Histone methylation is crucial for regulating chromatin structure, gene transcription and the epigenetic state of the cell. LSD1 is a lysine-specific histone demethylase that represses transcription by demethylating histone H3 on lysine 4 (ref. 1). The LSD1 complex contains a number of proteins, all of which have been assigned roles in events upstream of LSD1-mediated demethylation 2-4 apart from BHC80 (also known as PHF21A), a plant homeodomain (PHD) finger-containing protein. Here we report that, in contrast to the PHD fingers of the bromodomain PHD finger transcription factor (BPTF) and inhibitor of growth family 2 (ING2), which bind methylated H3K4 (H3K4me3) 5,6 , the PHD finger of BHC80 binds unmethylated H3K4 (H3K4me0), and this interaction is specifically abrogated by methylation of H3K4. The crystal structure of the PHD finger of BHC80 bound to an unmodified H3 peptide has revealed the structural basis of the recognition of H3K4me0. Knockdown of BHC80 by RNA inhibition results in the de-repression of LSD1 target genes, and this repression is restored by the reintroduction of wild-type BHC80 but not by a PHD-finger mutant that cannot bind H3. Chromatin immunoprecipitation showed that BHC80 and LSD1 depend reciprocally on one another to associate with chromatin. These findings couple the function of BHC80 to that of LSD1, and indicate that unmodified H3K4 is part of the 'histone code' 7 . They further raise the possibility that the generation and recognition of the unmodified state on histone tails in general might be just as crucial as post-translational modifications of histone for chromatin and transcriptional regulation. © 2007 Nature Publishing GroupCorrespondence and requests for materials should be addressed to Y.S. (E-mail: yshi@hms.harvard.edu) and X.C. (E-mail: xcheng@emory.edu).. † Present address: Telethon Institute of Genetics and Medicine (TIGEM), Via P. Castellino 111, 80131 Naples, Italy. * These authors contributed equally to this work.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author InformationThe X-ray structure of the BHC80 PHD domain in complex with the H3 tail peptide has been deposited to PDB as 2PUY.Reprints and permissions information is available at www.nature.com/reprints.The authors declare no competing financial interests. Recent studies have identified a subset of PHD fingers that bind methyl lysine 5,6,8,9 . To investigate the role of BHC80, a PHD finger-containing protein (Fig. 1a) of the LSD1 corepressor complex, in transcriptional repression, we determined whether BHC80 also binds histone tails through its PHD finger. As shown in Fig. 1b, BHC80 binds the first 21 residues of histone H3 (lane 3), but not residues 21-44 or histones H4, H2A or H2B (lanes 4, 8-11). Unexpectedly, the BHC80-H3 interaction is disrupted by methylation of K4, but is insensitive to modifications at K9 or K14 (Fig. 1b, lanes 5-7 and Supplementary Fig. 1a). Native BHC80 in the LSD1 complex also binds H3K4me0, and this binding is similarly ...

With genome sequencing nearing completion for the model organisms used in biomedical research, there is a rapidly growing appreciation that proteomics, the study of covalent modification to proteins, and transcriptional regulation will likely dominate the research headlines in the next decade. Protein methylation plays a central role in both of these fields, as several different residues (Arg, Lys, Gln) are methylated in cells and methylation plays a central role in the "histone code" that regulates chromatin structure and impacts transcription. In some cases, a single lysine can be mono-, di-, or trimethylated, with different functional consequences for each of the three forms. This review describes structural aspects of methylation of histone lysine residues by two enzyme families with entirely different structural scaffolding (the SET proteins and Dot1p) and methylation of protein arginine residues by PRMTs.

Histone modifications have important roles in transcriptional control, mitosis and heterochromatin formation. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by mono- and dimethylating histone H3 at Lys9 (H3K9). Here we demonstrate that the ankyrin repeat domains of G9a and GLP bind with strong preference to N-terminal H3 peptides containing mono- or dimethyl K9. X-ray crystallography revealed the basis for recognition of the methylated lysine by a partial hydrophobic cage with three tryptophans and one acidic residue. Substitution of key residues in the cage eliminated the H3 tail interaction. Hence, G9a and GLP contain a new type of methyllysine binding module (the ankyrin repeat domains) and are the first examples of protein (histone) methyltransferases harboring in a single polypeptide the activities that generate and read the same epigenetic mark.

