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Histone H3R2 symmetric dimethylation and histone H3K4 trimethylation are tightly correlated in eukaryotic genomes - PubMed

  • ️Sun Jan 01 2012

Histone H3R2 symmetric dimethylation and histone H3K4 trimethylation are tightly correlated in eukaryotic genomes

Chih-Chi Yuan et al. Cell Rep. 2012.

Abstract

The preferential in vitro interaction of the PHD finger of RAG2, a subunit of the V(D)J recombinase, with histone H3 tails simultaneously trimethylated at lysine 4 and symmetrically dimethylated at arginine 2 (H3R2me2sK4me3) predicted the existence of the previously unknown histone modification H3R2me2s. Here, we report the in vivo identification of H3R2me2s . Consistent with the binding specificity of the RAG2 PHD finger, high levels of H3R2me2sK4me3 are found at antigen receptor gene segments ready for rearrangement. However, this double modification is much more general; it is conserved throughout eukaryotic evolution. In mouse, H3R2me2s is tightly correlated with H3K4me3 at active promoters throughout the genome. Mutational analysis in S. cerevisiae reveals that deposition of H3R2me2s requires the same Set1 complex that deposits H3K4me3. Our work suggests that H3R2me2sK4me3, not simply H3K4me3 alone, is the mark of active promoters and that factors that recognize H3K4me3 will have their binding modulated by their preference for H3R2me2s.

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Figures

Figure 1
Figure 1

H3R2me2s colocalizes with H3K4me3 at the IgH locus in Rag2−/− pro-B cells. Chromatin from Rag2−/− Abelson-transformed pro-B cells was immunoprecipitated with the α-H3K4me3 (upper panel), α-H3R2me2sK4me3 (middle panel), or α-pan-H3R2me2s (lower panel) antibodies. The enrichment of each modification relative to histone H3 was examined by qPCR using primers that span the IgH and TCRβ loci. Results represent the mean ± S.D. of at least two independent experiments.

Figure 2
Figure 2. H3R2me2s, H3R2me2sK4me3, and H3K4me3 are colocalized throughout the mouse genome

(A) H3R2me2s is colocalized with H3K4me3 across a broad 350 kb region of murine chromosome 19. ChIP-seq analysis of H3K4me3 (top), H3R2me2sK4me3 (middle), and pan-H3R2me2s (bottom) enrichment in Rag2−/− Abelson-transformed pro-B cells. The transcription start sites, exons, introns, and relative orientations of the genes present in this 350 kb region are shown below the three panels. (B) H3R2me2s is tightly colocalized with H3K4me3 near the transcriptional start site of Dpf2. A higher-resolution view of H3K4me3 (top), H3R2me2sK4me3 (middle), and pan-H3R2me2s (bottom) enrichment across a 1.75 kb region of murine chromosome 19. The transcription start site (arrow), first exon (thick block), and part of the first intron (thin line) are shown below the three panels. (C) H3R2me2s is present in two peaks flanking transcriptional start sites and is correlated with gene expression. The signal intensity of H3K4me3 (left), H3R2me2sK4me3 (middle), and H3R2me2s (right), averaged for all annotated murine genes, and plotted over a 4 kb window centered on the transcription start site. The genes are further stratified into four quartiles according to their expression levels in pro-B cells. (D) H3R2me2s colocalizes with H3K4me3 at the murine IgH locus. H3K4me3 (top), H3R2me2sK4me3 (middle), and pan-H3R2me2s (bottom) enrichment across a 1.75 Mb region spanning the murine IgH locus. The relative positions of V, D, and J segments are indicated below the three panels. (E) qPCR validation of the deep sequencing data. Chromatin from Rag2−/− Abelson-transformed pro-B cells was immunoprecipitated with the α-H3K4me3, α-H3R2me2sK4me3, or α-pan-H3R2me2s antibodies. The enrichment of each modification relative to histone H3 was examined by qPCR using primers specific for 60 randomly selected promoters. The enrichment levels of H3R2me2s vs. H3K4me3 (left panel), H3R2me2sK4me3 vs. H3K4me3 (middle), and H3R2me2sK4me3 vs. H3R2me2s (right panel) were plotted for each of the 60 promoters. The Spearman’s correlation coefficient and p-value for each combination are shown.

Figure 3
Figure 3. H3R2me2s exists in S. cerevisiae and is intimately connected to H3K4me3

(A) Evolutionary conservation of H3R2me2s and H3R2me2sK4me3. Nuclear extracts from human (Hs), mouse (Mm), frog (Xl), fruit fly (Dm), and budding yeast (Sc) were subjected to Western blot analysis using the α-pan-H3R2me2s, α-H3R2me2sK4me3, and α-pan-Histone H4 antibodies. (B) H3R2me2s, H3R2me2sK4me3, and H3K4me3 colocalize at representative yeast genes. S. cerevisiae chromatin was immunoprecipitated with the α-H3K4me3 (top panel), α-H3R2me2sK4me3 (second panel), α-pan-H3R2me2s (third panel), or α-Pol II antibodies. The enrichment of each modification relative to histone H3 was examined by qPCR using primers to a highly expressed gene, two moderately expressed genes, and a silent gene. The schematic representations at the bottom of the graphs represent the genes that were analyzed and the locations of the qPCR primers within the genes. Results represent the mean ± SD of three independent experiments. (C) H3R2me2s is dependent upon Set1 and H3K4. Nuclear extract was prepared from wild type S. cerevisiae as well as several mutant strains, and subjected to Western blot analysis using the α-pan-H3R2me2s α-pan-H3 antibodies. (D) The α-pan-H3R2me2s antibody’s recognition of symmetrically dimethylated H3R2 is unaffected by an H3K4A mutation. Indicated amounts of unmodified histone H3 (1–21), H3R2me2s, and H3R2me2sK4A peptides were spotted on PVDF membrane and probed with the α-pan-H3R2me2s antibody. (E) H3R2me2s is dependent upon Set1, Spp1, and Cps30. Nuclear extract from either wildtype, set1, spp1, or cps30 deletion mutant strains of S. cerevisiae were analyzed by Western blotting with the α-pan-H3R2me2s, α-H3K4me3, α-H3K4me2, or α-pan-H3 antibodies.

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