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Series GSE37519 Query DataSets for GSE37519
Status Public on Jun 04, 2012
Title Threonine-4 of Mammalian RNA Polymerase II CTD is Targeted by Polo-like Kinase-3 and Required for Transcriptional Elongation
Organism Homo sapiens
Experiment type Genome binding/occupancy profiling by high throughput sequencing
Summary Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5’ and 3’ regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like-kinase-3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3’ region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells.
 
Overall design In this study, we investigated the function and ChIPseq genome-wide profiling of Thr4P residue (using the 6D7 antibody) of the Pol II CTD in Raji human B cells in comparison with either total Pol II profiling (N20 antibody, santa-cruz sc-899x), Ser5P CTD (3E8) or Ser2P (3E10) profiling in WT Raji cells. In another set of experiments, we also analysed total Pol II profiling (using an HA tag at the N-terminus of RPB1 and HA antibody Abcam ab9110) when endogenous enzyme is shut down by alpha-amanitin and replaced by either a recominant Pol II with 48 consensus repeats of the CTD (con48) or a mutated version where Thr4 residues were replaced by Ala (Thr4-Ala).In total 6 experimental sets (Pol IIt, Ser5P, Ser2P, Thr4P, con48, Thr4-Ala) were generated for our analysis and for each a biological replicate was performed. Biological replicates were merged when the data showed comparable signal noise ratio. Otherwise a unique replicate, showing the best noise ratio, was chosen for further analysis although the second replicate (for Ser2P and Thr4-Ala experiments). An input control (genomic DNA extracted after reverse crosslinking of the nuclear chip extracts) was performred and used for substraction to the ChIP experiments. One specific input material was used for wt cells, one for con48 and one for Thr4-Ala. Our data were processed to generate final wig files using our in house analysis pipeline essentially as described in Koch et al, (2011) NSMB 18 (8) p956.In brief, after alignment, sequence tags are: (i) artefact removed, (ii) elongated to an in silico optimized actual size of the initial fragments , (iii) input substracted, (iv) merged if applicable, (v) scaled for all experiments to correct for variation of tag number in between experiments.
Several of the raw data files were no longer available.
 
Contributor(s) Hintermair C, Heidemann M, Koch F, Descostes N, Gut M, Gut I, Fenouil R, Ferrier P, Flatley A, Kremmer E, Chapman RD, Andrau J, Eick D
Citation(s) 22549466
Submission date Apr 23, 2012
Last update date May 15, 2019
Contact name nicolas descostes
Organization name EMBL
Department Bioinformatics
Street address via Ramarini 32
City Monterotondo
ZIP/Postal code 00015
Country Italy
 
Platforms (1)
GPL9052 Illumina Genome Analyzer (Homo sapiens)
Samples (9)
GSM920942 PolII_N20_ChIPSeq
GSM920943 PolII_Ser5P_ChIPSeq
GSM920944 PolII_Ser2P_ChIP-Seq
Relations
BioProject PRJNA160979
SRA SRP012460

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SOFT formatted family file(s) SOFTHelp
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Supplementary file Size Download File type/resource
GSE37519_RAW.tar 2.5 Gb (http)(custom) TAR (of BED, WIG)
SRA Run SelectorHelp
Raw data are available in SRA
Raw data not provided for this record
Processed data provided as supplementary file

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