures of PHD domains in PDB PDB code Main view Title WSTF-PHD SOLUTION STRUCTURE OF THE PHD DOMAIN FROM THE KAP-1 COREPRESSOR Solution structure of the 2nd PHD domain from Mi2b Solution structure of the 2nd PHD domain from Mi2b with C-terminal loop replaced by corresponding loop from WSTF Solution structure of PHD domain in nucleic acid binding protein-like NP_197993 Solution structure of PHD domain in PHD finger family protein Solution structure of PHD domain in death inducer-obliterator 1(DIO-1) Solution structure of PHD domain in ING1-like protein BAC25079 Solution structure of PHD domain in PHF8 Solution structure of PHD domain in inhibitor of growth family, member 1-like Solution structure of PHD domain in ING1-like protein BAC25009 Solution structure of PHD domain in protein NP_082203 Solution structure of PHD domain in DNA-binding family protein AAM98074 Solution structure of PHD domain in inhibitor of growth protein 3 (ING3) NMR structure of the first phd finger of autoimmune regulator protein (AIRE1): insights into apeced Crystal Structure Analysis of the PHD domain of the Transcription Coactivator Pygophus Solution structure of the PHD domain in SmcY protein Solution structure of the PHD domain in RING finger protein 107 Crystal structure of PHD finger-linker-bromodomain fragment of human BPTF in the H3(1-15)K4me3 bound state Crystal structure of PHD finger-linker-bromodomain fragment of human BPTF in the free form Crystal structure of PHD finger-linker-bromodomain fragment of human BPTF in the H3(1-15)K4ME2 bound state NMR solution structure of PHD finger fragment of human BPTF in free state NMR solution structure of the PHD domain from the human BPTF in complex with H3(1-15)K4me3 peptide Crystal structure of ING2 PHD finger in complex with H3K4Me3 peptide NMR solution structure of PHD finger fragment of Yeast Yng1 protein in free state NMR solution structure of the PHD domain from the yeast YNG1 protein in complex with H3(1-9)K4me3 peptide Solution structure of the free TAF3 PHD domain Solution structure of the TAF3 PHD domain in complex with a H3K4me3 peptide Plan homeodomain finger of tumour supressor ING4 Molecular Basis of non-modified histone H3 tail Recognition by the First PHD Finger of Autoimmune Regulator NMR Solution structure of the first PHD finger domain of human Autoimmune Regulator (AIRE) in complex with Histone H3(1-20Cys) Peptide Solution Structure of JARID1A C-terminal PHD finger Solution structure of JARID1A C-terminal PHD finger in complex with H3(1-9)K4me3 Solution structure of BRD1 PHD1 finger Solution structure of MLL1 PHD3-Cyp33 RRM chimeric protein Solution structures of the double PHD fingers of human transcriptional protein DPF3 bound to a histone peptide containing acetylation at lysine 14 Solution structures of the double PHD fingers of human transcriptional protein DPF3b bound to a H3 peptide wild type Solution structure of the double PHD (plant homeodomain) fingers of human transcriptional protein DPF3b bound to a histone H4 peptide containing acetylation at Lysine 16 Solution structure of the double PHD (plant homeodomain) fingers of human transcriptional protein DPF3b bound to a histone H4 peptide containing N-terminal acetylation at Serine 1 The solution structure of the PHD3 finger of MLL Structural basis for histone code recognition by BRPF2-PHD1 finger Structure of the first PHD finger (PHD1) from CHD4 (Mi2b) Solution structure of CHD4-PHD2 in complex with H3K9me3 Structure of PHD domain of UHRF1 in complex with H3 peptide NMR Structure of UHRF1 PHD domains in a complex with histone H3 peptide NMR structure of the UHRF1 PHD domain Structure of MOZ Solution structure of BRD1 PHD2 finger NMR structure of the second PHD finger of AIRE (AIRE-PHD2) Solution NMR structure of the PHD domain of human MLL5, Northeast structural genomics consortium target HR6512A PHD domain of ING4 N214D mutant Structure of Dido PHD domain PHD Domain from Human SHPRH Solution NMR structure of PHD type Zinc finger domain of Lysine-specific demethylase 5B (PLU-1/JARID1B) from Homo sapiens, Northeast Structural Genomics Consortium (NESG) Target HR7375C NMR structure of human Sp140 PHD finger trans conformer NMR structure of Sp140 PHD finger cis conformer Solution NMR Structure of PHD Type 1 Zinc Finger Domain 1 of Lysine-specific Demethylase Lid from Drosophila melanogaster, Northeast Structural Genomics Consortium (NESG) Target FR824J 2MNY 2MNZ 2MUM 2NAA The PHD finger of ING4 in complex with an H3K4Me3 histone peptide Crystal Structure of the BHC80 PHD finger Crystal Structure of the ING1 PHD Finger in complex with a Histone H3K4ME3 peptide Crystal structure of PHD finger-linker-bromodomain Y17E mutant from human BPTF in the H3(1-9)K4ME2 bound state NMR Solution Structures of Human KAP1 PHD finger-bromodomain Solution structure of the plant homeodomain (PHD) of the E3 SUMO ligase Siz1 from rice MOLECULAR BASIS OF HISTONE H3K4ME3 RECOGNITION BY ING4 Decoding of methylated histone H3 tail by the Pygo-BCL9 Wnt signaling complex Decoding of methylated histone H3 tail by the Pygo-BCL9 Wnt signaling complex Decoding of methylated histone H3 tail by the Pygo-BCL9 Wnt signaling complex Decoding of methylated histone H3 tail by the Pygo-BCL9 Wnt signaling complex Decoding of methylated histone H3 tail by the Pygo-BCL9 Wnt signaling complex Crystal structure of the human Pygo2 PHD finger in complex with the B9L HD1 domain Solution structure of the PHD domain in PHD finger protein 21A Solution structure of the first and second PHD domain from Myeloid/lymphoid or mixed-lineage leukemia protein 3 homolog Solution structure of the PHD domain of Metal-response element-binding transcription factor 2 Structural analysis of PHD domain of Pygopus complexed with trimethylated histone H3 peptide Structure of UHRF1 in complex with histone tail Structure of UHRF1 in complex with histone tail Crystal structure of the ING5 PHD finger in complex with H3K4me3 peptide Crystal structure of JARID1A-PHD3 complexed with H3(1-9)K4me3 peptide crystal structure of PHF2 PHD domain complexed with H3K4Me3 peptide Structure of PHF8 in complex with histone H3 Structure of KIAA1718, human Jumonji demethylase, in complex with N-oxalylglycine Structure of KIAA1718, human Jumonji demethylase, in complex with alpha-ketoglutarate Crystal structure of MLL1 PHD3-Bromo in the free form Crystal structure of MLL1 PHD3-Bromo complexed with H3(1-9)K4me2 peptide Crystal structure of MLL1 PHD3-Bromo complexed with H3(1-9)K4me3 peptide ceKDM7A from C.elegans, complex with H3K4me3 peptide and NOG ceKDM7A from C.elegans, alone ceKDM7A from C.elegans, complex with H3K4me3K9me2 peptide and NOG ceKDM7A from C.elegans, complex with H3K4me3 peptide, H3K9me2 peptide and NOG ceKDM7A from C.elegans, complex with H3K4me3K27me2 peptide and NOG ceKDM7A from C.elegans, complex with H3K4me3 peptide, H3K27me2 peptide and NOG Crystal structure of TRIM24 PHD-Bromo in the free state Crystal structure of TRIM24 PHD-Bromo complexed with H3(13-32)K23ac peptide Crystal structure of TRIM24 PHD-Bromo complexed with H3(23-31)K27ac peptide Crystal structure of TRIM24 PHD-Bromo complexed with H4(14-19)K16ac