THE SYMMETRIC DIMETHYLATION OF HISTONE H3 ARGININE 2: A NOVEL HISTONE MARK INVOLVED IN EUCHROMATIN MAINTENANCE VALENTINA MIGLIORI (Master in Molecular Biotechnology (Hons), Alma Mater Studiorum Bologna, Italy) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2012 ! ABSTRACT The asymmetric dimethylation of histone H3 arginine acts as a repressive mark that antagonizes trimethylation of H3 lysine 4. In this study, I report that H3R2 is also symmetrically dimethylated (H3R2me2s) by PRMT5 and PRMT7, and is present in euchromatic regions. Profiling of H3-tail interactors by SILAC-Mass Spectrometry revealed that H3R2me2s excludes binding of RBBP7, a central component of co-repressor complexes Sin3a, NURD and PRC2. Conversely H3R2me2s enhances binding of WDR5, a common component of the MLL/Set1a/b, the NLS1 and the ATAC co-activator complexes. The interaction with WDR5 distinguishes H3R2me2s from H3R2me2a, which impedes its recruitment to chromatin. The crystallographic structure of WDR5 and the H3R2me2s peptide in a stable complex elucidates the molecular determinants of this high affinity interaction (collaboration with Marina Mapelli IFOM IEO Milan). My findings provide insight into H3R2me2s as a novel mark that keeps genes poised in euchromatin for transcriptional activation upon cell cycle withdrawal and differentiation. ! ! ! ! ! ! ! ! TO ERNESTO WHO STARTED ME ON THIS JOURNEY AND TO SEMIL WHO MADE EVERY BIT WORTHWHILE ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ""! ! ACKNOWLEDGMENTS I would like to thank my mentor, Dr Ernesto Guccione for taking me into his lab. Dr Guccione has been an incredible teacher and friend and I am immensely grateful to have been able to train with him. The first time I met him, I told him I would have followed him wherever he would have gone and so I did, finding myself in Singapore! I would like to thank the members of my thesis committee- Dr Philipp Kaldis and Dr FU Xin Yuan for helping me navigate a path through the complexities of scientific research. I am indebted to all the members of the Guccione’s lab through the years- in particular Marco, Sameer, Sleem, Diana, Julius, Shun Xie and Cheryl, for their generosity not just in terms of sharing samples, materials and equipment, but also for sharing their invaluable scientific skills, experience and knowledge with me. I would also like to thank the friends that I have made during my years in Singapore, and my “family”: Taz, Edgar and in particular Semil, who, alone, has made worthwhile this entire journey. Finally, I would like to thank my parents, Silvi and Giordi, my sister Elena, not to mention Max and La Pucci, for their constant and unconditional support throughout the years.! ! """! TABLE OF CONTENTS ACKNOWLEDGEMENTS iii! TABLE OF CONTENTS iv! SUMMARY viii! LIST OF FIGURES . ix! LIST OF TABLES xii! LIST OF PUBLICATIONS .xii! PREFACE .xiii! CHAPTER – INTRODUCTION 1! 1.1 CHROMATIN AND TRANSCRIPTIONAL REGULATION 1! 1.2 HISTONE MARKS AND HISTONE CODE HYPOTHESIS . 5! 1.3 ARGININE METHYLATION . 10! 1.3.1 THE BIOCHEMISTRY OF PROTEIN ARGININE METHYLATION 13 1.3.2 HISTONE ARGININE METHYLATION 13 1.3.1 THE BIOCHEMISTRY OF PROTEIN ARGININE METHYLATION 13 1.3.2 HISTONE ARGININE METHYLATION 13 1.3.3 ARGININE METHYLATIONS LINKED TO TRANSCRIPTIONAL ACTIVATION: H4/H2AR3me2a, H3R17me2a, H3R26me2a 13 1.3.3.1 H4R3me2a and H2AR3me2a . 13 1.3.3.2 H3R17me2a 15 1.3.3.3 H3R26me2a 17 1.3.4 REPRESSIVE ARGININE METHYLATIONS: H4/H2AR3me2s, H3R8me2s and H3R2me2a . 18 1.3.4.1 H4/H2AR3me2s 18 1.3.4.2 H3R8me2s . 21 1.3.4.3 H3R2me2a 22 1.3.5 CITRULLINATION AND ARGININE DEMETHYLATION . 24 1.3.6 METHYLATED AND UNMETHYLATED ARGININE AS PROTEIN DOCKING SITES: ADDING ANOTHER LAYER OF COMPLEXITY TO THE “HISTONE CODE” . 28 1.3.6.1 METHYLATED ARGININES AS DOCKING SITES . 31 1.3.6.2 METHYLATED ARGININES AS EXCLUSION MARKS 33 CHAPTER – OBJECTIVES . 36 CHAPTER – MATHERIALS AND METHODS . 39! 3.1 ANTIBODIES . 39! 3.2 ANTIBODY PURIFICATION . 39! 3.3 QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION (qChIP) 41 3.4 ChIPseq LIBRARY PREPARATION 42! 3.5 DATA PROCESSING 44! 3.6 QUANTITATIVE PCR 44! 3.7 PEPTIDE PULL DOWN ASSAY 45! 3.8 METHYLATION ASSAY . 46! v 3.9 GST-WDR5 PROTEIN PREPARATION 47! 3.10 CRYSTALIZATION, CRYSTAL STRUCTURE DETERMINATION . 49! 3.11 MICROARRAY ANALYSIS . 50! 3.12 IMMUNOBLOTTING . 50 3.13 PRODUTION OF LENTIVIRAL PARTICLES 52! 3.14 PRODUCTION OF RETROVIRAL PARTICLES 53! 3.15 LENTIVIRAL AND RETROVIRAL INFECTION . 54 3.16 HISTONE ACID EXTRACTION 55! 3.17 SILAC (Stable Isotopic Labeling using Amino acids in Cell culture) . 56! 3.18 MASS SPECTROMETRY AND DATA ANALYSIS (SILAC) . 57! 3.19 ChIP-seq BINDING SITE IDENTIFICATION AND CLASSIFICATION 59! 3.20 De-Novo MOTIV DISCOVERY 59! 3.21 CELL LINES 60! CHAPTER – RESULTS 61! 4.1 H3R2 IS SYMMETRICALLY DIMETHYLATED IN VIVO 61! 4.2 H3R2me2s LOCALISES TO EUCHROMATIC REGIONS IN HUMAN CELLS 65! 4.3 H3R2me2s IMPEDES CO-REPRESSORS BINDING 74! 4.4 H3R2me2s FAVORS THE BINDING OF THE CO-ACTIVATOR WDR5. ……………………………………………………………………………… 79 vi 4.5 THE WD40 DOMAIN OF WDR5 IS A NOVEL METHYL ARGININE “READER” . 83! 4.6 H3R2me2s RECRUITS WDR5 IN VIVO UPON CELL CYCLE EXIT . 88! 4.7 H3R2me2s RECRUITS WDR5 IN VIVO 92! 4.8 H3R2me2s DISTRIBUTION IN GROWING AND RETINOIC ACID DIFFERENTIATED MOUSE EMBRYONIC STEM CELLS. . 93! 4.9 PRMT5/WDR77 AND PRMT7 METHYLATE H3R2me2s . 100! 4.10 PRMT5 IS NUCLEAR AND BINDS TO H3R2ME2S TARGETS IN CANCER CELL LINE HST746 . 105 CHAPTER – DISCUSSION . 109! 5.1 H3R2me2s ON THE -1 NUCLEOSOME 110 5.2 H3R2me2s AT DISTAL SITES IN P493-6 112 5.3 WDR5 AS A METHYL-ARGININE “READER”…………………… 116 5.4 PRMT5/WDR77 AND PRMT7 METHYLATE H3R2me2s . 123! BIBLIOGRAPHY . 130! vii 5(9,(: Migliori, Phalke, Bezzi & Guccione PADI family, promiscuously deiminates arginines on histone H3 (H3R2, H3R8, H3R17 and H3R26) and that deimination by PADI4 counteracts arginine methylation by CARM1. The authors also concluded that dimethylation of arginines prevents deimination by PADI4 [98] . In a parallel paper, Wang and colleagues detected the release of methylamine from CARM1 or PRMT1 asymmetrically dimethylated arginines [97] on both H3 and H4, suggesting that asymmetric dimethylation could be permissive for the deimination reaction carried out by PADI4. High levels of citrullinated histones are detected on decondensed chromatin in both HL-60 granulocytes and blood neutrophils during the formation of neutrophil extracelluar traps (NETs). This seems to be a global effect, similar to, but distinct from, that observed for H4K16ac [99] . Citrullinated histones directly impair the compaction of in vitro assembled histones, thus defining a biochemical mechanism associated with the presence of this unconventional amino acid on histones [100] . A recent paper strengthens the link between PADI4-mediated citrullination and histone deacetylation, specifically studying the pS2 promoter, and the mutually exclusive presence of citrullinated H3 and methylated