Dataset Information

Anti-Pol II pS5 co-immunoprecipitation in DIDO WT, KO and ΔSPOC HEK293T cells

ABSTRACT: The analysis of differential interactome of Pol II pS5 in DIDO WT, KO and ΔSPOC HEK293T cells using Pol II pS5 (3E8) antibody coupled to Protein G beads (no crosslinking).

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: WeiQiang Chen

LAB HEAD: Dea Slade

PROVIDER: PXD039567 | Pride | 2023-10-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20220524_E3_RSLC1_Benedum_Slade_MPL_UniWien_pSer5_DIDO_KO1_s13_40p.raw	Raw
	20220524_E3_RSLC1_Benedum_Slade_MPL_UniWien_pSer5_DIDO_KO2_s14_40p.raw	Raw
	20220524_E3_RSLC1_Benedum_Slade_MPL_UniWien_pSer5_DIDO_KO3_s15_40p.raw	Raw
	20220524_E3_RSLC1_Benedum_Slade_MPL_UniWien_pSer5_DIDO_WT1_s10_40p.raw	Raw
	20220524_E3_RSLC1_Benedum_Slade_MPL_UniWien_pSer5_DIDO_WT2_s11_40p.raw	Raw

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Project description: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 5M-bM-^@M-^Y and 3M-bM-^@M-^Y 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 3M-bM-^@M-^Y 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. 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.

Project description:Cellular responses to signalling pathways are often highly dynamic, however most analyses of developmental signalling pathways focus on a single endpoint. We have analyzed the temporal changes in transcription following a short Notch activation treatment and related these to the recruitment of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and to the state of RNA Polymerase II (Pol II) binding. A total of 154 genes showed significant differential expression over time and their expression profiles stratified into 14 clusters based on temporal and quantitative differences in their responses. These differences were partially reflected in the profiles of Pol II and Su(H) binding. However, neither could fully account for the different response profiles. Furthermore, the timing of the different responses was unaffected by more prolonged Notch activation. Instead our data suggest that regulatory relationships between genes that segregate into different response clusters can partially account for the stratification. Thus, feed-forward repression, where products of early responding Enhancer of split bHLH genes (E(spl)bHLH) inhibit expression of endogenous repressors, is one mechanism that explains the profile of genes that exhibit delayed up-regulation. E(spl)bHLH genes may therefore be responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining their critical functions in many developmental and disease contexts. DmD8 cells were collected at 7 time points (0M-bM-^@M-^Y, 10M-bM-^@M-^Y, 20M-bM-^@M-^Y, 30M-bM-^@M-^Y, 40M-bM-^@M-^Y, 60M-bM-^@M-^Y and 100M-bM-^@M-^Y) after a 5 minute Notch stimulation as 3 independent replicates. Immunoprecipitation was performed with Su(H) antibody and compared to the total input DNA. Samples from replicates #1 and #2 were labelled with Cy5, and replicate #3 was labelled with Cy3 as a dye-swap. For time point 10M-bM-^@M-^Y, only 2 biological replicates were obtained.

Dataset Information

Anti-Pol II pS5 co-immunoprecipitation in DIDO WT, KO and ΔSPOC HEK293T cells

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