peptide Crystal structure of TRIM24 PHD-Bromo complexed with H3(1-10)K4 peptide PHD-type zinc finger of human PHD finger protein 13 Crystal structure of PHF13 in complex with H3K4me3 CEKDM7A from C.Elegans, complex with alpha-KG CEKDM7A from C.Elegans, complex with D-2-HG Crystal Structure of BPTF PHD-linker-bromo in complex with histone H4K12ac peptide Crystal Structure of PHD Domain of UHRF1 Structure of UHRF1 PHD finger in complex with histone H3 1-9 peptide Structure of UHRF1 PHD finger in complex with histone H3K4me3 1-9 peptide Structure of UHRF1 PHD finger in the free form Structure of UHRF1 in complex with unmodified H3 N-terminal tail Crystal structure of TRIM33 PHD-Bromo in the free state Crystal structure of the complex of TRIM33 PHD-Bromo and H3(1-20)K9me3K14ac histone peptide Crystal structure of the complex of TRIM33 PHD-Bromo and H3(1-22)K9me3K14acK18ac histone peptide Crystal structure of the complex of TRIM33 PHD-Bromo and H3(1-28)K9me3K14acK18acK23ac histone peptide Crystal structure of MOZ Crystal structure of Drosophila Pygo PHD finger in complex with Legless HD1 domain PHD finger of human UHRF1 in complex with unmodified histone H3 N- terminal tail PHD finger of human UHRF1 Crystal structure of the CXXC and PHD domain of Human Lysine-specific Demethylase 2A (KDM2A)(FBXL11) RNA Polymerase II-Bye1 complex Crystal structure of the PHD-Bromo-PWWP cassette of human PRKCBP1 Crystal Structure of NSD3 tandem PHD5-C5HCH domains Crystal Structure of NSD3 tandem PHD5-C5HCH domains complexed with H3 peptide 1-7 Crystal Structure of NSD3 tandem PHD5-C5HCH domains complexed with H3 peptide 1-15 Crystal Structure of NSD3 tandem PHD5-C5HCH domains complexed with H3K9me3 peptide Crystal structure of the tandem tudor domain and plant homeodomain of UHRF1 with Histone H3K9me3 Crystal structure of the MLL5 PHD finger in complex with H3K4me3 Crystal structure of the DIDO PHD finger in complex with H3K4me3 Crystal Structure of MOZ double PHD finger Crystal Structure of MOZ double PHD finger histone H3 tail complex Crystal Structure of MOZ double PHD finger histone H3K9ac complex Crystal Structure of MOZ double PHD finger histone H3K14ac complex Protein Crystal Structure of Human Borjeson-Forssman-Lehmann Syndrome Associated Protein PHF6 Zinc fingers of KDM2B Crystal structure of human SP100 PHD-Bromodomain in the free state Crystal structure of human BAZ2A PHD zinc finger in complex with unmodified H3K4 histone peptide 4QF2 4QF3 4TVR 4UP0 4UP5 4YAB 4YAD 4YAT 4YAX 4YBM 4YBS 4YBT 4YC9 4ZQL 5B73 5B75 5B76 5B77 5B78 5B79 5C11 5C13 5CEH 5DAG 5DAH 5ERC 5FB0 5FB1 5HH7 5I3L 5K4L 5TAB 5TBN 5TDR 5TDW Links (links to other resources describing this domain) INTERPRO PFAM     Function of PHD Domain Proteins in Chromatin Regulation Gozani, Or P.    Stanford University, Stanford, CA, United States Institution Related projects Publications  Recent in Grantomics: Your institution vs. funders. Who wins? Read more... How should you pick the next fundable research topic? Read more...  Recently viewed grants: MARC Honors Undergraduate Research Training Program Molecular Origins and Evolution of Meiosis PU1 Transcription Factor and Osteoclastogenesis Systematic Approach for Defining Shape of A Hazard Curve Cardiovascular Mechanisms of Angiotensin in Birds  Recently added grants: Proteins important for dense-core vesicle function Determining the subcellular and molecular players in neutrophil trogocytic killing of the sexually-transmitted parasite Trichomonas vaginalis Membrane-Mediated Interactions of the Bcl-xL/Bid/Bax Triad of Apoptotic Regulators Pre- and Peri-natal Predictors of Childhood Obesity From GWAS loci to blood pressure genes, variants & mechanisms   Abstract The PHD finger (Plant Homeodomain) module is a signature chromatin-associated protein motif. This module is present throughout eukaryotic proteomes, and mutations in the PHD fingers of many proteins are associated with cancers, immunodeficiency and mental retardation syndromes, and other genetic disorders. We previously demonstrated that a subset of PHD fingers act as high affinity binding modules for histone H3 trimethylated at lysine 4 (H3K4me3). We linked H3K4me3 to multiple different functions via its recognition by discrete PHD finger nuclear proteins, including providing the firs evidence that disrupting the read-out of a histone modification can cause an inherited human disease. Our long-term goal is to develop a comprehensive understanding of how PHD domain-containing proteins impact on chromatin dynamics and the relationship of such activities to fundamental nuclear functions and human disease processes. Here we focus on the multiple PHD domain-containing protein NSD2 (also named MMSET and WHSC1), a histone lysine methyltransferase implicated in the pathogenesis of the hematologic malignancy multiple myeloma. However, the molecular mechanism by which NSD2 regulates chromatin and the relationship of its enzymatic activity to disease pathogenesis is not well understood. Our preliminary work indicates that the primary physiologic activity at chromatin of NSD2 is dimethylation of histone H3 at lysine 36 (H3K36me2), and that NSD2 - via H3K36me2 catalysis - drives oncogenic programming in myeloma cells. In Aim 1, we propose to extend our genomic studies and determine the genome-wide distribution of NSD2 in cancer and normal cells, and investigate the role of NSD2 activity and chromatin targeting in determining the H3K36me2 chromatin landscape. We also aim to elucidate the molecular mode of action for the NSD2 PHD domains and their role in the regulation of NSD2 cellular functions. In Aim 2, we will characterize the mode of action for H3K36 methylation. We will identify proteins that preferentially recognize H3K36me2 and test the hypothesis that these proteins transduce NSD2 activity at chromatin to downstream biological outcomes. We will also explore the broader hypothesis that exquisite level of biological regulation can be achieved by subtle changes in histone methylation. The goal of Aim 3 is to identify new substrates of NSD2 using a novel chemical biological-proteomic strategy we have developed for proteome-wide discovery of functionally-relevant NSD2 substrates. The role of the most promising targets in regulation of nuclear pathways will be investigated using a combination of molecular and cellular approaches. These studies will identify new nuclear signaling pathways that are regulated by NSD2 and that may play a role in human disease. Together these studies will provide important new insights into how histone methylation regulates fundamental nuclear processes and the relationship of these activities to the pathogenesis of human diseases. Public Health Relevance We propose to investigate the molecular mode of action for the lysine methyltransferase and epigenetic regulator NSD2 in mammalian cells. Numerous human diseases, including cancer arise from epigenetic abnormalities. This proposal will provide new insight into how epigenetic mechanisms regulate important cellular functions, and has the potential to identify new targets for therapeutic intervention for human disease.   