arginine 17 [101] . The cyclical appearance of citrullination and arginine methylation on the estrogen-activated promoter suggests that citrullines have to be quickly converted into newly methylatable arginines [101] . Given that several papers have indeed questioned whether methylated arginines are a physiological substrate for PADI4, as both MMA and ADMA are either poor substrates or completely refractory to deimination [102,103] , the most likely explanation is that the unmethylated arginine ground-state is re-established either by histone substitution or by yet to be identified enzymes able to convert citrullines back to arginines (FIGURE 2A) . Direct histone demethylation Demethylation by FAD-dependent monoamine oxydases such as LSD1 and members of the Jumonji family has been extensively studied in the past few years (see [27] for a recent review). So far, only one member of the Jumonji family, JMJD6, seems to possess an arginine demethylase activity [104] . This, however, has been recently challenged by Webby and coworkers, who claim that JMJD6 has a stronger lysine-hydroxylase activity than an arginine demethylase activity [105] . The authors link JMJD6 to the regulation of gene expression via the post-translational hydroxylation of the splicing factor U2-small 130 Epigenomics (2010) 2(1) nuclear ribonucleoprotein auxiliary factor-65 (U2AF65), which in turn affects the regulation of RNA splicing of a subset of genes [105] . Chang and colleagues had also previously reported a similar activity, but as they were enriching for the arginine monomethylated pepides in their mass spectrometric analysis, they might have overestimated the demethylase function with respect to the hydroxylation function [104] . Moreover, the fact that JMJD6 shows no preference, in vitro, for either asymmetrically or symmetrically dimethylated substrates, poses a question regarding the specificity of this reaction. Further work on this enzyme is needed to assess which reaction is preferentially catalyzed in vivo and whether cofactors can confer substrate specificity. Future perspective A few areas in the PRMT field will need to be explored in the near future. These will be discussed in the following sections. „ In vivo functions of PRMTs We have learned much about the biochemistry of PRMTs by studying their activity in vitro. Now we need to progress to studying the function of PRMTs in cells and animal systems. To that we can take advantage of three main approaches: genomics, proteomics and drug discovery. Genomics Our knowledge of which genes are controlled by different PRMTs is very limited and is mainly based on single-gene analyses [38,55,61,69,76,80] . Chromatin immunoprecipitation (ChIP) is an essential technique for analyzing protein–DNA interactions in vivo, and when combined with technologies such as microarrays (ChIP-onchip) or deep sequencing (ChIP-PET and ChIP-Seq), it gives rise to powerful platforms for high-throughput genomic analysis. These ChIPbased approaches allow genome-wide epigenetic studies and have already greatly advanced our understanding of the ‘epigenetic landscape’ in different systems. As an example, we now have a very clear picture of which genomic loci are occupied by different transcription factors in mouse embryonic stem cells [106] and whether they overlap with defined histone PTMs [107] or with DNA methylation [108] . Similarly, large-scale mapping studies of the genomic sites occupied by different PRMTs, in different cell lines or tissues, will give us a better understanding of the regulatory pathways controlled by these enzymes. Moreover, this information, together with the mapping of histone arginine future science group Arginine/lysine–methyl/methyl switches methylation in the same systems, will advance our understanding of how these PTMs control transcription. Further biochemical validation will nevertheless be necessary to ultimately define the role of each methylated arginine on histones. Finally, with the generation of conditional knockout animals lacking specific PRMTs, it will be possible to address the consequences, in terms of global and promoter specific chromatin changes, of targeted deletions of PRMTs in a specific cell type and at a given time during development. Proteomics Structural determination of the endogenous complexes containing the different PRMTs will give us insight into how these enzymes target specific substrate arginines on different histones. So far, PRMTs have been studied as isolated proteins, but evidence suggests that they act in large macromolecular complexes in vivo [109] . As an example, PRMT4 within the NUMAC is able to methylate nucleosomes, while the isolated protein preferentially methylates histones [70] . As for PRMT5, different interacting proteins are able to control its specificity. When complexed with COPR5, it preferentially methylates H4 over H3 [85] , and this change in specificity, which can also redirect PRMT5 to methylate nonhistone substrates, might also explain why PRMT5 has been associated with both transcriptional repression and activation [88] . Drug discovery Targeting arginine methyltransferase activity with a high degree of specificity will be instrumental in characterizing the role of PRMTs in vivo, and will hopefully be effective in treating malignancies such as certain subsets of lymphomas and hormone responsive tumors [80,110] . In the past, two categories of compounds have been used to block methyltransferase activity: ƒ Molecules that inhibit the activity of adenosyl homocysteine hydrolase, like adenosine dialdehyde, resulting in the accumulation of the methyltransferases natural inhibitor, adenosylhomocysteine; ƒ Analogs of S-adenosyl methyionine (SAM or AdoMet), like sinefungin, which compete with the cofactor to hamper methylation. These classes of inhibitors are, however, highly aspecific, and cannot discriminate between arginine- and other kind of methyltransferases. Indeed, both arginine and lysine methyltransferases use SAM as a methyl donor. More recently, future science group 5(9,(: new small molecules, termed arginine methyltransferase inhibitors (AMIs) have been demonstrated to more selectively target and block PRMT, as opposed to lysine methyl transeferases activity [110] . AMI-1, one of the most effective compounds isolated by this screen, does not bind solely to the SAM pocket, but is, however, still not able to selectively inhibit the different PRMTs family members. Nonetheless, hormone dependent transcription can be negatively affected by the presence of the drug [110] . The same authors also used AMI-5 chemical structure to further design a set of simplified analogs, obtaining compounds with higher potency, and in some cases higher specificity, to either PRMT1 or PRMT4/CARM1 [111] . A recent attempt to increase the specificity has been described by Osborne and colleagues who have developed a bisubstrate analog by combining a histone