Mono-, di- and trimethylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centred on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular 'effectors' involved in specific recognition of H3K4me3 tails remain poorly understood. Here we demonstrate the molecular basis for specific recognition of H3(1-15)K4me3 (residues 1-15 of histone H3 trimethylated at K4) by a plant homeodomain (PHD) finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). We report on crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1-15)K4me3-bound states. H3(1-15)K4me3 interacts through anti-parallel beta-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, we identified key specificity-determining residues from binding studies of H3(1-15)K4me3 with PHD finger point mutants. Our findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins.

Human L3MBTL1, which contains three malignant brain tumor (MBT) repeats, binds monomethylated and dimethylated lysines, but not trimethylated lysines, in several histone sequence contexts. In crystal structures of L3MBTL1 complexes, the monomethyl- and dimethyllysines insert into a narrow and deep cavity of aromatic residue-lined pocket 2, while a proline ring inserts into shallower pocket 1. We have also engineered a single Y to E substitution within the aromatic cage of the BPTF PHD finger, resulting in a reversal of binding preference from trimethyl- to dimethyllysine in an H3K4 sequence context. In both the "cavity insertion" (L3MBTL1) and "surface groove" (PHD finger) modes of methyllysine recognition, a carboxylate group both hydrogen bonds and ion pairs to the methylammonium proton. Our structural and binding studies of these two modules provide insights into the molecular principles governing the decoding of lysine methylation states, thereby highlighting a methylation state-specific layer of histone mark readout impacting on epigenetic regulation.

WDR5 is a core component of SET1-family complexes that achieve transcriptional activation via methylation of histone H3 on Nζ of Lys4 (H3K4). The role of WDR5 in the MLL1 complex has recently been described as specific recognition of dimethyl-K4 in the context of a histone H3 amino terminus; WDR5 is essential for vertebrate development, Hox gene activation and global H3K4 trimethylation. We report the high-resolution X-ray structures of WDR5 in the unliganded form and complexed with histone H3 peptides having unmodified and mono-, di-and trimethylated K4, which together provide the first comprehensive analysis of methylated histone recognition by the ubiquitous WD40-repeat fold. Contrary to predictions, the structures reveal that WDR5 does not read out the methylation state of K4 directly, but instead serves to present the K4 side chain for further methylation by SET1-family complexes.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ Correspondence should be addressed to G.L.V. (verdine@chemistry.harvard.edu).. AUTHOR CONTRIBUTIONS A.J.R. is responsible for the X-ray studies of the apo structure and the H3K4me1, H3K4me2 and H3K4me3 structures, W.-K.W. and H.L. are responsible for the unmodified peptide structure and an additional H3K4me2 structure (complex II), and D.M.G. performed the binding studies with some assistance from A.J.R. and W.-K.W. G.L.V., D.J.P. and C.D.A. supervised the structural and biochemical aspects of the project and take overall responsibility for their joint research. All authors discussed the results and commented on the manuscript. Accession codes. Protein Data Bank: coordinates have been deposited with the accession codes 2H68, 2CO0, 2H6K, 2H6N, 2CNX and 2H6Q, representing the apo structure, unmodified H3 complex, H3K4me1 complex, H3K4me2 complex crystal form I, H3K4me2 complex crystal form II and H3K4me3 complex, respectively. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.Supplementary information is available on the Nature Structural & Molecular Biology website. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe structure and dynamics of chromatin are increasingly recognized to be essential for mediating both global and local regulation of the genome 1,2 . Vital to maintaining and remodeling the various chromatin states are a collection of post-translational modifications of histones that are introduced in a highly regulated fashion and cause specific functional consequences 3 . In particular, post-translational introduction of methyl groups onto lysine side chains of histone proteins greatly affects chromatin function in complex and often opposing ways, resulting in either transcriptional repression or activation, contingent upon the precise location and degree of methylation [4][5][6][7] . For example, trimethylation of histone H3 at K9 or K27 is implicated in silencing of the underlying DNA 6,8 , whereas methylation at other s...