Funding Agency   institution,   related projects.   Publications Sankaran, Saumya M; Gozani, Or Epigenetics 12:917-922 Zhu, Li; Li, Qin; Wong, Stephen H K et al. Cancer Discov 6:770-83 Li, Sisi; Yang, Zhenlin; Du, Xuan et al. Structure 24:486-94 Huang, Wei-Hsiang; Guenthner, Casey J; Xu, Jin et al. Neuron 92:392-406 van Nuland, Rick; Gozani, Or Mol Cell Proteomics 15:755-64 Sankaran, Saumya M; Wilkinson, Alex W; Elias, Joshua E et al. J Biol Chem 291:8465-74 Chen, Shoudeng; Yang, Ze; Wilkinson, Alex W et al. Mol Cell 60:319-27 Zhang, Wei; Sankaran, Saumya; Gozani, Or et al. ACS Chem Biol 10:1176-80 Carlson, Scott M; Moore, Kaitlyn E; Sankaran, Saumya M et al. J Biol Chem 290:12040-7 Moore, Kaitlyn E; Gozani, Or Biochim Biophys Acta 1839:1395-403 Showing the most recent 10 out of 52 publications   Comments Visit registry.google for more domains Hats off to higher education More information about why you are seeing these registrars listed What is .phd, .prof, and .esq? Who’s on these domains? An official website of the United States government The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site. The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. Publications Account settings My Bibliography Collections Citation manager Save citation to file Email citation, add to collections. Create a new collection Add to an existing collection Add to My Bibliography Your saved search, create a file for external citation management software, your rss feed. Search in PubMed Search in NLM Catalog Add to Search The DPF Domain As a Unique Structural Unit Participating in Transcriptional Activation, Cell Differentiation, and Malignant Transformation Affiliations. 1 Institute of Gene Biology Russian Academy of Sciences, Moscow, 119334 Russia. 2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia. PMID: 33456978 PMCID: PMC7800603 DOI: 10.32607/actanaturae.11092 The DPF (double PHD finger) domain consists of two PHD fingers organized in tandem. The two PHD-finger domains within a DPF form a single structure that interacts with the modification of the N-terminal histone fragment in a way different from that for single PHD fingers. Several histone modifications interacting with the DPF domain have already been identified. They include acetylation of H3K14 and H3K9, as well as crotonylation of H3K14. These modifications are found predominantly in transcriptionally active chromatin. Proteins containing DPF belong to two classes of protein complexes, which are the transcriptional coactivators involved in the regulation of the chromatin structure. These are the histone acetyltransferase complex belonging to the MYST family and the SWI/SNF chromatin-remodeling complex. The DPF domain is responsible for the specificity of the interactions between these complexes and chromatin. Proteins containing DPF play a crucial role in the activation of the transcription of a number of genes expressed during the development of an organism. These genes are important in the differentiation and malignant transformation of mammalian cells. Keywords: BAF; DPF domains; DPF1; DPF2; DPF3; MOZ and MORF histone acetyltransferases; PBAF; PHF10; tandem PHD. Copyright ® 2020 National Research University Higher School of Economics. PubMed Disclaimer Proteins and complexes containing the… Proteins and complexes containing the DPF domains. ( A ) – Domain organization… Alignment of the amino acid… Alignment of the amino acid sequences of the DPF motifs of the MOZ,… MOZ/MORF or DPF1-3 protein. The… MOZ/MORF or DPF1-3 protein. The interaction between the DPF domains and histone modifications… Similar articles Neuronal and Muscle Differentiation of Mammalian Cells Is Accompanied by a Change in PHF10 Isoform Expression. Bayramova DO, Azieva AM, Feoktistov AV, Georgieva SG, Soshnikova NV. Bayramova DO, et al. Dokl Biochem Biophys. 2024 Feb;514(1):23-27. doi: 10.1134/S1607672923700643. Epub 2024 Jan 8. Dokl Biochem Biophys. 2024. PMID: 38189889 Free PMC article. Conserved Structure and Evolution of DPF Domain of PHF10-The Specific Subunit of PBAF Chromatin Remodeling Complex. Chugunov AO, Potapova NA, Klimenko NS, Tatarskiy VV, Georgieva SG, Soshnikova NV. Chugunov AO, et al. Int J Mol Sci. 2021 Oct 15;22(20):11134. doi: 10.3390/ijms222011134. Int J Mol Sci. 2021. PMID: 34681795 Free PMC article. Review. Selective recognition of histone crotonylation by double PHD fingers of MOZ and DPF2. Xiong X, Panchenko T, Yang S, Zhao S, Yan P, Zhang W, Xie W, Li Y, Zhao Y, Allis CD, Li H. Xiong X, et al. Nat Chem Biol. 2016 Dec;12(12):1111-1118. doi: 10.1038/nchembio.2218. Epub 2016 Oct 24. Nat Chem Biol. 2016. PMID: 27775714 Free PMC article. The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification. Dreveny I, Deeves SE, Fulton J, Yue B, Messmer M, Bhattacharya A, Collins HM, Heery DM. Dreveny I, et al. Nucleic Acids Res. 2014 Jan;42(2):822-35. doi: 10.1093/nar/gkt931. Epub 2013 Oct 22. Nucleic Acids Res. 2014. PMID: 24150941 Free PMC article. Structural and functional characteristics of plant PHD domain-containing proteins. Wang TY, Wang YX, You CJ. Wang TY, et al. Yi Chuan. 2021 Apr 20;43(4):323-339. doi: 10.16288/j.yczz.20-412. Yi Chuan. 2021. PMID: 33972207 Review. Mass Spectrometry-Based Protein Footprinting Defines the Binding Pocket of Crotonylated H3K14 in the PHD1 Domain of BAF45D within the BAF Chromatin Remodeling Complex. Martinez MR, Kiselar J, Wang B, Sadalge D, Zawadzke L, Taherbhoy A, Musser D, Davenport Y, Setser J, Chance MR, Bellon S. Martinez MR, et al. ACS Bio Med Chem Au. 2024 Jun 17;4(4):204-213. doi: 10.1021/acsbiomedchemau.4c00009. eCollection 2024 Aug 21. ACS Bio Med Chem Au. 2024. PMID: 39184054 Free PMC article. Histone acylation at a glance. Bhattacharya S, Tu BP. Bhattacharya S, et al. J Cell Sci. 2024 Jun 1;137(11):jcs261250. doi: 10.1242/jcs.261250. Epub 2024 Jun 6. J Cell Sci. 2024. PMID: 38842578 Review. A novel chromatin-remodeling complex variant, dcPBAF, is involved in maintaining transcription in differentiated neurons. Soshnikova NV, Azieva AM, Klimenko NS, Khamidullina AI, Feoktistov AV, Sheynov AA, Brechalov AV, Tatarskiy VV, Georgieva SG. Soshnikova NV, et al. Front Cell Dev Biol. 2023 Nov 14;11:1271598. doi: 10.3389/fcell.2023.1271598. eCollection 2023. Front Cell Dev Biol. 2023. PMID: 38033872 Free PMC article. New twists of a TAIL: novel insights into the histone binding properties of a highly conserved PHD finger cluster within the MLR family of H3K4 mono-methyltransferases. Zraly CB, Schultz R, Diaz MO, Dingwall AK. Zraly CB, et al. Nucleic Acids Res. 2023 Oct 13;51(18):9672-9689. doi: 10.1093/nar/gkad698. Nucleic Acids Res. 2023. PMID: 37638761 Free PMC article. Kwan A.H.Y., Gell D.A., Verger A., Crossley M., Matthews J.M., Mackay J.P.. Structure. 2003;11(7):803–813. - PubMed Sanchez R., Zhou M.M.. Trends Biochem. 