Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression

Abstract

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 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), 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'. 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.

Figure 1. BHC80 binds histone H3 through the PHD zinc finger

a, Diagram of BHC80 domain architecture. AT hook, a motif that binds AT-rich DNA. b, In vitro assays of binding of recombinant BHC80 to histone tail peptides. ac, acetyl group. c, Native BHC80 in purified LSD1 complex also preferentially binds unmodified H3 tail. WB, western blot, using antibodies against BHC80. d, PHD finger of BHC80 is necessary for H3 tail binding. e, The PHD finger is sufficient for H3 tail binding. f, Methylation of H3K4 inhibits PHD finger binding to the H3 tail. g, ITC measurement of binding of the BHC80 PHD finger to unmodified, mono-, or di-methylated H3K4 peptides (residues 1–10). K_D values are means (±s.d.) of at least three experiments using varied peptide and protein concentrations.

Figure 2. Structure of BHC80 PHD with H3 1–10

a, Cross-brace topology of the PHD domain. Residues involved in peptide binding are underlined. Bold residues are those involved in chelating Zn. b, The H3 peptide binds BHC80 as an anti-parallel β-strand (grey). c, Peptide binding is specified by the insertion of M502 between H3R2 and H3K4 and D489 between H3K4 and H3R8. D489 and E488 bind H3K4, and H487 (4.0 Å away) restricts methyl-lysine binding (Supplementary Fig. 4). The N-terminal amine of H3 is caged by carbonyl oxygens, and H3A1 is recognized by a shallow hydrophobic pocket. d, Mutation of D489 and M502 disrupt PHD binding to unmodified H3. WT, wild type.

Figure 3. Structural comparison of PHD fingers

a, Superimposition of PHD domains of BHC80 (PDB 2PUY, green, H3 grey), BPTF (PDB 2F6J, red, H3 pink) and ING2 (PDB 2GQ6, blue, H3 purple). b, Recognition of H3K4 by BHC80 (left) and binding of H3K4me3 by BPTF (middle) and ING2 (right); colours as in a. c, Sequence alignment of the PHD fingers of BHC80, BPTF and ING2. Zinc-binding residues are bold, K4-binding residues blue and the R2-binding pocket, red. Residues that form the N-amine and A1 binding pockets are green. Residues that form the anti-parallel β-sheet with peptide are underlined. Stars mark invariant residues, and colons denote conservation.

Figure 4. BHC80 binding to the H3 tail is important for LSD1-mediated repression

a, The occupancy of promoters by BHC80 is reduced in HeLa cells where LSD1 is knocked down by RNAi. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is used as a control. b, BHC80 was effectively depleted by BHC80 shRNAs and expression of SCN1A, SCN3A and SYN1 were de-repressed in the BHC80 RNAi cells. Ctrl, control. c, RNAi-resistant wild-type BHC80, but not the PHD point mutant D489A, restored target gene repression in BHC80 RNAi cells. d, Reduction of BHC80 in the LSD1 complex reduced the association of LSD1 with H3 peptide. IP, immunoprecipitate. e, Inhibition of BHC80 does not affect LSD1 complex formation in solution. f, RNAi of BHC80 results in decreased LSD1 occupancy at its target genes. Error bars in a and f represent s.e.m. calculated from three independent experiments.