Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27.[Keywords: Suz12; histone methylation; polycomb; Eed; RNA interference; chromatin immunoprecipitation] Supplemental material is available at http://www.genesdev.org.
Modifications on histones control important biological processes through their effects on chromatin structure [1][2][3] . Methylation at histone H3 lysine 4 (H3K4) is found at the 5′end of active genes and contributes to transcriptional activation by recruiting chromatin remodeling enzymes 4,5 . An adjacent arginine residue (H3R2) is also known to be asymmetrically dimethylated (H3R2me2a) in mammalian cells 6 , but its location within genes and its function in transcription are unknown. Here we show that H3R2 is also methylated in budding yeast and using an antibody specific for H3R2me2a in ChIP-on-Chip analysis we determine the distribution of this modification on the entire yeast genome. We find that H3R2me2a is enriched throughout all heterochromatic loci and inactive euchromatic genes and is present at the 3′-end of moderately transcribed genes. In all cases the pattern of H3R2 methylation is mutually exclusive with the trimethyl form of H3K4 (H3K4me3). We show that methylation at H3R2 abrogates trimethylation of H3K4 by the Set1 methyltransferase. The specific effect on H3K4me3 results from the occlusion of Spp1, a Set1-methyltranferase subunit necessary for trimethylation. Thus, the inability of Spp1 to recognise H3 methylated at R2 prevents Set1 from trimethylating H3K4 . These results provide the first mechanistic insight into the function of arginine methylation on chromatin.Methylation at lysine and arginine residues within histones has been linked to gene expression [1][2][3] . Studies in mammalian cells have demonstrated that histone arginine methylation can influence both gene activation and repression. However, the precise mechanism employed by arginine methylation to exert its effects on the chromatin template remains elusive. In contrast, increasing evidence shows that lysine methylation modulates gene expression by recruiting downstream effector proteins. Recent findings showed that methylation at H3K4 (H3K4me) controls transcription activation by recruiting chromatin remodeling activities 4,5 . This recruitment can be specific for the 7,8 trimethyl form of H3K4 (H3K4me3) indicating that the three different methyl states of H3K4 (mono-, di-, or tri-) Correspondence and requests for materials should be addressed to T.K. (t.kouzarides@gurdon.cam.ac.uk). . Supplementary Information accompanies the paper on www.nature.com/nature. Author InformationThe microarray data sets are available from GEO (Gene Expression Omnibus) under accession number GSE8626, and from http://www.gurdon.cam.ac.uk/%7Ekouzarideslab/H3R2methylation.html.The authors declare competing financial interests. To investigate the role of methylation at histone H3 arginine 2 (H3R2) in gene expression we raised an antibody against the asymmetric dimethylated form of H3R2 (H3R2me2a). This modification is known to be catalysed by the mammalian CARM1/PRMT4 in vitro 12 and is affected by deletion of this methyltransferase in mouse embryonic fibroblasts 6 . Immunoblot analysis reveals that H3R2me2a is present in vivo, on mammalian ...
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