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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: 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.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Csaba Koncz

PROVIDER: E-GEOD-27687 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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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

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Super-enhancer-associated oncogenes as novel therapeutic targets in osteosarcoma (Microarray gene expression data)

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Project description: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.

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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.

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Project description:In fission yeast, the nuclear-localized Lsk1p-Lsc1p-Lsg1p cyclin dependent kinase complex is required for the reliable execution of cytokinesis and is also required for Ser-2 phosphorylation RNA pol II carboxy terminal domain. To address whether alterations in CTD phosphorylation might selectively alter expression of cytokinesis genes, expression profiling of site-directed CTD mutants was performed.

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