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So that we can show you the most relevant information, please select the option that most closely relates to you. Looking for undergraduate studies Looking for postgraduate studies Student but not looking for further education at the moment Parent or Guardian University administrator Professional Thanks for sending your response. Your input will help us improve your experience. You can close this popup to continue using the website or choose an option below to register in or login. Already have an account? Sign in What is a PhD? Hasna Haidar Share this Page Table of contents Introduction PhD admission requirements Phd applications, can i apply for a phd without relevant qualifications, phds through mphil, starting a phd, alternatives to a phd, degrees higher than a phd. Do you have a love of wisdom and a clear field of academic interest? If so, a PhD might be the right choice for you. But what is a PhD, and how can you get one? PhD is short for Doctor of Philosophy. This is an academic or professional degree that, in most countries, qualifies the degree holder to teach their chosen subject at university level or to work in a specialized position in their chosen field. The word ‘philosophy’ comes from the Ancient Greek philosophia , literally translated as ‘love of wisdom’. It originally signified an individual who had achieved a comprehensive general education in the fundamental issues of the present world. Today, the Doctor of Philosophy still requires a love of wisdom but applies to individuals who have pursued knowledge in a much more specialized field.      A PhD is a globally recognized postgraduate academic degree awarded by universities and higher education institutions to a candidate who has submitted a thesis or dissertation, based on extensive and original research in their chosen field. The specificities of PhD degrees vary depending on where you are and what subject you’re studying. In general, however, the PhD is the highest level of degree a student can achieve (with some exceptions). It usually follows a master’s degree, although some institutions also allow students to progress straight to a PhD from their bachelor’s degree. Some institutions also offer the opportunity to ‘upgrade’ or ‘fast-track’ your master’s degree to a PhD, provided you are deemed to possess the necessary grades, knowledge, skills and research abilities. Traditionally, a PhD involves three to four years of full-time study in which the student completes a substantial piece of original research presented as a thesis or dissertation. Some PhD programs accept a portfolio of published papers, while some countries require coursework to be submitted as well. Students must also complete a ‘viva voce’ or oral defense of their PhD. This can be with just a small number of examiners, or in front of a large examination panel (both usually last between one to three hours). While PhD students are traditionally expected to study on campus under close supervision, distance education and e-learning schemes have meant a growing number of universities are now accepting part-time and distance-learning PhD students.  Generally speaking,   PhD admission requirements   relate to the candidate’s grades (usually at both bachelor’s level and master’s level) and their potential research capabilities. Most institutions require that candidates hold an honors degree or a master’s degree with high academic standing, along with a bachelor’s degree with at least upper second-class honors. In some cases, you can also apply for a PhD simply on the basis of your master’s degree grades. Grades-based PhD admission requirements may also be based on the type of funding you will be using – you may be able apply with lower grades if you self-fund your PhD (read more on PhD funding  here ). Some institutions and subjects (such as psychology and some humanities and science subjects) stipulate that you must find a tenured professor in your chosen institution to serve as your formal advisor and supervisor throughout your PhD program before you can be formally accepted into the program. In other cases, you will be assigned a supervisor based on your research subject and methodology once you have been accepted into the PhD program. Either way, it is a good idea to approach a faculty member in your chosen institution before applying for a PhD, in order for them to determine whether your research interests align well with the department, and perhaps even help you to brainstorm PhD research options. Language proficiency Some PhD applications require proof of proficiency in the language in which you intend to study. You can either provide the results of an approved standardized language exam or show evidence of having completed undergraduate or postgraduate study in the relevant language. Employment/academic references Some institutions may also ask for a record of your employment such as a résumé, and/or all your academic transcripts, including details of course modules and module content as part of your PhD application. Details of other research projects you have completed and any publications you have been featured in can also help your application. Many PhD applicants are also asked to provide references from two or three people who know them well in an academic setting, such as their undergraduate or postgraduate tutors or professors. These references must have a particular focus on your academic performance, coursework and research abilities, your research potential and your interest in your chosen field of study. Personal statements Many institutions ask for a personal statement - a short essay which you can use to demonstrate your passion for your chosen subject. You can outline your reasons for wanting to study a PhD, personal motivations for doing so, any extracurricular activities that are particularly relevant or should be highlighted, and any flexibility in your chosen area(s) of research. If you need help, many institutions have a guide to personal statements on their website, which can also help you tailor your personal statement to each institution. PhD research proposals Finally, in order to be considered for a place on a PhD program, applicants are expected to submit a PhD research proposal. A research proposal: Outlines your proposed research topics in the context of previous work, Highlights your awareness of current debates within the field, Demonstrates a suitable level of analysis, Identifies relevant gaps in current knowledge, Suggests a relevant research hypothesis to fill some of these gaps, Explains your intended research methodology in sufficient detail, Discusses the implications to real-world policy that your PhD proposal may invite. This will help admissions tutors to assess your aptitude for PhD research, and also to determine whether your research interests align with their own research priorities and available facilities. They will also consider whether they have the relevant staff to provide you sufficient supervisory expertise. For this reason in particular, it is important to research institutions thoroughly before applying for a PhD. Not only will you be happier if your research interests fit in with those of your chosen institution, but institutions may be forced to reject your application simply on the basis of discrepancies between their research interests and yours. Note that this initial research proposal is not necessarily binding – it is usually a starting point from which to further develop your research idea. Some subject areas (such as science and engineering) do not ask for original research proposals. Instead, the institution presents a selection of PhD research projects which are formulated by the supervisor(s) concerned and peer reviewed. This may be done at a certain time of year or year-round, depending on the institution. Students can then submit a statement demonstrating a clear understanding of the research to be undertaken and their suitability to undertake it. These PhD research projects may also have been formulated in consultation with another organization that may provide funding/scholarships for the successful candidate. These pre-defined PhD projects are less common in arts, humanities and social sciences subjects, where it’s more common for students to submit their own proposals. If you wish to do a PhD but do not have the relevant qualifications or their equivalent, you may still be able to apply for a PhD program by fulfilling additional requirements as stipulated by your institution of choice. Some possible requirements could be to undertake specified extra study or passing a qualifying examination. You may also be able to make a special case to your chosen institution, either on the basis of a non-degree professional qualification and considerable practical experience, or on the basis of foreign qualifications. Special case PhD applications will require the strong backing of your potential supervisor, so you will need to seek his/her advice and support before applying in this manner. Another option available for potential PhD candidates is to apply as a general research student or for an  MPhil degree . This is a common path taken by PhD candidates. The MPhil is an advanced master’s degree awarded for research and can be suitable for students who do not have a strong research background. You will be required to take some taught courses to get you up to speed with things like research methods. The successful completion of a one-year taught program may lead to the award of the MRes degree, which includes more taught components than the MPhil and can be awarded in lieu of a PhD for students who have not completed the required period of study for a PhD. Alternatively, the successful completion of original research may lead to the award of the MPhil degree, which can be awarded without the candidate having to present a defense of their dissertation (a requirement to achieve a PhD). If, after the first or second year of your research (i.e. during your MPhil), the institution is satisfied with the progress of your work, you may then be able to apply for full PhD registration. Usually, your supervisor or tutor will be in charge of determining whether you are ready to progress to a PhD. If you’re deemed to be ready, you will then need to develop a title for your thesis and choose your PhD program. Testimonials "CUHK’s MBA programme provided me with the stepping stone into a larger sports Asian market wherein I could leverage the large alumni network to make the right connections for relevant discussions and learning." Read my story Abhinav Singh Bhal Chinese University of Hong Kong graduate "I have so many wonderful memories of my MBA and I think, for me, the biggest thing that I've taken away was not what I learned in the classroom but the relationships, the friendships, the community that I'm now part of." Alex Pitt QS scholarship recipient "The best part of my degree is getting to know more about how important my job as an architect is: the hidden roles I play, that every beautiful feature has significance, and that even the smallest details are well thought out." Rayyan Sultan Said Al-Harthy University of Nizwa student "An MBA at EAHM is superior due to  the nature of the Academy’s academic and  industry strength. The subject  matter, the curriculum structure and the  access to opportunities within the hospitality industry is remarkable." Sharihan Al Mashary Emirates Academy of Hospitality Management graduate When registration has been completed you should be officially informed of: your supervisor(s) and their area(s) of expertise; the topic or field of PhD research for which you have been accepted; the minimum length of time required before submission of your thesis; the formal assessment methods preferred by the institution. Most institutions will also provide you with a comprehensive list of provisions and available facilities for PhD and research students at the university. They will also include a detailed outline of the milestones you must reach on your journey to achieve a PhD. Your supervisor will be in charge of going through these milestones with you, making reports on your progress, and advising you on your next steps. You will need to make adequate progress each year in order to continue your PhD studies. When looking for PhD programs, keep in mind that there are several types of degrees which have the term “doctor” in their title, such as the Juris Doctor (common in the US, Canada, Australia, Mexico and parts of Asia), the Doctor of Physical Therapy (DPT) or the Doctor of Pharmacy (DPharm) and the US and Canada version of the Doctor of Medicine (MD). These degrees are generally not classified as PhDs as they lack that vital component that really defines the PhD: academic research. These other types of doctorate degrees are instead referred to as entry-level doctorate degrees. Candidates who wish to pursue a PhD may do so afterwards, and this may be known as a ‘post-professional doctorate’. Neither the JD nor the US/Canada MD programs universally require students to complete a specified academic research component in order to be awarded the degree title. However, there are also many research degrees, such as the MD, which conduct scholarly research (medical in the case of the MD) which is published in peer-reviewed journals. This makes them very similar to PhDs, and some countries consider them equivalent. Some institutions therefore offer combined professional and research training degrees, such as the MD-PhD dual program, which is useful for medical professionals looking to pursue a research career. In addition to various degrees which may be considered equivalent to a PhD, there are also some ‘higher doctorate’ courses considered to be a step above the Doctor of Philosophy (PhD). These are most common in UK universities and in some European countries, although they are increasingly awarded as honorary degrees. The US does not have a system of higher doctorates and offer the titles solely as honorary degrees. Honorary degrees are sometimes signified by adding ‘hc’ (for  honoris causa ) to the end of the degree title. Some higher doctorate degrees include: Doctor of Science (DS/SD): Awarded in recognition of a substantial and sustained contribution to scientific knowledge beyond that required for a PhD. Doctor of Literature/Letters (DLit/DLitt/LitD): Awarded in recognition of achievement in the humanities or for original contribution to the creative arts. Doctor of Divinity (DD): Awarded above the Doctor of Theology (DTh), usually to recognize the recipient’s ministry-oriented accomplishments. Doctor of Music (DMus): Awarded in the UK, Ireland and some Commonwealth countries on the basis of a substantial portfolio of compositions and/or scholarly publications on music. Doctor of Civil Law (DCL): Highest doctorate excepting the DD, offered on the basis of exceptionally insightful and distinctive publications that contain significant and original contributions to the study of law or politics in general. This article was first published in February 2014 and most recently updated in January 2020. Want more content like this?   