H4 peptide, a known substrate of PRMT1, with N-mustard, 5´-(diaminobutyric acid)-N-iodoethyl-5´-deoxyadenosine ammonium hydrochloride, to generate a PRMT1 specific inhibitor [112] . Virtual screening and chemical docking studies [113–115] have also been attempted and the common conclusion from these very promising studies is that larger libraries and more refined docking tools will be necessary before these compounds will reach the desired specificity and potency to be used in vivo. „ Arginine methylation signaling cascade This last point leads directly to one of the crucial challenges in the arginine methylation field; so far many methylation events on histones have been associated with either gene activation or repression. We must now attempt to understand the biochemical mechanism, and the causal role, behind this association, and to this we need to gain a better understanding of the proteins that act both upstream and downstream of the arginine methylation events. Upstream If we think of arginine methylation as yet another way of transducing the signal in a cascade, then we need to find out how these enzymes are regulated in different tissues and during key physiological and pathological events such as differentiation, development and cancer. Conditional knockout mice will be instrumental in this effort, as they will allow us to study the effects of loss of different PRMTs in a spatially and temporally restricted manner. www.futuremedicine.com 131 132 Peptide arrays have been successfully developed and used recently for high-throughput identification of effector-modules that bind to histone marks [134] In a recent paper the authors describe a HEMP, to screen the binding of a large library of Royal domain family members to modified histone peptides Alternative to peptide arrays, this technology allows the spotting of purified proteins [20,135] or of cDNAs on glass slides [136] , followed by in vitro transcription and translation (NAPPA) Protein arrays Epigenomics (2010) 2(1) Involves the complete in vivo labeling of the whole proteome of two to three different cell lines or experimental conditions: Proteins derived from the different sources can later be distinguish based on the different molecular weight:charge ratio of the heavy, intermediate or light amino acids used to culture them [137] SILAC* Not quantitative, often background binding to the unmodified histone tail can create a list of false-positive candidates Only one or a small pool of peptides can be tested at any given time Only one or small pools of known proteins can be used at a time Sensitivity might be low, allowing for the study of high-affinity interactions only The derivatization step involved in chemical strategies is often not complete (limits in sensitivity) In order to allow the relative quantification of two samples, the purification and fractionation steps for the different proteomes have to be carried out separately but precisely in the same way (higher chances of experimental errors) Quantitative, mixing labeled Living cells are required, expensive and unlabelled cells allows one to avoid the introduction of errors in the purification and fractionation step, expected mass differences are known before the identification of the peptide, reduces false-positive interactions High-throughput method, it is a flexible platform and can be implemented and updated as new modifications are identified on histones It is relatively easy to set up High-throughput method, it is a flexible platform and high-density arrays are capable of covering the entire proteome Allows the identification of novel proteins, with novel histone binding modules Quantitative, any proteome can be chemically labeled A limited number of candidates can be tested Good antibodies against the endogenous candidates are necessary Choosing a cell line that expresses the proteins of interest is crucial Disadvantages The identification and characterization of downstream chromatin effectors and readers is essential to better understand the biochemical role of histone modifications. The proteins known to bind histones recognize, on average, ten amino acids of the histone sequence, and post-translational modifications occur predominantly at the N- and C-terminal tails. Therefore, synthetic peptides that mimic histone tails and that are chemically modified on different residues are particularly useful as baits to fish out potential histone binders. *These techniques, involving peptide pull downs from nuclear extracts followed by mass spectrometry, allow coupling of the simple peptide pull-down assay to an unbiased identification of binding partners. HEMP: Human epigenome peptide microarray platform; ICAT: Isotope-coded affinity tags; ITRAC: Isobaric tag for relative and absolute quantitation; NAPPA: Nucleic acid programmable protein arrays; SILAC: Stable-isotope labeling by amino acids in cell culture. These are gel-free methods, which rely on chemical labeling reagents for quantitative proteomics ITRAQ* Standard mass spectrometry* Advantages Potential interaction can be The use of C-terminal biotin tagged peptides allows incubation with a cellular or nuclear cell extract, followed by immunoblotting scored by western blot Easy to establish and antibody-based detection. The use of streptavidinconjugated agarose or magnetic beads allows for the easy recovery of potential binders Description Peptide arrays High throughput Peptide pull downs Low throughput Assay Table 2. Biochemical assays that allow for the analysis of protein–peptide interactions. 5(9,(: Migliori, Phalke, Bezzi & Guccione future science group Arginine/lysine–methyl/methyl switches Downstream It will be essential to identify new arginine binding modules in different proteins: so far only four proteins containing a Tudor domain (SMN, SPF30 and TDRD3) or a PHD finger (Dnmt3a) have been demonstrated to directly interact with dimethylated arginine residues. Additional studies are needed to obtain the full repertoire of interactors in each tissue. This is particularly important because not all PRMTs are ubiquitously expressed, and in addition, different proteins can recognize the same methylated residue in different contexts. Several high-throughput techniques will allow for the rapid screening of potential methyl–arginine interactors: see TABLE for details. Acknowledgements Owing to space limitations, we have often referred to reviews instead of original articles. We would therefore like 5(9,(: to apologize to those whose work could not be cited directly. We also would like to thank Cinza Crociani for help in creating the artwork and for figure preparation. Financial & competing interests disclosure The work is funded by A-Star (Singapore’s Agency for Science Technology and Research) an agency of the Singapore Government. Valentina Migliori and Marco Bezzi are graduate students funded by the SINGA (Singapore International Graduate Award) fellowship. Sameer Phalke is a post-doctoral fellow funded by an A-STAR fellowship. The laboratory research is supported by the Institute of Molecular and Cell Biology (Singapore) and by A-Star (Singapore’s Agency for Science Technology and Research). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Executive summary Arginine methylation is catalyzed by protein arginine methyltransferases ƒ There are two classes of protein arginine methyltransferases (PRMTs): Type I PRMTs promote the formation of asymmetrically dimethylarginine (:-NG,NG-dimethylarginine), Type II PRMTs catalyze the formation of symmetrically dimethylarginine (:-NG,N’Gdimethylarginine). Both types, and a third type (Type III), are capable of forming :-NG-monomethylarginine. Arginine methylation on histones ƒ Histones H3, H4 and H2A have been reported to be monomethylated (H3R2me1, H4R3me1), asymmetrically dimethylated (H4/H2AR3me2a, H3R2me2a, H3R17me2a and H3R26me2a), and symmetrically dimethylated (H4/H2AR3me2s and H3R8me2s). These can be divided into two groups, based on their role in transcription regulation: – Activating arginine methylations: H4/H2AR3me2a, H3R17me2a and H3R26me2a. – Repressive arginine methylations: H4/H2AR3me2s, H3R8me2S and H3R2me2a. Methylated & unmethylated arginines can act in two major ways to increase the complexity of the ‘histone code’ ƒ Methylated arginines can act as docking sites for other proteins on histone tails (examples include the plant homeodomain finger of DNMT3a binding to H4R3me2s and the plant homeodomain finger of RAG2 binding to H3K4me3/H3R2me2s). ƒ Methylated arginines can act as exclusion marks, impeding the binding of proteins to histones (examples include the WD40 domain containing protein WDR5, the PHD finger of ING2, of TAF3 and of BPTF, the double chromodomain of CHD1 and the double Tudor domain of JMJD2A, which are all excluded from binding to histone H3 when H3R2 is asymmetrically dimethylated [H3R2me2a]). Research areas in the protein arginine N-methyltransferase field in the near future ƒ To explore the in vivo functions of PRMTs, using high-throughput genomics and proteomics techniques and by developing specific PRMT inhibitors. ƒ To characterize the signaling cascade both upstream and downstream of histone arginine methylation. Upstream events controlling enzymatic activity of PRMTs need to be elucidated, and we still need to uncover the full repertoire of downstream effectors, which are able to ‘read’ the methylated arginine and interpret them in terms of transcriptional output. Bibliography Papers of special note have been highlighted as: „ of interest „„ of considerable interest Barski A, Cuddapah S, Cui K et al.: High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007). Kouzarides T: Chromatin modifications and their function. 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On WD40 proteins Propelling our knowledge of transcriptional control? Valentina Migliori,1 Marina Mapelli2,* and Ernesto Guccione1,* Institute of Molecular and Cell Biology; Singapore; 2Department of Experimental Oncology; European Institute of Oncology; IFOM-IEO Campus; Milano, Italy A direct effect of post-translational modifications (PTMs) on nucleosomes is the formation of a dynamic platform able to assemble the transcriptional machinery and to recruit chromatin modifiers. The histone code hypothesis suggests that histone PTMs can act as binding sites for chromatin readers and effector proteins, such as the bromodomains, that selectively interact with acetylated lysines, or the “Royal family” and the PHD finger domains, which are able to recognize methylated arginines and lysines. In this review we will discuss recent data describing the function of WD40 proteins as a new class of histone readers, with particular emphasis on the ones able to recognize methylated arginine and lysine residues. We will discuss how WDR5, a classical sevenbladed WD40 propeller, is able to bind with similar affinities both the catalytic subunit of the Trithorax-like complexes, and the histone H3 tail either unmodified or symmetrically dimethylated on arginine (H3R2me2s). Furthermore, we will speculate on how these mutually exclusive interactions of WDR5 may play a role in mediating different degrees of H3K4 methylations at both promoters and distal regulatory sites. Finally, we will summarize recent literature elucidating how other WD40 proteins such as NURF55, EED and LRWD1 recognize methylated histone tails, highlighting similarities and differences among them. WD40-Containing Proteins are Adaptor Proteins Mediating Protein-Protein Interactions WD40 repeats (also known as WD repeats) are 40–60 amino acid motifs that preferentially end with a tryptophan and an aspartic acid (WD). They are highly conserved from bacteria to mammals, and are often found as part of multisubunit complexes, where they play a role in mediating protein-protein interactions. The first WD40 domain-containing protein was identified as part of the heterotrimeric G protein transducin complex.1 The crystal structure of the β subunit of the complex showed the characteristic seven blade β propeller fold, with a central cavity (Fig. 1A and B), and each blade comprising a four-stranded antiparallel β-sheet (Fig. 1C).2-4 It was later shown that β propeller can be assembled with a number of repeats varying between and 16 (SMART:SM00320; INTER PRO :IPR001680 ; PFAM:PF00400; PROSITE:PS00678). As highlighted by the crystallographic structures determined to date, WD40 domain proteins have several surfaces for the interaction with multiple binding partners, and it is no surprise that they are crucial for maintaining the integrity of the complexes that they are part of. They serve as interaction hubs and are associated with a wide variety of physiological pathways such as vesicle biogenesis,5 cytokinesis,6 control of © 2012 Landes Bioscience. Do not distribute. Keywords: Arginine Methylation, WD40, chromatin, histone modifications, WDR5, MLL Submitted: 05/03/12 Revised: 06/11/12 Accepted: 06/15/12 http://dx.doi.org/ *Correspondence to: Marina Mapelli and Ernesto Guccione; Email: marina.mapelli@ieo.eu and eguccione@imcb.a-star.edu.sg www.landesbioscience.com Epigenetics Figure 1. Topology of WD40 β propellers. (A and B) Ribbon diagrams of a seven-bladed WD40 β propeller viewed from the side and from the top. The blades are organized sequentially, and pack on each other counterclockwise around a central channel. The closure of the ring-like structure is achieved in blade by the contribution of the β-strand 7d deriving from the N-terminal residues of the domain. (C) Each blade of the β propeller consists of a β-sheet formed by four antiparallel β-strands, which are denoted a-b-c-d starting from the innermost strand to the most peripheral. Notably, the strand d of each blade corresponds to the N-terminal strand of the subsequent WD40 repeat. protein stability,7 RNA processing,8 control of