Unknown,Transcriptomics,Genomics,Proteomics

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The C-Terminal Domain of RNA Polymerase II is Modified by Site-Specific Methylation

ABSTRACT: The Carboxy-terminal domain (CTD) of RNA Polymerase II (RNAPII) in mammals undergoes extensive post-translational modification, which is essential for transcriptional initiation and elongation. Here, we show that the CTD of RNAPII is methylated at a single arginine (R1810) by the transcriptional co-activator CARM1. Although methylation at R1810 is present on the hyper-phosphorylated form of RNAPII in vivo, Ser-2 or Ser-5 phosphorylation inhibit CARM1 activity towards this site in vitro, suggesting that methylation occurs before transcription initiation. Mutation of R1810 results in the mis-expression of a variety of snRNAs and snoRNAs, an effect that is also observed in Carm1-/- MEFs. These results demonstrate that CTD methylation facilitates the expression of select RNAs, perhaps serving to discriminate the RNAPII-associated machinery recruited to distinct gene types. To address the function of RNAPII methylation, we generated Raji cell lines expressing an RNA Polymerase II resistant to Î±-amanitin and carrying either wild-type R1810 or an arginine to alanine substitution at that same residue, abolishing R1810 methylation of the CTD. In cells cultured in Î±-amanitin, the Î±-amanitin-resistant mutants fully replaced the functions of endogenous RNAPII, allowing us to study if gene-expression is affected by the absence of R1810me

ORGANISM(S): Homo sapiens

SUBMITTER: Roberto Bonasio

PROVIDER: E-GEOD-27315 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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The C-terminal domain of RNA polymerase II is modified by site-specific methylation.

Sims Robert J RJ Rojas Luis Alejandro LA Beck David B DB Bonasio Roberto R Schüller Roland R Drury William J WJ Eick Dirk D Reinberg Danny D

Science (New York, N.Y.) 20110401 6025

The carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) in mammals undergoes extensive posttranslational modification, which is essential for transcriptional initiation and elongation. Here, we show that the CTD of RNAPII is methylated at a single arginine (R1810) by the coactivator-associated arginine methyltransferase 1 (CARM1). Although methylation at R1810 is present on the hyperphosphorylated form of RNAPII in vivo, Ser2 or Ser5 phosphorylation inhibits CARM1 activity toward this si ...[more]

PMID: 21454787

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Project description:During development histone modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of embryonic epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using Î±-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3 and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300-recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences. We have performed ChIP-sequencing of eight histone modifications, RNA polymerase II (RNAPII) and the enhancer protein p300 at five stages of development: blastula (st. 9), gastrula (st. 10.5, 12.5), neurula (st. 16) and tailbud (st. 30). These experiments allow identification of enhancers (H3K4me1, p300), promoters (H3K4me3, H3K9ac), transcribed regions (H3K36me3, RNAPII) and repressed and heterochromatic domains (H3K27me3, H3K9me2, H3K9me3, H4K20me3). In addition we generated pre-MBT (st. 8) maps for three histone modifications (H3K4me3, H3K9ac, H3K27me3) and single-base resolution DNA methylome maps using whole genome bisulfite sequencing of blastula and gastrula (st. 9 and 10.5) embryos. To determine the maternal and zygotic contributions to chromatin state, we used alpha-amanitin to block embryonic transcription. Fertilised eggs were injected with 2.3 nl of 2.67 ng/ul alpha-amanitin and developed until the control embryos reached mid-gastrulation. Alpha-amanitin and control embryos were used for RNA-seq and ChIP-seq of RNAPII, H3K4me3, H3K27me3 and p300. For all ChIP-seq samples of the epigenome reference maps and RNAPII ChIP-seq samples of the Î±-amanitin experiments three biological replicates of different chromatin isolations of 45 embryos were pooled. Two biological replicates for H3K4me3 (Î±-amanitin injected: resp. 90 and 56 embryo equivalents (eeq); control: resp. 45 and 67 eeq), H3K27me3 (Î±-amanitin injected: resp. 90 and 180 eeq; control: resp. 45 and 202 eeq) and p300 (Î±-amanitin injected: resp. 112 and 56 eeq; control: resp. 112 and 67 eeq) ChIP-seq samples of the Î±-amanitin experiments were generated. For RNA-seq samples of the Î±-amanitin experiments RNA from 5 embryos from one biological replicate was isolated and depleted of ribosomal RNA

Dataset Information

The C-Terminal Domain of RNA Polymerase II is Modified by Site-Specific Methylation

Publications

The C-terminal domain of RNA polymerase II is modified by site-specific methylation.

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