Register for free site membership  to get regular updates and your own personal content feed. + 28 others saved this article + 29 others saved this article Recommended articles Last year Oxford or Cambridge? Top law schools Top business schools Discover top-ranked universities! universities events every year Sign up to continue reading Ask me about universities, programs, or rankings! Our chatbot is here to guide you. QS SearchBot What Is a PhD Thesis? Doing a PhD This page will explain what a PhD thesis is and offer advice on how to write a good thesis, from outlining the typical structure to guiding you through the referencing. A summary of this page is as follows: A PhD thesis is a concentrated piece of original research which must be carried out by all PhD students in order to successfully earn their doctoral degree. The fundamental purpose of a thesis is to explain the conclusion that has been reached as a result of undertaking the research project. The typical PhD thesis structure will contain four chapters of original work sandwiched between a literature review chapter and a concluding chapter. There is no universal rule for the length of a thesis, but general guidelines set the word count between 70,000 to 100,000 words . What Is a Thesis? A thesis is the main output of a PhD as it explains your workflow in reaching the conclusions you have come to in undertaking the research project. As a result, much of the content of your thesis will be based around your chapters of original work. For your thesis to be successful, it needs to adequately defend your argument and provide a unique or increased insight into your field that was not previously available. As such, you can’t rely on other ideas or results to produce your thesis; it needs to be an original piece of text that belongs to you and you alone. What Should a Thesis Include? Although each thesis will be unique, they will all follow the same general format. To demonstrate this, we’ve put together an example structure of a PhD thesis and explained what you should include in each section below. Acknowledgements This is a personal section which you may or may not choose to include. The vast majority of students include it, giving both gratitude and recognition to their supervisor, university, sponsor/funder and anyone else who has supported them along the way. 1. Introduction Provide a brief overview of your reason for carrying out your research project and what you hope to achieve by undertaking it. Following this, explain the structure of your thesis to give the reader context for what he or she is about to read. 2. Literature Review Set the context of your research by explaining the foundation of what is currently known within your field of research, what recent developments have occurred, and where the gaps in knowledge are. You should conclude the literature review by outlining the overarching aims and objectives of the research project. 3. Main Body This section focuses on explaining all aspects of your original research and so will form the bulk of your thesis. Typically, this section will contain four chapters covering the below: your research/data collection methodologies, your results, a comprehensive analysis of your results, a detailed discussion of your findings. Depending on your project, each of your chapters may independently contain the structure listed above or in some projects, each chapter could be focussed entirely on one aspect (e.g. a standalone results chapter). Ideally, each of these chapters should be formatted such that they could be translated into papers for submission to peer-reviewed journals. Therefore, following your PhD, you should be able to submit papers for peer-review by reusing content you have already produced. 4. Conclusion The conclusion will be a summary of your key findings with emphasis placed on the new contributions you have made to your field. When producing your conclusion, it’s imperative that you relate it back to your original research aims, objectives and hypotheses. Make sure you have answered your original question. Finding a PhD has never been this easy – search for a PhD by keyword, location or academic area of interest. How Many Words Is a PhD Thesis? A common question we receive from students is – “how long should my thesis be?“. Every university has different guidelines on this matter, therefore, consult with your university to get an understanding of their full requirements. Generally speaking, most supervisors will suggest somewhere between 70,000 and 100,000 words . This usually corresponds to somewhere between 250 – 350 pages . We must stress that this is flexible, and it is important not to focus solely on the length of your thesis, but rather the quality. How Do I Format My Thesis? Although the exact formatting requirements will vary depending on the university, the typical formatting policies adopted by most universities are: Font Any serif font e.g. Times New Roman, Arial or Cambria Font Size 12pt Vertical Line Spacing 1.5 Lines Page Size A4 Page Layout Portrait Page Margins Variable, however, must allow space for binding Referencing Variable, however, typically Harvard or Vancouver What Happens When I Finish My Thesis? After you have submitted your thesis, you will attend a viva . A viva is an interview-style examination during which you are required to defend your thesis and answer questions on it. The aim of the viva is to convince your examiners that your work is of the level required for a doctoral degree. It is one of the last steps in the PhD process and arguably one of the most daunting! For more information on the viva process and for tips on how to confidently pass it, please refer to our in-depth PhD Viva Guide . How Do I Publish My Thesis? Unfortunately, you can’t publish your thesis in its entirety in a journal. However, universities can make it available for others to read through their library system. If you want to submit your work in a journal, you will need to develop it into one or more peer-reviewed papers. This will largely involve reformatting, condensing and tailoring it to meet the standards of the journal you are targeting. Browse PhDs Now Join thousands of students. Join thousands of other students and stay up to date with the latest PhD programmes, funding opportunities and advice. An official website of the United States government The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site. The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. Publications Account settings The PMC website