Characterization of the Plant Homeodomain (PHD) Reader Family for Their Histone Tail Interactions Kanishk Jain1,2, Caroline S
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Jain et al. Epigenetics & Chromatin (2020) 13:3 https://doi.org/10.1186/s13072-020-0328-z Epigenetics & Chromatin RESEARCH Open Access Characterization of the plant homeodomain (PHD) reader family for their histone tail interactions Kanishk Jain1,2, Caroline S. Fraser2,3, Matthew R. Marunde4, Madison M. Parker1,2, Cari Sagum5, Jonathan M. Burg4, Nathan Hall4, Irina K. Popova4, Keli L. Rodriguez4, Anup Vaidya4, Krzysztof Krajewski1, Michael‑Christopher Keogh4, Mark T. Bedford5* and Brian D. Strahl1,2,3* Abstract Background: Plant homeodomain (PHD) fngers are central “readers” of histone post‑translational modifcations (PTMs) with > 100 PHD fnger‑containing proteins encoded by the human genome. Many of the PHDs studied to date bind to unmodifed 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 fngers have gone uncharacterized; thus, our understanding of how these domains contribute to chromatin biology remains incomplete. Results: We expressed and screened 123 of the annotated human PHD fngers 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 unmodifed or methylated at H3K4. These H3 readers were further characterized by histone peptide microarrays and/or AlphaScreen to comprehensively defne 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. Keywords: Chromatin, Histone methylation, PHD fngers, Histone peptide microarray, Protein domain microarray Background chromatin function: some are believed to directly impact Histone proteins are fundamental to genome organiza- chromatin organization through biophysical means, but tion and packaging, and are chemically modifed by a the vast number likely function through their ability to wide range of “writer” or “eraser” enzymes that, respec- recruit efector or “reader” domain-containing proteins tively, install or remove histone post-translational modi- to chromatin. Tese reader proteins, which are often fcations (PTMs) [1, 2]. Tese PTMs play a central role in found in large multi-subunit complexes and in additional chromatin-modifying machines, interact with histone tails and chromatin in various ways that regulate gene *Correspondence: [email protected]; [email protected] transcription and other chromatin functions [2, 3]. Te 1 Department of Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, NC 27599, USA varied and diverse patterns of histone PTMs that exist 5 Department of Epigenetics and Molecular Carcinogenesis, The in vivo are referred to as the ‘histone code’, which is still University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA poorly understood [2, 3]. Full list of author information is available at the end of the article © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Jain et al. Epigenetics & Chromatin (2020) 13:3 Page 2 of 11 Histone PTMs often have either activating or repres- diseases such as breast cancer and leukemia [7, 20–24], sive efects on gene transcription depending on the type these fndings enhance our overall understanding of of PTM (acetylation, methylation, etc.) and the position PHD reader–histone interactions and should serve as a being modifed (H3K4, H3S10, etc.). In general, distinct resource and platform for future studies. classes of reader domains bind to specifc types of PTMs; for example, bromodomains recognize lysine acetylation Results [4], chromodomains recognize methyl-lysine [5], and Analysis of the PHD fnger proteome via protein domain the PHD fngers characterized to date generally recog- microarrays nize unmodifed or methylated lysine residues [6]. Fur- To defne the histone binding preferences of the PHD fn- thermore, many chromatin-associated proteins contain ger proteome, we expressed and purifed 123 annotated multiple reader domains, either multiples of the same human PHD-containing domains as GST-tagged recom- type [7] or a variety of diferent domains [8], potentially binant fusions from E. coli. Te recombinant proteins meaning that the in vivo engagement with chromatin is consisted of either PHD fngers in isolation, or as tandem multivalent. Signifcantly, increasing