replication9,10 and transcriptional regulation.11-14 In terms of transcriptional regulation, proteins containing WD-domains, such as EED, LRWD1, WDR77, RbBP4/7 and the homolog Drosophila NURF55, have been shown to mediate the localization of chromatin modifiers to specific sites on the genome by directly binding to histones and their methylated tails. Here, we will summarize recent literature elucidating how WDR5, EED and NURF55 bind to histone tails, highlighting similarities and differences between them. methyltransferases (PRMTs) and sequentially asymmetrically (by Type I PRMTs) or symmetrically (by Type II PRMTs) dimethylated.15 Only a few proteins have been shown to interact specifically with methylated arginines on histones. For example, TDRD3 is a transcriptional co-activator which directly interacts with H3R17me2a16 (where “a” stands for asymmetric), and the ADD domain of DNMT3A could possibly bind to H4R3me2s (where “s” refers to symmetric), though this is still controversial.17,18 Recently, it has been shown that the methylation on H3R2 critically affects the binding of the transcriptional coactivator protein WDR5, to histone H3. Specifically, the symmetric dimethylation leads to WDR5 recruitment,14 while the asymmetric dimethylation excludes its binding.13,19,20 Wysocka and coworkers showed that WDR5 could bind to unmodified histone H3, and that the affinity was stronger between WDR5 and a dimethylated H3K4 peptide (H3K4me2).21 Three later studies independently were able to determine the structure of WDR5, which folds as a classical seven-bladed β-propeller (Fig. 2A),22-24 bound to H3. Analysis of the structure revealed that Ala1, Arg2 and Thr3 of H3 are important for the specificity of binding, and that the side chain of Arg2 inserts into the central channel of the β-propeller.22-24 Depending on the assay used, the three groups reported an affinity of WDR5 toward unmodified H3 ranging from 3.3 to 35 μM (K D). Moreover, despite an increased protein stability of WDR5 bound to H3K4me2 over unmodified H3, as measured by differential static light scattering,24 none of the groups detected the increase in affinity toward K4 methylated peptides, that was initially reported.21 Recently, it was shown that WDR5 binds H3 peptides symmetrically dimethylated on arginine (H3R2me2s) with a higher affinity, likely due to a reorganization of the water-mediated interaction network within the central channel. Specifically H3R2me2s is hydrogen-bonded to only one water molecule, as opposed to two waters bridging the interaction of WDR5 with unmodified H3R2me.14 The crystallographic structure of WDR5 in complex with H3R2me2s revealed a marginal shift of the methylated, hydrophobic guanidinium group of H3R2 away from the single water molecule present in the direction of a hydrophobic pocket contributed by the Phe219 of WDR5 (Fig. 2A). Phe219 is thus an important determinant of the high affinity interaction between WDR5 and H3R2me2s. Following this line of reasoning, mutation of Phe219 into more hydrophilic residues (WDR5-F219H) reduces the binding affinity between H3R2me2s and WDR5 from a K D of 0.1 μM to 0.2–1.1 μM, without severely affecting the binding to the unmodified H3R2me0 (KD of 5.6 ± 1.5μM to 7.0 ± 2.0μM) (Migliori and Guccione, unpublished). © 2012 Landes Bioscience. WD40 Containing Proteins as Chromatin Readers WDR5. Unlike phosphorylation or acetylation, methylation of histones does not change the overall charge of the modified amino acids, but it does render them bulkier and more hydrophobic. It is thus thought that methylation at specific sites, either on the histone globular domain or on the tails, can lead to either transcriptional activation or repression, depending on downstream proteins recognizing the specific methylation event. Methylation can occur either on lysines, which can be mono-, di- or trimethylated by lysine methyltransferases (KMTs) or on arginines, which can be monomethylated by class I, II and III protein arginine Do not distribute. Epigenetics Volume Issue © 2012 Landes Bioscience. Do not distribute. Figure 2. Structural organization of WD40-domains in complex with histone tails and other ligands. (A) Structure of WDR5 (colored in light blue) in complex with the N-terminal fragment of histone H3 (in yellow balls-and-sticks) symmetrically di-methylated on Arg2 (PDB id 4A7J). The H3 peptide lines on the top surface of the β propeller inserting the Arg2 side chain into the central channel, in between two conserved Phe aromatic rings. At the bottom of the cavity, the interaction is further stabilized by hydrophobic interactions with Phe219 and a water-mediated hydrogen bond to Ser175. In this configuration, the side chain of Lys4 points away from the propeller. (B) The MLL2 Win peptide encompassing Arg5065 (shown in pink) binds to WDR5 in much the same way as the histone H3 N-terminus, implying that the two ligands are mutually exclusive interactors of WDR5 (PDB id 3UVK). (C) Architecture of the EED WD40 propeller (displayed in green) in complex with histone H3 trimetyhylated on Lys27 (PDB id 3IIW). The methylated H3K27 side chain is docked on the propeller surface by hydrophobic interactions contributed by Phe97, Tyr148 and Tyr365. The presence on EED of bulky residues such as Tyr308 and Trp364 restricts the binding to histone peptides coding for small residues in position -2 from the methylated Lys. (D) Topology of NURF55 (colored in lilac) bound to histone H4 (PDB id 2XYI). The histone H4 peptide folds as a α-helix that fits into a groove on the lateral surface of the propeller. The amphipathic character of the helix with positively charged residues on the left side and hydrophobic side chains on the opposite, matches the chemical environment of the propeller’s groove (left panel). (E) The PRC2 subunit Su(z)12 (shown in red) recognizes the same lateral groove of the NURF55 propeller occupied by histone H4 (PDB id2YB8). (F) Structure of NURF55 in complex to the N-terminus of histone H3 (PDB id 2YBA). The H3 peptide runs on the top of the propeller in an extended conformation, inserting the Arg2 side chain between Tyr185 and Phe325. This geometry is reminiscent of the complex with WDR5, although Arg2 does not go as deep into the central channel, and additional polar interactions contributed by Glu130NURF55, Glu183 NURF55 and Asn132 NURF55 anchor the Lys4H3 side chain on the propeller surface. Intriguingly, works by other groups25-27 have shown that the binding pocket of WDR5, which interacts with H3R2me2s, can also interact with the SET domain of MLL1 with comparable affinity, via the so called Win (WDR5 interacting) motif.14,25-27 This association is not specific for MLL1, as initially thought, and it is indeed conserved among all SET domain members: MLL2–4, SET1A and SET1B.28 www.landesbioscience.com The topology of the WDR5:MLLWin interaction is reminiscent of what observed for WDR5:H3R2 (Fig. 2B). Very