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Kinetics of RNA polymerase 2 recruitment and H3K36me3 deposition after stimulation of murine T-cells

ABSTRACT: Activation of immune cells results in extremely rapid functional changes, yet it remains enigmatic how this is accomplished. By combining time-courses of RNA-polymerase-II (RNAPII) Chip-Seq, 4sU-Seq, RNA-Seq and ribosome profiling during Th1 activation we provide a genome-wide view on temporal dynamics for ~10.000 genes. Gene responses vary in time and magnitude and allow the identification of unknown immediate early (IEG) and posttranscriptionally regulated genes. However, >90% are regulated transcriptionally with coupled changes in translation. Surprisingly, not poised, but rather re-positioned RNAPII correlates with transcriptional changes and coincides with fluctuations in cotranscriptional splicing, which lags behind at first. Unspliced transcripts remain here at the chromatin, but are already poly-adenylated. Later splicing readjusts, correlating with progressive Ser-2 phosphorylation of the CTD domain of RNAPII and activation of the elongation-complex pTEFb. Thus, rapid re-positioning of RNAPII and regulating the phosphorylation state of its CTD dictates almost all events during immune responses, including post-transcriptional processes.

ORGANISM(S): Mus musculus

PROVIDER: GSE94698 | GEO | 2017/03/31

SECONDARY ACCESSION(S): PRJNA371839

REPOSITORIES: GEO

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Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing and Translation Events during T Cell Responses

Project description:Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time-courses of 4sU-Seq, RNA-Seq, ribosome profiling and RNA polymerase II (RNAPII) ChIP-Seq during T cell activation, we illustrate genome-wide temporal dynamics for ~10,000 genes. This approach reveals immediate-early as well as posttranscriptionally-regulated genes, but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of paused RNAPII, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNAPII carboxy-terminal domain (CTD) and activation of the positive transcription elongation factor complex (pTEFb). Thus, rapid de novo recruitment of RNAPII dictates the course of events during T cell activation, in particular transcription, splicing and consequently translation.

2017-03-31 | GSE83351 | GEO

RNA polymerase II is trapped in initiation states on non-coding RNA transcripts

Project description:The interaction between RNA polymerase II (RNAPII) and RNA processing and packaging factors is strongly influenced by the C-terminal domain (CTD), which consists of multiple heptad repeats that can be differentially phosphorylated at five positions. Here we report strand-specific, high-resolution profiling of the five types of RNAPII CTD phosphorylation in yeast using crosslinking and analysis of modified polymerase (CLAMP). The 5’ regions of protein coding genes showed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P and Ser7P. CTD phosphorylation pattern boundaries were associated with known sites of RNAPII pausing, splicing, and nucleosome positioning. To integrate the distribution of the RNAPII modifications across all transcription units, we developed an eight-state, strand-specific Hidden Markov Model. This identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the Transcription Start Site (TSS) of mRNAs and ncRNAs, and persisted throughout the 1st exon of intron-containing genes. Notably, unstable ncRNAs failed to transition into the elongation states seen on protein coding genes, and their early termination and rapid degradation probably reflect this failure.

2016-06-08 | GSE69676 | GEO

Expanding the CTD code: methylation of non-consensus Lysine residues marks early transcription in mammalian cells

Project description:Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and co-transcriptional processing of nascent RNA. Extensive phosphorylation of serine residues occurs at the structurally-disordered C-terminal domain (CTD) of the largest RNAPII subunit, which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe for the first time mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. Genome wide mapping of 2 novel RNAPII post-translational modifications (CTD-K7me1 and CTD-K7me2) in mouse ES cells.

2015-12-22 | E-GEOD-72876 | biostudies-arrayexpress

Early effects of cdkf;1 RNAPII CTD Serine-7 kinase mutation on regulation of transcription in 7 days old Arabidopsis seedlings

2011-09-01 | GSE27687 | GEO

Distribution of RNA polymerase II and changes of carbo-terminal phosphorylation sites after INTS11 knockdown

Project description:As Integrator is tightly associated with RNAPII-CTD, it is critical to understand how the RNAPII engaged and conducted within the active gene promoter for divergent transcription. We thus employed RNAPII ChIP-seq (Chromatin Immuno-precipitation with RNAPII antibody) to determine the distribution of the total RNAPII and its phosphorylation isoforms. Total RNA polymerase II and its carbon terminal phosphorylation(RNA polymerase II-ctd-Tyr-1，RNA polymerase II-ctd-ser-2) before and after INTS11 knockdown in HCT116-INTS11-AID cells changes chromatin immunoprecipitation DNA sequencing (ChIP-seq).

