ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression

Abstract

Dynamic regulation of diverse nuclear processes is intimately linked to covalent modifications of chromatin. Much attention has focused on methylation at lysine 4 of histone H3 (H3K4), owing to its association with euchromatic genomic regions. H3K4 can be mono-, di- or tri-methylated. Trimethylated H3K4 (H3K4me3) is preferentially detected at active genes, and is proposed to promote gene expression through recognition by transcription-activating effector molecules. Here we identify a novel class of methylated H3K4 effector domains--the PHD domains of the ING (for inhibitor of growth) family of tumour suppressor proteins. The ING PHD domains are specific and highly robust binding modules for H3K4me3 and H3K4me2. ING2, a native subunit of a repressive mSin3a-HDAC1 histone deacetylase complex, binds with high affinity to the trimethylated species. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD domain stabilizes the mSin3a-HDAC1 complex at the promoters of proliferation genes. This pathway constitutes a new mechanism by which H3K4me3 functions in active gene repression. Furthermore, ING2 modulates cellular responses to genotoxic insults, and these functions are critically dependent on ING2 interaction with H3K4me3. Together, our findings establish a pivotal role for trimethylation of H3K4 in gene repression and, potentially, tumour suppressor mechanisms.

Figure 1. The ING2 PHD domain specifically binds to H3K4me3 in vitro

a, Coomassie-blue stain of glutathione S-transferase (GST)–ING2_PHD and GST control pull downs from calf thymus histones. b, Gel shift showing ING2_PHD-binding to purified native nucleosomes. Ethidium-bromide stain of nucleosomal DNA on a non-denaturing gel. c, Western blot analysis of GST pull-down assays as in a. d, The ING2 PHD domain preferentially binds H3K4me3 peptides. Western analysis of histone peptide pull downs with the indicated GST fusion proteins and biotinylated peptides. Peptide integrity was confirmed with known methyl-lysine-binding domains (Supplementary Fig. S1d, e). aa, amino acids. e, Methylation at H3K4 is required for ING2_PHD-binding to chromatin in vitro. Western analysis of GST–ING2_PHD pull-down assays of chromatin purified from wild-type or Set1-null S. cerevisiae strains. f, PHD domains of yeast and human ING family proteins preferentially bind to H3K4me2/3 in histone peptide binding assays.

Figure 2. Methylated H3K4 recognition by the ING2 PHD domain enhances ING2-associated HDAC1 histone deacetylase activity in vitro

a, Western analysis of affinity-purified wild-type and mutant Flag–ING2 complexes. Mock, empty vector control immunoprecipitation. b, Binding of ING2 complexes to methylated H3K4 peptides requires an intact PHD domain. Anti-ING2 western analysis of histone peptide pull downs from the indicated HDAC1–ING2 complexes. c, Histone deacetylation by HDAC1 in wild-type, but not mutant, ING2 complexes is increased by binding of the ING2 PHD domain to methylated H3K4. Western analysis of in vitro histone deacetylation reactions by ING2 complexes in the presence or absence of methylation by SET7. Histone deacetylase inhibition with trichostatin A (TSA) is shown as a control. d, Quantification of relative histone deacetylase activity of ING2 complexes as in c from three independent experiments. Error bars indicate the s.e.m.

Figure 3. The ING2 interaction with trimethylated H3K4 occurs in vivo and requires an intact PHD domain

a, b, In vivo association of wild-type, but not PHD domain mutant, ING2 proteins with H3K4me3 at chromatin. Western analysis of proteins crosslinked in situ to wild-type or mutant Flag–ING2 proteins in protein–protein ChIPs. Note equal binding of mutant and wild-type ING2 to SAP30 (a) and HDAC1 (b). Input loaded represents 5% of total. c, Decreased H3K4me3 levels following WDR5 knockdown. Western analysis of HEK293T cells transfected with WDR5 or control RNA interference (RNAi) vectors. d, Flag–ING2 association with endogenous H3 requires H3K4me3. Western analysis of protein–protein ChIPs as in a with or without WDR5 knockdown. e, Endogenous association of H3 and ING2 requires H3K4me3. Western analysis of H3-bound proteins by protein–protein ChIP, with or without WDR5 knockdown. f, WDR5 knockdown decreases ING2 occupancy at the cyclin D1 promoter. Levels (per cent input = ChIP/input × 100) of cyclin D1 promoter and 3′ coding region sequences in the indicated ChIPs, with or without WDR5 knockdown. Error bars indicate the s.e.m. from three independent experiments.

Figure 4. Recognition of H3K4me3 by ING2 PHD domain in vivo is critical for ING2 function

a, Western analysis of ING2 expression in the indicated cell lines. *, endogenous ING2. b, ING2 is required for repression of cyclin D1 expression in response to doxorubicin (dox). Quantitative PCR of cyclin D1 mRNA, with or without doxorubicin (0.2μg ml⁻¹, 24 h), in wild-type (“WT”), knockdown (“KD”) and knockdown reconstituted with ING2 (“KD+ING2”) cell lines. c, Impaired activity of mutant ING2 proteins in reconstitution of doxorubicin-dependent cyclin D1 repression, as in b, relative to wild-type ING2 (“KD+ING2”). In b and c, error bars indicate the s.e.m. of triplicate experiments; P-values < 0.01. d-f, DNA-damage-dependent increase of ING2-HDAC1 occupancy at the cyclin D1 promoter requires methylated H3K4-binding. ChIPs with antibodies to Flag (d), HDAC1 (e) and H4K8ac (f) at the cyclin D1 promoter in the indicated cell lines, with or without doxorubicin (2μM, 1 h). Error bars indicate the s.e.m. of at least three independent experiments. P-values < 0.05. g, Western analysis of cyclin D1 protein in the indicated cell lines with or without doxorubicin (0.2μg ml⁻¹, 24 h). h, Impaired doxorubicin sensitivity following ING2 knockdown or reconstitution with H3K4me3-binding-defective ING2. Relative cell viability is normalized to untreated cells. Error bars indicate the s.e.m. of 3–6 independent experiments.