importantly there seem to be a direct competition between H3 and the Win motif peptides in binding to WDR5, implying that the Win motif acts as dose dependent inhibitor of complex formation and catalytic activity toward H3K4 methylation.29 Epigenetics How is it possible to reconcile these biochemical data with the high degree of co-localization observed in vivo between H3R2me2s and methylated H3K414,30 ? The presence of H3K4me1 at distal regulatory sites has been long observed by several groups,31-33 but the mechanism by which H3K4me1 is preferentially catalyzed over H3K4me2/3 is still largely unknown. One possibility is the © 2012 Landes Bioscience. Do not distribute. Figure 3. Regulation of the MLL/SET complexes at distal regulatory sites (Enhancers) and at Promoters. (A) H3K4 is efficiently di- and tri-methylated (H3K4me2/3) in vivo by MLL/SET complexes. At distal regulatory sites H3K4me1 is more abundantly detected, either because of a constant demethylation by Jumonji-containing enzymes or by LSD1/2 (KDMs) or because alternative enzymes (SETx) directly monomethylate H3K4. (B) In the absence of the MLL/SET catalytic subunit, the WRAD complex could potentially monomethylate H3K4me1. Alternatively, the competition between WDR5, H3 and the Win Motif peptide on MLL/SET proteins could lead to reduced efficiency in H3K4 tri-methylation and a consequent enhanced monomethylation at these sites. (C) Active promoters that are not enriched for H3R2me2s will recruit the MLL/SET complex to trimethylate H3K4 (H3K4me3). (D) When the -1 nucleosome is methylated on H3R2 (H3R2me2s) WDR5 will bind with high affinity, most likely in the absence of the catalytic MLL/SET subunit. In order to be tri-methylated on H3K4, the histone tail will then translocate to the catalytic pocket of the MLL/SET (conformational change). (E) WDR5 binding to H3 could be unrelated to the WRAD and MLL/SET complexes. WDR5 is part of the ATAC, the MOF and the CHD8 remodeler complex among possibly others. recruitment of histone demethylases that can demethylate H3K4me2/3 (KDMs)34 or, alternatively, enzymes other than MLL could be involved in promoting H3K4 monomethylation (Fig. 3A). At those distal regulatory sites marked by H3R2me2s, the competitive binding of WDR5 to both H3R2me2s and the SET catalytic subunit of the Lys4-methylating complexes might have a regulatory function on the latter. The first possibility is that the WDR5/ASH2L/RBBP5/DPY30 (WRAD) sub-complex could be recruited to chromatin via the high affinity interaction between WDR5 and H3R2me2s, in the absence of MLL/SET recruitment. Interestingly, this sub-complex has been proposed to have direct H3K4 monomethylation activity, which is dependent on the ability of the ASH2L-SPRY domain to bind the cofactor S-adenosyl methionine (SAM).35,36 The WRAD enzymatic activity would fit with the observed colocalization of H3R2me2s and H3K4me1 Epigenetics at enhancer sites distant from TSSs14 (Fig. 3B). These hypotheses are provocative, but it must be said that the mechanistic details of the WRAD mediated methylation are still largely missing and it is still unclear whether a dual catalytic site (one on MLL and the second on WRAD) is used toward an efficient tri-methylation of the substrates. Moreover, Takahashi and colleagues have recently shown that the reconstituted yeast WRAD complex does not have a substantial HMT activity. Volume Issue Indeed, when they use the WRAD complex, less than 1% of H3K4me1 and no H3K4me2–3 were detected in their methylation assays, much less than what observed with the wt Set1/COMPASS or MLL/COMPASS-like complexes.37 These discrepancies could be due to the different protocols used to purify the complex subunits or the quality of the substrate, thus we reckon additional experiments will be necessary to clarify the issue. A second role for the presence of H3R2me2s at distal regulatory sites could be to favor the dissociation of the MLL/ SET complex itself from the WRAD, by directly competing for the Win motif. This would lead to a preferential monoover trimethylation of H3K4 (H3K4me1 > H3K4me3) (Fig. 3B). At promoters H3K4me3 is typically enriched over H3K4me138,39 (Fig. 3C). It is possible that at the subset of promoters marked by both H3K4me3 and H3R2me2s, the topology of the interactions among subunits of the WDR5/ Ash2/Rbbp5/DPY30 sub complex is rearranged by the direct binding of the SET catalytic subunit to WDR5, via the Win motif. If so, the WRAD complex could be recruited first by the high affinity interaction with H3R2me2s, and then undergo a conformational change leading to assembly of the full complex that trimethylates histone H3 (Fig. 3D). Detailed time course studies will have to be performed in order to clarify whether this is the case. Interestingly, a recent study on H3R2me2s both in yeast and eukaryotic cells reports a much higher degree of overlap between H3R2me2s and H3K4me3 at promoters. By taking advantage of yeast genetics, the authors further prove that the interdependency between H3R2me2s and H3K4me3 is mutual, since the arginine methylation mark is not present in a SET1 mutant background.30 Although no formal prove has been provided to date, it is possible that the Win motif of the MLL proteins can also be symmetrically methylated on the arginine required for the binding to WDR5, and that this methylation could enable MLL to better compete with H3R2me2s for binding to WDR5. Alternatively, the Win motif could possibly be monoor asymmetrically dimethylated, a modification that would be predicted to destroy the binding, as observed in the case of H3R2me2a/me1 and WDR5.13,20,22 More experiments are needed to clarify this point. It is worth mentioning that the initial proposed model of WDR5 as the protein responsible to present the histone H3 tail to the MLL complex for methylation, is probably not accurate.21,40,41 On the contrary, it has been reported that the N-terminal of the histone tail has to be free to allow methylation by MLL,37,42 while WDR5 plays a role in maintaining the integrity of the complex. An alternate role for the observed binding of WDR5 to histone H3 has to finally be considered. WDR5 is part of several other complexes, for example the ATAC complex43 and the MOF complex,44 which possess acetyltransferase activity, and could in turn act to promote H3K4me3,45 or the CHD8-containing chromatin remodeling complex,46 which could be similarly important to promote accessibility for further histone modifications (Fig. 3E). EED. The interplay between WDR5, H3 (either unmodified or symmetrically dimethylated on R2) and the other components of the SET complexes is reminiscent of another important WD40 domain protein: EED. EED, together with EZH2 and SU(z)12, form the PRC2 complex, a transcriptional co-repressor complex whose activity depends on its ability to trimethylate H3K27. While EZH2 is the catalytic subunit of the PRC2 complex, EED binds directly to H3K27me3 