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PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression

ABSTRACT: Histone methylation occurs on both lysine and arginine residues and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD domain (PHDUHRF1), an important regulator of DNA CpG methylation, as an unanticipated histone H3 unmodified arginine 2 (H3R2)-recognition modality. This conclusion is based on binding studies and co-crystal structures of the PHDUHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1’s ability to repress its direct target gene expression is dependent on PHDUHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.

ORGANISM(S): Homo sapiens

PROVIDER: GSE30478 | GEO | 2011/07/07

SECONDARY ACCESSION(S): PRJNA143463

REPOSITORIES: GEO

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PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression

Project description:Histone methylation occurs on both lysine and arginine residues and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD domain (PHDUHRF1), an important regulator of DNA CpG methylation, as an unanticipated histone H3 unmodified arginine 2 (H3R2)-recognition modality. This conclusion is based on binding studies and co-crystal structures of the PHDUHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1’s ability to repress its direct target gene expression is dependent on PHDUHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function. UHRF1 protein was depleted in HCT116 cells by shRNA treatment. Total RNA was purified and used to determine the global gene transcription profiles by microarray assays. The UHRF1-regulated genes were identified by comparing the gene expression profiles of control and UHRF1-depleted HCT116 cells.

2011-07-07 | E-GEOD-30478 | biostudies-arrayexpress

Homo sapiens

Project description:PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression

| PRJNA143463 | ENA

Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation

Project description:Modifications on histones control important biological processes through their effects on chromatin structure. Methylation at histone H3 lysine 4 (H3K4) by Set1p is found at the 5’end of active genes and contributes to transcriptional activation by recruiting chromatin remodeling enzymes. An adjacent arginine residue (H3R2) is also known to be asymmetrically dimethylated (H3R2me2a) in mammalian cells6, but its location within genes and its function in transcription are unknown. Here we show that H3R2 is also methylated in budding yeast. 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 at all heterochromatic loci, at inactive euchromatic genes and at the 3’-end of moderately transcribed genes. In all cases the pattern of H3R2 methylation is mutually exclusive with the presence of trimethylation at H3K4 (H3K4me3). The inverse correlation reflects the fact that methylation at H3R2 disrupts the ability of the Set1-complex to methylate H3K4. H3R2m2a inhibits specifically the trimethylation of H3K4 because it prevents Spp1 (a Set1-methyltranferase subunit) from binding to histone H3. These results indicate that methylation at H3R2 controls the global distribution of H3K4me3 and provides the first mechanistic insight into the function of arginine methylation on chromatin. Keywords: ChIP-chip

2007-07-31 | GSE8626 | GEO

Hemi-methylated DNA regulates DNA methylation inheritance through allosteric control of H3 ubiquitylation by UHRF1

Project description:DNA methylation is a heritable chromatin modification essential to mammalian development that functions with histone post-translational modifications to regulate chromatin structure and gene expression programs. The epigenetic inheritance of DNA methylation requires the combined actions of DNMT1 and UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that facilitates DNMT1 recruitment to sites of newly replicated DNA through the ubiquitylation of histone H3. UHRF1 binds DNA with modest selectivity towards hemi-methylated CpG dinucleotides (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation inheritance but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. We further show that HeDNA functions as an allosteric regulator of UHRF1 ubiquitin ligase activity, directing ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies define a highly orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance.

2016-08-25 | PXD003983 | Pride

PRMT6 substrate specificity analysis

Project description:Protein arginine methyltransferase 6 (PRMT6) catalyses the asymmetric dimethylation of arginines on numerous substrate proteins within the human cell. In particular, PRMT6 methylates histone H3 arginine 2 (H3R2) which affects both gene repression and activation. However, the substrate specificity of PRMT6 has not been comprehensively analysed. Here we have systematically characterised the substrate recognition motif of PRMT6 in context of the histone H3 tail, finding that it has broad specificity and recognises the RG motif. Working with a H3 tail peptide as a template, on which we made 204 amino acid substitutions, we use targeted mass spectrometry to measure the effect on PRMT6 in vitro activity. We first show that PRMT6 methylates R2 and R8 in the H3 peptide, although H3R8 is methylated with lower efficiency and is not an in vivo PRMT6 substrate. Then, using parallel reaction monitoring (PRM) with electron transfer dissociation (ETD), we quantify the effect of 194 of these amino acid substitutions on methylation at both H3R2 and H3R8. In both cases, we find that PRMT6 is capable of tolerating essentially any amino acid substitution in the H3 peptide, but that positively charged and bulky residues are preferred near the target arginine. We show that PRMT6 prefers glycine in the position immediately following the target arginine, indicating that PRMT6 recognises the RG motif. We confirm this preference for the RG motif on another PRMT6 substrate, histone H4R3. This broad specificity, along with recognition of RG rather than RGG, is distinctive among the PRMT family and has implications for the development of drugs to selectively target PRMT6.

2021-03-31 | PXD016711 | panorama

Distinct transcriptional outputs associated with mono- and di-methylated histone H3 arginine 2

2009-01-20 | GSE14453 | GEO

Distinct transcriptional outputs associated with mono- and di-methylated histone H3 arginine 2

Project description:Dimethylation of histone H3 arginine 2 (H3R2me2) maintains transcriptional silencing by inhibiting Set-1 mediated trimethylation of H3K4. Here we demonstrate that arginine 2 is also monomethylated (H3R2me1) in yeast but that its functional characteristics are distinct from H3R2me2: (a) H3R2me1 does not inhibit H3K4 methylation, (b) it is present throughout the coding region of genes and (c) it correlates with active transcription. Collectively, these results indicate that different H3R2 methylated states have defined roles in gene expression. hIP-chip analysis was performed to determine the genomic distribution of histone modifications H3R2me1, H3R2me2a, H3K4me3, H3K36me3, and H3K79me3 in the BY4741 yeast strain. ChIP samples were amplified, labelled, and hybridized to 50-mer tiling arrays covering the entire yeast genome at a 64 bp resolution.

2010-05-06 | E-GEOD-14453 | biostudies-arrayexpress

Critical role of the UBL-domain in stimulating the E3 ubiquitin ligase activity of UHRF1 towards chromatin

Project description:The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1, however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RINGmediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays and recombinant chromatin substrates we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the “backside” of the E2 to stabilise the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBLdomain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.

2018-11-04 | PXD011510 | Pride

Saccharomyces cerevisiae

Project description:Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation

| PRJNA101805 | ENA

Ubiquitome analysis of UHRF1 and UHRF2-deficient mESCs

Project description:The E3 ligase UHRF1 is an essential epigenetic cofactor for DNMT1 dependent maintenance DNA methylation, which provides a binding platform for DNMT1 by both cooperative binding of histones and hemi-methylated DNA as well as by ubiquitinating histone H3. Here, we conduct a comprehensive screen to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2 by comparing the ubiquitome of wildtype (wt), UHRF1- and UHRF2-deficient mouse embryonic stem cells. With an antibody-dependent enrichment of ubiquitin remnant motif-containing peptides followed by isobaric-labeling based quantitative mass spectrometry, we find both known and novel E3 ligase substrates of UHRF1 involved in a variety of biological processes such as RNA processing, DNA methylation and DNA damage repair.

2017-10-24 | PXD006593 | Pride

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