Estrogen receptor alpha (ER) is a ligand-dependent transcription factor. Upon binding estrogen, ER recruits coactivator complexes with histone acetyltransferase or methyltransferase activities to activate downstream target genes. In addition to histones, coactivators can modify ER itself and other proteins in the transactivation complex. Here, we show that ER is directly methylated at lysine 302 (K302) by the SET7 methyltransferase. SET7-mediated methylation stabilizes ER and is necessary for the efficient recruitment of ER to its target genes and for their transactivation. The SET7-ER complex structure reveals the molecular basis for ER peptide recognition and predicts that modifications or mutations of nearby residues would affect K302 methylation. Indeed, a breast cancer-associated mutation at K303 (K303R) alters methylation at K302 in vitro and in vivo. These findings raise the possibility that generation, recognition, and removal of modifications within the ER hinge region generate "ER modification cassettes" that yield distinct patterns for signaling downstream events.

The functional significance of mono-, di-, and trimethylation of lysine residues within histone proteins is under investigation. Evidence from several model organisms suggests that different methylated states of H3 Lys 9 (H3K9) are generated by specific histone methyltransferases (MTases) to mark distinct types of silent chromatin. Sequence alignment of all histone lysine MTases with known product specificity suggested that a key residue in the active site determines how many methyl groups they add. We examined this possibility both in vitro and in vivo and found that a Phe at the position equivalent to Phe 281 of Neurospora crassa DIM-5 or Phe 1205 of human G9a allows the enzyme to perform di and tri-methylation, whereas a Tyr at this position is restrictive, inhibiting tri-methylation and thus yielding a mono-or di-MTase. Phe to Tyr mutants of both DIM-5 and G9a restrict product specificity in vitro and in vivo without compromising overall catalysis. These mutants were employed to probe the biological significance of mono-, di-, and tri-methylation of H3K9 in both mouse embryonic stem cells and N. crassa. G9a F1205Y, when expressed in G9a (؊/؊) embryonic stem cells, rescued only H3K9 mono-methylation, but not di-methylation, to wild-type levels yet silenced Mage-a gene expression. When expressed in dim-5 strains, DIM-5 F281Y generated significant levels of mono-and di-H3K9 methylation (which are not observed in wild type Neurospora) as well as tri-methyl H3K9. The altered DIM-5 rescued the growth defect characteristic of dim-5 N. crassa but did not fully rescue the gross DNA hypomethylation of dim-5 strains.Histones are subject to diverse post-translational modifications including acetylation, phosphorylation, ubiquitination, methylation, and sumoylation. Evidence accumulated over the past few years suggests that such modifications constitute a "histone code" that directs a variety of processes involving chromatin (1, 2). Considering just methylation of lysines, there are at least six modification sites (Lys 4 , Lys 9 , Lys 27 , Lys 36 , and Lys 79 of histone H3 and Lys 20 of histone H4), and in principle each site can have zero, one, two, or three methyl groups. It has been suggested that methylation at these sites, in combination with other nearby modifications, generates "modification cassettes" (3), yielding distinct patterns on chromatin for signaling downstream events (reviewed in Refs. 3 and 4).With only one known exception, histone lysine methyltransferases (HKMTs) 1 contain a SET domain of ϳ130 amino acids. SET proteins can be grouped into families according to the sequences surrounding this distinctive domain (5, 6) (see Table I). In this study we focus on two members of the SUV family that methylate Lys 9 of histone H3, G9a characterized in mammals and DIM-5 characterized in the filamentous fungus Neurospora crassa. DIM-5 provided the first evidence that histone methylation can direct DNA methylation (7). DIM-5 normally generates primarily tri-methyl-Lys 9 on histone H3 (8, 9). G9a is essential for...