through its C-terminal domain, leading to the allosteric activation of the methyltransferase activity of PRC2 and, as such, to the propagation of the repressive mark.47,48 Similarly, H3R2me2s may favor H3K4 methylation by its interaction with WDR5 (Fig. 3D). This interaction displaces the Win motif from the WDR5 binding cleft, and could cause an allosteric change in the complex, leading to its activation or favoring its processivity. The structure of EED in complex with H3K27me3 shows that the interaction occurs through a hydrophobic/aromatic cavity formed mainly by three residues: Phe97, Tyr148 and Tyr365. The peculiarity of this cage is that it is able to prevent the binding of large or charged amino acids, specifically at position-2, that typically flank activation marks on histones. This could provide a molecular explanation for the evidence that EED binds to H3K27me3 and H3K9me3 (ARKS), but not to H3K4me3 (RTKQ) (Fig. 2C) While EED has only been observed to bind H3K27me3 in vivo, in vitro it binds to most of the repressive marks, such as H3K9me3 or H4K20me3, with an affinity that decreases with the degree of methylation (for example, H3K27me3 > me2 > me1). This poor specificity observed in vitro could be explained by the topology of the interaction between EED and the H3K27 histone marks that occur only on the top surface of the β-propeller and not in the central channel (Fig. 2C). Instead, in the case of WDR5, the prominent feature of the association of the H3 tail with the β-propeller is the insertion of the Arg2 side chain into the central cavity, where the presence of two phenylalanines favors the formation of a specific hydrophobic interaction with H3R2me2s14 (Fig. 2A). As a result, the interaction between WDR5 and H3R2me2s appears to be extremely specific. In sharp contrast to EED that can bind several differentially methylated residues on histone H3 in vitro, WDR5 has not been observed to interact with any other symmetrically di-methylated arginines on histones (Migliori and Guccione, unpublished). NURF55 and RbBP4/7. Besides EED, the PRC2 complex contains a second WD40 protein termed NURF55 in Drosophila and RBBP4/7 in humans. This seven-blade β-propeller was originally identified as an interacting partner of the Retinoblastoma protein.49 Subsequently it was co-purified with the HAT1 acetyltransferase complex50 and several co-repressor complexes, including PRC2,51 NURD52 and CAF1.53 While NURF55 has been demonstrated to bind both H3 and H4,54,55 the precise mechanism by which the binding of this multifunctional chromatin reader is modulated by the crosstalk among histone modifications was only recently described. The crystal structure of NURF55 bound to histone H4 revealed that the binding occurs with the first helix of the histone fold, a region normally buried © 2012 Landes Bioscience. www.landesbioscience.com Do not distribute. Epigenetics in the nucleosome structure (Fig. 2D).25 Consistently, mutations that disrupted such interactions led specifically to the loss of HAT1 acetyltransferase activity, which is directed toward newly synthetized and non-nucleosomal H4. Unlike the binding between WDR5 and histone H3, which occurs deep in the central cavity of the WD40 propeller, NURF55 interacts with H4 on the side surface of the protein. The binding surface is hydrophobic on one side, and negatively charged on the other, thus perfectly matching the H4-helix, which has alternating hydrophobic and positively-charged amino acids (Fig. 2D). The extensive hydrogen bond network that stabilizes the binding is unique to the surface of this β-propeller, and is absent in WDR5 and other members of the family. RBBP4/7 is also part of the NURF complex, and it remains to be addressed whether the interaction surface on H4 is made accessible during the remodeling process by the ISWI-containing ATPdependent chromatin-remodeling complex. By determining the crystallographic structure of NURF55 bound to H3, a more recent publication reconciled the binding data with the direct activity of NURF55containing complexes on chromatin.56 The authors show that in the context of the PRC2 complex, NURF55 is able to bind to both Su(z)12, which binds in the same binding pocket as the H4 peptide (Fig. 2E), and to H3, which instead binds on the top-flat surface of the β-propeller (Fig. 2F). Unlike WDR5, the arginine at position does not interact with residues deep in the central cavity, but is nonetheless very important in recognizing NURF55, together with H3K4 whose side chain is docked on the propeller surface by polar interactions with negatively charged residues of NURF55. Consistently, methylation on H3K4 impairs NURF55/Su(z)12 binding.56,57 More specifically, H3K4me3 methylation on nucleosomes reduces the catalytic turnover of PRC2, resulting in an overall reduction in H3K27 methylation. Interestingly, despite an involvement of H3R2 in binding to the surface of NURF, the presence of the asymmetric dimethylation (H3R2me2a) of this residue does not impair binding.56 This is in line with more recent data showing that the opposite symmetricity (H3R2me2s) has a negative effect on this interaction.14 WDR77. The cofactor WDR77, also called Mep50, is another WD40 protein, which directly interacts with histones. WDR77 binds specifically to histone H2A, most likely favoring the specific methylation of H2AR3 by PRMT5.58 The exact surface of interaction and how WDR77 stimulates this methylation event is however, still unknown. Solving the crystal structure of PRMT5/WDR77 in combination with H2A will prove extremely interesting and provide further understanding into the role of this WD40 protein in aiding PRMT5 activity. It is also important to note that PRMT5 methylates histones both in the nucleus and in the cytoplasm. Specifically the symmetrical dimethyation of H2AR3 seems to occur in the cytoplasm, contributing to the repression of pro-differentiation genes in embryonic stem cells.59 Whether this holds true for the other histone methylation events catalyzed by PRMT5, such as H3R2me2s or H3R8me2s, remains to be addressed. LRWD1. In two recent proteomic screens, LRWD1, another WD40 repeat-containing protein, was identified to interact with repressive trimethyl marks (H3K9me3, H3K27me3 and H4K20me3) and with the ORC complex.60,61 Further studies have shown that LRWD1 stabilizes ORC binding to chromatin, specifically heterochromatin.9 While the ability of LRWD1 to bind heterochromatin was accurately described by multiple groups, none addressed whether this interaction occurred directly or indirectly. A more recent study proved that LRWD1 is recruited to pericentric heterochromatin by its direct binding to H3K9me3. The localization of the WD40 protein is not altered in cells lacking H4K20me3 (Suv420h1h2-/-), while it is compromised in cells lacking H3K9me3 (Suv39h1h2-/-). Functionally, this interaction helps to maintain heterochromatin silencing, as Lrwd1 knockdown