is updating on October 15, 2024. Learn More or Try it out now . Advanced Search Journal List v.7(11); 2006 Nov What is a PhD? This might seem like an unusual topic, as most scientists seem to know exactly what a PhD is and for what it stands. But on closer inspection, a PhD has as many meanings as there are educational systems. It is not—and has never been—a single, well-defined qualification. As research practices and funding change, the situation becomes even more confused, with consequences for the quality of both scientific training and research. I received my PhD from a British university. After three years of research, I submitted a three-centimetre-thick thesis that addressed a specific problem. Being awarded my doctorate meant that I knew my topic, I understood enzymology, I could work with proteins and I was able to navigate the complexities of enzyme kinetics. I was not qualified for the title until I was able to demonstrate all these things. In essence, my PhD showed that I developed from a dependent student into an independent scientist. Since then, PhDs in the UK seem to have changed. More often than not, a PhD is now awarded after the completion of a fixed term of research. Of course, there is an overall topic, but if the student does not reach a hypothesis-based conclusion within a timeframe of about three years, this is no longer a hindrance to earning the degree. Increasingly, the thesis has become a report with an emphasis on training rather than the detailed description of a scientific project. Other countries have different systems. In the USA, the PhD phase is a genuine period of postgraduate training that includes both theory and research, with a greater emphasis on course work and the possibility of rotating through different laboratories. In Nordic countries, the situation is more complex: some universities adopt the US model, whereas some focus on publication output, and others are variations of these. In Germany, it is necessary to spend up to two years on a diploma degree before moving on to a PhD. Many other countries require their PhD students to teach undergraduates. In some systems, the final examination is a mere formality with an inevitably positive outcome; in others, it is a rigorous cross-examination by jury. Against this background of different systems, new aspects have arisen that are moulding the PhD into a different entity to what it was. For example, the concept that a student must carry out an individual piece of research seems outdated. Most publications list many authors, each of whom contributed to the overall content of the paper. In fact, scientific research increasingly demands teamwork, and the PhD system must adapt accordingly; indeed, an important lesson for a young scientist is to learn how to work in a team. But if the thesis is a cooperative effort, then it becomes even more difficult to judge the input of each individual—yet a PhD is awarded to an individual. Finances are another matter. In some countries, there is only a limited amount of money available to support a PhD student. Once that is spent, the student must survive by the most precarious means: relying on parents or partners to cover the gap, finding a grant to stay afloat, or taking a part-time job, even if this eats into the precious time and energy needed to complete the thesis. If we accept these realities, it makes sense that a PhD is awarded on the basis of time and effort spent, rather than on scientific work alone. But in that case, a PhD is merely an apprenticeship and no longer represents a stamp of achievement. Is this really a cause for concern? Even if all PhD programmes followed the same rules and regulations, there would still be many theses chronicling failure rather than achievement. But if we collectively become unconcerned about what a PhD is, then we have little basis for expecting the pre-doc students in our laboratories to go through the diligent work that ultimately enables experiments to work and provides robust results. The ‘three years and out' mentality concentrates on time and investment rather than quality, and runs the risk of producing substandard scientists. Thus, there might be real consequences for research if we lower the standards for earning a PhD. Perhaps one of the reasons behind the success of the US research system is the quality and structure of their PhD training. Maybe one reason why European countries produce such a high number of papers of more moderate quality is the frequent requirement for a defined number of first-author publications to complete a PhD. Perhaps the concept of writing a thesis on the basis of a well-defined body of work is so foreign to today's students that they prefer the easier route of collating a few papers on which they contributed. If we change the standards and requirements for obtaining a PhD, this will inevitably shape the next generation of scientists. Thus, we should know more and ask more about what a PhD really means. Instead of treating the degree as an ‘access card' to the laboratory, we should ask for more information: how the candidate was examined, who sat on the jury, and what comprises training in the applicant's country or university. Most importantly, we should insist that a PhD is not merely a vague title but actually means what it implies: it is an award to an expert who has proven their scientific worth and not to someone who stayed in a tolerant group for long enough. 279 IMAGES The PHD domain of the human MLL protein. A. The complete structure of The PHD domain of the human MLL protein. A. The complete structure of Breakpoints within the MLL gene influence the PHD domain structure. A Molecular surface representation of the PHD domain shown from two PhD Studies Domain vs. Non-Domain Characteristics Exploring The New .PHD Domain Name For Business Growth VIDEO Dearest (You're the Nearest to My Heart) WHEN TO USE CLASS DIAGRAM & more examples class and domain diagram 43989 boardwalk loop, Babcock Ranch, FL Presented by Meyers Fowler & Associates Is PhD End PhD Let's make LAMORDIA in 5e! COMMENTS PHD finger The PHD finger was discovered in 1993 as a Cys 4-His-Cys 3 motif in the plant homeodomain (hence PHD) proteins HAT3.1 in Arabidopsis and maize ZmHox1a. [1] The PHD zinc finger motif resembles the metal binding RING domain (Cys 3-His-Cys 4) and FYVE domain.It occurs as a single finger, but often in clusters of two or three, and it also occurs together with other domains, such as the ... The PHD Finger: A Versatile Epigenome Reader Ligand recognition. The PHD finger is a small protein domain of 50-80 amino acid residues of diverse sequences containing a zinc-binding motif that appears in many chromatin-associated proteins [] (Figure 1A).The conserved PHD fold consists of a two-strand anti-parallel β-sheet and a C-terminal α-helix (not present in all PHDs) that is stabilized by two zinc atoms anchored by the Cys4-His ... The PHD finger, a nuclear protein-interaction domain The PHD finger is a common structural motif found in all eukaryotic genomes. It is a Zn(2+)-binding domain and its closest structural relative is the RING domain. Many