2023-07-25 | GSE207269 | GEO

Early effects of cdkf;1 RNAPII CTD Serine-7 kinase mutation on regulation of transcription in 7 days old Arabidopsis seedlings

Project description:Serine phosphorylation of conserved Y1S2P3T4S5P6S7 repeats of RNA polymerase II carboxy-terminal domain (RNAPII CTD) plays a central role in the regulation of transcription and co-transcriptional RNA processing. Maintenance of CTD phosphoserine-7 mark in Arabidopsis requires the CDKF;1 kinase, which mediates in vivo activation of downstream-acting CDKD CTD kinase family. Knockout mutations of CDKF;1 lead to over 50% reduction of RNAPII CTD Ser-7 phosphorylation as early as 7 days after germination in seedlings. The transcript profiling experiment aimed at determining how early changes in CTD Ser-7 phosphorylation affect global regulation of transcription. We used Affymetrix ATH1-121501 Genome Array to compare global transcript levels in wild type and cdkf;1-2 mutant seedlings 7 days after germination. Wild type and cdkf;1-2 mutant Arabidopsis (Col-0) seedlings were grown in solid MSAR medium (Koncz, C., Martini, N., Szabados, L., Hrouda, M., Bachmair, A., and Schell, J. (1994). Specialized vectors for gene tagging and expression studies. In: Plant Molecular Biology Manual, Gelvin,S., and Schilperoort, B. (eds.), Kluwer Academic Publishers, Dordrecht-Boston-London, B2, 1-22.) containing ½ concentration of macro elements and 0.5% sucrose in Petri-dishes at 22°C, 120 mol/m2s light intensity and 8h light/16h dark period. Seedling were collected at 4h after the start of the light period in liquid nitrogen for RNA preparation.

2011-08-31 | E-GEOD-27687 | biostudies-arrayexpress

ENCODE: two phosphorylation states of RNAP II ChIP-chip from HeLa cells

Project description:Two phosphorylation states of RNAPII in HeLa cells Keywords: ChIP-chip Study of RNAPII binding patterns to ENCODE regions. 3 arrays using 8wg16 antibody, hypophosphoryated PolII, 3 arrays using 4H8 antibody, phosphorylated PolII CTD, and 6 input DNA arrays.

2010-06-10 | E-GEOD-2735 | biostudies-arrayexpress

Regulation of pre-mRNA splicing by m6A through the low-complexity protein hnRNPG and co-transcriptional pausing

Project description:Co-transcriptional processing of nascent transcripts is coupled with transcription through the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). The mRNA modification N6-methyladenosine (m6A) occurs co-transcriptionally and impacts pre-mRNA processing. How alternative splicing of pre-mRNA is co-transcriptionally regulated in an m6A-dependent manner is not well understood. Furthermore, splicing regulation through RNAPII pausing has been frequently suggested but the underlying control mechanism remains unclear. Here, we show that the m6A reader protein hnRNPG directly binds to the phosphorylated CTD of RNAPII using Arg-Gly-Gly (RGG) motifs in its low-complexity region. This RGG-phospho-CTD interaction and nascent RNA binding by hnRNPG enables its co-transcriptional association with RNAPII, resulting in transcriptome-wide regulation of alternative splicing and transcript abundance. m6A sites near exon splice sites in nascent mRNA further modulate hnRNPG binding and exon splicing. Exon inclusion is associated with RNAPII pausing downstream of the m6A site. Our results reveal an integrated nuclear mechanism of m6A-mediated gene regulation, in which an m6A reader protein regulates gene expression by using RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA, while the interplay between hnRNPG binding and RNAPII pausing modulates alternative splicing regulation.

2019-09-05 | GSE114311 | GEO

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

Project description: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

2011-04-01 | E-GEOD-27315 | biostudies-arrayexpress

Spliceosome specifically associates with RNA polymerase II CTD Ser5-P isoform to promote co-transcriptional splicing

Project description:Co-transcriptional splicing of introns is a defining feature of eukaryotic gene expression. We show that the mammalian spliceosome specifically associates with the S5P CTD isoform of RNA polymerase II (Pol II) as it elongates across spliced exons of protein coding genes, both in human Hela and murine lymphoid cell lines. Immuno-precipitation of MNase digested chromatin with phospho CTD specific antibodies reveals that components of the active spliceosome (both snRNA and proteins) form a specific complex with S5P CTD Pol II. Furthermore a dominant splicing intermediate formed by cleavage at intron 5’ss results in the tethering of upstream exons to this complex at all spliced exons. These are invariably connected to upstream spliced constitutive and less frequently to alternative exons. Finally S5P CTD Pol II accumulates over spliced exons but not adjacent introns. We propose that mammalian splicing employs a rapid, co-transcriptional splicing mechanism based on CTD phosphorylation transitions.

2018-10-06 | GSE106881 | GEO

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