results in failure to silence the major satellite repeats. However, the authors fail to explain the mechanisms by which the WD40 motifs simultaneously interact with H3K9me3, ORC and other possible co-repressors to stabilize heterochromatin in cis.62 This will be an interesting aspect to address with both biochemical and structural biology studies. WD40 Domains as Flexible Scaffolds for Intracellular Interactions A recent report by Wang and colleagues identified HOTTIP, a long non-coding RNA transcribed from the 5' of the HOXA locus, as a direct interactor of WDR5. In vivo, HOTTIP has been shown to coordinate the activation of several HOXA genes and its binding to WDR5 targets the MLL complex activity to all genes in the HOXA cluster.63 WDR5 is also the first member of the Trithorax complex to be shown to directly interact with Oct4 and to regulate selfrenewal in mouse embryonic stem cells.64 The authors observed a strong correlation between WDR5 and Oct4 binding to chromatin on a genome-wide scale. Moreover, they proved that WDR5 is required in the initial phase of somatic cell reprogramming to iPS cells and propose a model in which Oct4 first binds and promotes WDR5 expression in somatic cells, then interacts with WDR5 and confers DNA specificity to the WDR5/MLL complex. This re-establishes H3K4me3 on self-renewal genes such as Nanog or Pou5f1, promoting subsequent strong binding of Oct4 to these promoters and directing a robust transcriptional activation of the pluripotency network.64 To conclude, WD40-containing proteins are versatile proteins, which can simultaneously mediate interactions between histones, protein- and RNApartners, thus integrating complex signaling pathways and ultimately controlling gene transcription. © 2012 Landes Bioscience. Do not distribute. Epigenetics Acknowledgments We are grateful to Cheryl Mei-Yi Koh for critically reading the manuscript. Volume Issue References 1. Fong HK, Hurley JB, Hopkins RS, Miake-Lye R, Johnson MS, Doolittle RF, et al. Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs. Proc Natl Acad Sci U S A 1986; 83:2162-6; PMID:3083416; http://dx.doi. org/10.1073/pnas.83.7.2162. 2. 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Epigenetics Volume Issue [...]...SUMMARY The Asymmetric dimethylation of histone H3 Arginine 2 acts as a repressive mark that antagonizes trimethylation of H3 lysine 4 In this study, I report that H3R2 is also symmetrically dimethylated (H3R2me2s) by PRMT5 and PRMT7 and present in euchromatic regions Profiling of H3- tail interactors by SILAC-Mass Spectrometry revealed that H3R2me2s excludes binding of RBBP7, a central component of. .. events on histones or with DNA methylation will also be addressed 1.3.3 ARGININE METHYLATIONS LINKED TO TRANSCRIPTIONAL ACTIVATION: H4/H2AR3me 2a, H3R17me 2a, H3R26me 2a 1.3.3.1 H4R3me 2a and H2AR3me 2a Methylation of arginine at position 3 on the histone H4 tail was the first to be described (Strahl et al., 20 01) and it is of particular interest as H4R3 is the only site on which both an asymmetric and a symmetric. .. (Migliori et al., 20 10) ! "%! 1.3 .2 HISTONE ARGININE METHYLATION To date, only a few methylated arginines have been described on histones: asymmetric and symmetric dimethylation on arginine 3 of histone 4 (respectively H4/H2AR3me 2a catalyzed by PRMT1, and possibly by PRMT8, H4/H2AR3me2s catalyzed by PRMT5, and possibly by PRMT7), H3R2me 2a by PRMT6, H3R17me 2a and H3R26me 2a by PRMT4/CARM1, and H3R8me2s by PRMT5... development and cancer 1.3 ARGININE METHYLATION 1.3.1 THE BIOCHEMISTRY OF PROTEIN ARGININE METHYLATION In eukaryotes, from S cerevisiae to humans, arginine methylation is an abundant PTM (Najbauer et al., 1993), occurring on both histones and other nuclear and cytoplasmic proteins (Bedford and Clarke, 20 09) Arginine side chains have two terminal guanidino-groups (NH2), which are often involved in hydrogen... histone H3 binders H3R17me 2a could either interfere with or stimulate the binding of bromodomains, such as the one contained in PCAF, to the histone H3 tail The PCAF bromodomain has been shown to specifically bind to H3K14 when acetylated, and may contact the surrounding amino acids, including H3R17 (Zeng et al., 20 08) Similarly, H3R26 is predicted to affect the binding of PC/CBX proteins (Bernstein... et al., 20 06) or of the mammalian PRC2 complex to H3K27me3 (Hansen et al., 20 08) Whether H3R26 methylation plays a modulating role in Polycomb repressive functions still remains to be explored ! "*! 1.3.4 REPRESSIVE ARGININE METHYLATIONS: H4/H2AR3me2s, H3R8me2s and H3R2me 2a 1.3.4.1 H4/H2AR3me2s The symmetric dimethylation of arginine 3 on histone H4 can be catalyzed by at least two enzymes: PRMT5 and... for an enzyme catalyzing the remaining step of converting citrulline back to arginine JMJD6, a member of the second class of enzymes, is part of the large Jumonji family of demethylases The demethylase activity of this protein has thus far only been characterized in vitro and does not seem to discriminate between the demethylation of ADMA or SDMA back to arginine (B) PRMT family members The mammalian... complexes Sin 3a, NURD and PRC2 Conversely H3R2me2s enhances binding of WDR5, a common component of the MLL/Set 1a/ b, the NLS1 and the ATAC co-activator complexes The interaction with WDR5 distinguishes H3R2me2s from H3R2me 2a, which impedes its recruitment to chromatin The crystallographic structure of WDR5 and the H3R2me2s peptide in a stable complex elucidates the molecular determinants of this high affinity... proteins In agreement with this notion, the bromodomain was the first protein module shown to selectively interact with a covalent mark (acetylated lysine) in the histone NH2-terminal tail Chromodomains, on the other hand, appear to be targeting modules for methylation marks (Jenuwein and Allis, 20 01) Recently, in addition to chromodomain, other protein domains have been shown to be capable of binding... tetramer H3- H4 (Fig .2) Histones are highly positively charged proteins, rich in arginines and lysines, a characteristic that allows them to interact strongly with the negatively charged DNA They are defined by two separate functional domains: a histone- fold” motif that is sufficient for both histone- DNA and histone- histone contacts within the nucleosome, and NH2-terminal and COOH-terminal “tails” that are . H3R26me 2a 13 1.3.3.1 H4R3me 2a and H2AR3me 2a 13 1.3.3 .2 H3R17me 2a 15 v 1.3.3.3 H3R26me 2a 17 1.3.4 REPRESSIVE ARGININE METHYLATIONS: H4/H2AR3me2s, H3R8me2s and H3R2me 2a 18 1.3.4.1 H4/H2AR3me2s. 1.3.4 .2 H3R8me2s 21 1.3.4.3 H3R2me 2a 22 1.3.5 CITRULLINATION AND ARGININE DEMETHYLATION 24 1.3.6 METHYLATED AND UNMETHYLATED ARGININE AS PROTEIN DOCKING SITES: ADDING ANOTHER LAYER OF COMPLEXITY. THE SYMMETRIC DIMETHYLATION OF HISTONE H3 ARGININE 2: A NOVEL HISTONE MARK INVOLVED IN EUCHROMATIN MAINTENANCE VALENTINA MIGLIORI (Master in Molecular Biotechnology (Hons), Alma