RING fingers bind to E2 ligases to mediate the ubiquitination of proteins. Whether PHD fingers share a common function is unclear. N … Characterization of the plant homeodomain (PHD) reader family for their Analysis of the PHD finger proteome via protein domain microarrays. To define the histone binding preferences of the PHD finger proteome, we expressed and purified 123 annotated human PHD-containing domains as GST-tagged recombinant fusions from E. coli.The recombinant proteins consisted of either PHD fingers in isolation, or as tandem domains if a given PHD finger was located adjacent to ... PHD Finger The PHD finger is a small protein domain of 50-80 amino acid residues of diverse sequences containing a zinc-binding motif that appears in many chromatin-associated proteins [15] (Figure 1 a).The conserved PHD fold consists of a two-strand anti-parallel β-sheet and a C terminal α-helix (not present in all PHDs) that is stabilized by two zinc atoms anchored by the Cys4-His-Cys3 motif in ... The PHD finger, a nuclear protein-interaction domain The PHD finger is a common structural motif found in all eukaryotic genomes. It is a Zn2+-binding domain and its closest structural relative is the RING domain. Many RING fingers bind to E2 ligases to mediate the ubiquitination of proteins. Whether PHD fingers share a common function is unclear. Notably, many if not all PHD fingers are found in nuclear proteins whose substrate tends to be ... Structural and functional characteristics of plant PHD domain Plant homeodomain (PHD) is a class of transcription factor in the Zinc finger domain family. The most important function of which is to recognize various histone modifications, including histone methylation and acetylation, etc. They can also bind to DNA. Proteins with PHD domains, some of which possess histone modification enzyme activity, or ... Genome wide survey, evolution and expression analysis of PHD finger Plant homeodomain (PHD) finger proteins are widely present in all eukaryotes and play important roles in chromatin remodeling and transcriptional regulation. The PHD finger can specifically bind a number of histone modifications as an "epigenome reader", and mediate the activation or repression of underlying genes. Many PHD finger genes have been characterized in animals, but only few ... PHD Fingers: Epigenetic Effectors and Potential Drug Targets The PHD fingers of BPTF (bromodomain and PHD domain transcription factor) and ING2 (inhibitor of growth 2) were first identified as histone code readers within the PHD family when they were found to recognize H3K4me3 (11-14). BPTF is a subunit of the ATP-dependent chromatin-remodeling NURF (nucleosome remodeling factor) complex that promotes ... Non-histone binding functions of PHD fingers The PHD finger is found in eukaryotic nuclear proteins involved in the regulation of gene transcription and chromatin (see Glossary) remodeling. This small, ~65-residue evolutionarily conserved cysteine-rich domain can be distinguished by its canonical C4HC3 motif that coordinates two zinc ions in a cross-braced topology. The typical PHD finger folds into a short double-stranded antiparallel ... SMART: PHD domain annotation Description: The plant homeodomain (PHD) finger is a C4HC3 zinc-finger-like motif found in nuclear proteins thought to be involved in epigenetics and chromatin-mediated transcriptional regulation. The PHD finger binds two zinc ions using the so-called 'cross-brace' motif and is thus structurally related to the RING finger and the FYVE finger. Function of PHD Domain Proteins in Chromatin Regulation Function of PHD Domain Proteins in Chromatin Regulation. The PHD finger (Plant Homeodomain) module is a signature chromatin-associated protein motif. This module is present throughout eukaryotic proteomes, and mutations in the PHD fingers of many proteins are associated with cancers, immunodeficiency and mental retardation syndromes, and other ... What will you do with your .phd? .Phd is a secure domain for anyone who has earned their doctorate. You've earned your diploma, now show off what you can do. Please use suggested copy when talking about .phd on your website or in any marketing materials..Phd is a secure domain for anyone who has earned their doctorate. You've earned your diploma, now show off what you can do. Hats off to higher education The domains are automatically included on the HSTS preload list, meaning security is built in. .Phd is for anyone who has earned their doctorate. You've earned your diploma, now show off what you can do. .Prof is for professors. Profess your accomplishments on a .prof domain. .Esq is for lawyers. Show off your BAR admissions, alma mater ... How to Choose a PhD Research Topic Consider several ideas and critically appraise them: You must be able to explain to others why your chosen topic is worth studying. You must be genuinely interested in the subject area. You must be competent and equipped to answer the research question. You must set achievable and measurable aims and objectives. Exploring The New .PHD Domain Name For Business Growth The .phd domain is a generic top-level domain (gTLD) that is open to anyone to register. There are no restrictions on who can register a .phd domain, regardless of their educational background. What is the registration term for .phd domain names? .phd domain names can be registered for a minimum period of 1 year and a maximum of 10 years. The DPF Domain As a Unique Structural Unit Participating in The DPF (double PHD finger) domain consists of two PHD fingers organized in tandem. The two PHD-finger domains within a DPF form a single structure that interacts with the modification of the N-terminal histone fragment in a way different from that for single PHD fingers. Several histone modificatio … What is a PhD? PhD is short for Doctor of Philosophy. This is an academic or professional degree that, in most countries, qualifies the degree holder to teach their chosen subject at university level or to work in a specialized position in their chosen field. The word 'philosophy' comes from the Ancient Greek philosophia, literally translated as 'love ... PhD The 7 terms your supervisor/adviser have given you form a useful continuum for focusing your research. However it is important that you personalise the approach for yourself. Research Domain ... What Is a PhD Thesis? A PhD thesis is a concentrated piece of original research which must be carried out by all PhD students in order to successfully earn their doctoral degree. The fundamental purpose of a thesis is to explain the conclusion that has been reached as a result of undertaking the research project. The typical PhD thesis structure will contain four ... What is a PhD? Other countries have different systems. In the USA, the PhD phase is a genuine period of postgraduate training that includes both theory and research, with a greater emphasis on course work and the possibility of rotating through different laboratories. In Nordic countries, the situation is more complex: some universities adopt the US model ... essay homework paper phd project resume review speech study thesis