evidence shows that domains if a given PHD fnger was located adjacent to dysregulation of the epigenetic machinery, most notably another reader domain (e.g., one or more PHD fngers, the readers, writers, and erasers of the histone code, is Tudor, chromo and/or bromodomains) (Additional fle 1: causal for a wide range of human disease, including can- Table S1). Tese GST fusions were printed in duplicate on cer [9]. nitrocellulose-coated microarray slides and probed with Plant homeodomain fngers comprise one of the larg- biotinylated peptides that represented the N-termini of est families of reader domains, with over 100 human H3, H4, H2A or H2B (Fig. 1a and Additional fle 2: Figure proteins containing this module [6]. PHD fngers are Zn- S1). As the majority of PHD readers thus far character- coordinating domains that generally recognize unmodi- ized are H3K4me0/3 readers [6], we included additional fed or methylated lysines. To date, the majority of those peptides (H3K4 as either mono-, di-, or trimethylated) to characterized bind to histone H3 tails either methylated further determine any H3K4 methyl preference (Addi- at K4 [7], or unmodifed in that position (i.e., KDM5B tional fle 2: Figure S2 and Fig. 1b). As a control, we also PHD3 versus KDM5B PHD1 [10, 11] or PHF21A, also probed these microarrays with an α-Tubulin peptide (a.a. known as BHC80 [12]). A smaller number of PHD fn- 30–50) that would not be predicted to interact with PHD gers are reported as readers of H3K9 trimethylation fngers (Additional fle 2: Figure S1). As in Fig. 1a, b, 31 of (H3K9me3; e.g., CHD4) [13, 14] and H3K36me3 (e.g., the 123 PHD-containing fusions showed positive binding budding yeast Nto1) [15]. Intriguingly, the dual PHD to the H3 N-terminus, with the majority of these interac- fnger region of DPF3b has been reported as a reader of tions showing preference for trimethylated H3K4. In con- H3K14ac [16], while PHD6 of MLL4 has been reported trast, the H2A, H2B, H4, and tubulin peptides showed to recognize H4K16ac [17]. Additionally, a number of little to no positive interactions, suggesting that the PHD these PHD fngers occur in tandem (e.g., MLL1-4 [7] and fnger family broadly prefers the histone H3 tail (Addi- PZP-containing proteins [18, 19]) or next to additional tional fle 2: Figure S1). We note that the absence of bind- reader domain types (e.g., bromodomains and chromo- ing in these experiments does not rule out the possibility domains) [20–22], suggesting combinatorial interaction of PHD-fnger:histone PTM recognition under diferent capabilities. hybridization conditions. We also cannot exclude the Despite great progress in uncovering the role of a sub- possibility that some PHD fngers might not be function- set of PHD fngers, many (over 100) of the annotated ally active on the microarrays (perhaps due to misfolding domain family remain uncharacterized. In this report, or the lack of an important adjacent region). we set out to close the gap in our understanding of this Based on the above, we were able to classify the [PHD– reader domain class. Using a combination of comple- H3 tail] interactions into three groups, namely PHD fn- mentary approaches (reader domain microarrays, pep- gers that: (1) bound specifcally with methylated H3K4; tide microarrays, pulldowns, and AlphaScreen peptide (2) interacted only with unmethylated H3K4; or (3) assays), we show (31/123) of the PHD-containing query bound without preference to the H3K4 methylated state. proteins to bind histone H3 N-terminal peptides, with Many of the PHD fngers found to only bind H3K4 meth- the majority of these preferring H3K4me3 over unmodi- ylation have previously been described and include the fed H3K4. Furthermore, a number of unreported histone well characterized domains from the ING and PHF pro- PTM–PHD protein interactions were uncovered, with tein families [6, 24]. Te PHD fnger of MLL5, a mem- the PHD regions of PHRF1 and TRIM66 binding pref- ber of the MLL/KMT2 family [25–28], showed strong erentially to an unmodifed H3 N-terminal tail peptide. preference for H3K4me2 and H3K4me3. Tis fnding Given that many of these PHD fngers are mutated in adds to the relatively small number of MLL5-histone Jain et al.