Dataset Information

Heptad-specific phosphorylation of RNA polymerase II CTD

ABSTRACT: The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Monoclonal antibodies have been used to study in vivo the potentially phosphorylated CTD amino acids (Y1, S2, T4, S5 and S7). However, the epitopes detected by antibodies can be masked by proteins or modifications at neighbouring sites. Therefore, the effectiveness of antibodies in western blot or ChIP analysis reflects the number of accessible CTD phosphorylation marks, but not the total number of phosphorylations. Most importantly, CTD phospho-specific antibodies do not provide any heptad - (location) specific information of CTD phosphorylation. Due to these limitations, the principles and patterns of CTD phosphorylation remained elusive. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5 and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast) Mus Musculus (mouse)

TISSUE(S): B Cell, Cell Culture

DISEASE(S): Lymphoma

SUBMITTER: Ignasi Forne

LAB HEAD: Axel Imhof

PROVIDER: PXD003159 | Pride | 2016-01-25

REPOSITORIES: Pride

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Publications

Heptad-Specific Phosphorylation of RNA Polymerase II CTD.

Schüller Roland R Forné Ignasi I Straub Tobias T Schreieck Amelie A Texier Yves Y Shah Nilay N Decker Tim-Michael TM Cramer Patrick P Imhof Axel A Eick Dirk D

Molecular cell 20160101 2

The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures domin ...[more]

PMID: 26799765

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Project description:Missense mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines. In contrast to wild-type p53, the mutant p53 (mutp53) protein has lost the transcriptional activity towards pro-apoptotic and growth arrest genes, but retained the property to interact with DNA in a structure-specific fashion. Expression of mutp53 is advantageous for tumor cells, however the molecular mechanism of mutp53 action is still not known. We used the glioblastoma-derived U-251 MG human cell line to analyze DNA binding of mutant p53 (R273H mutation) on a Nimblegen custom 135k tiling array and to correlate mutp53 binding regions with the epigenetic state and occupation by other transcription factors (ETS1 and SP1). We found that mutp53-binding regions are G/C-rich and are located around transcriptional start sites (TSS) of many protein-coding genes, which in most cases are active, but are not always regulated upon transient mutp53 depletion. We propose a model which does not only rely on interactions of mutp53 with diverse transcriptional regulators at active promoters, but primarily is based on a DNA binding activity of mutp53. We designed a Nimblegen custom 135k tiling array that covers a large set of putative and known p53 (wild-type and mutant) target genes in the human genome. For analysis of the epigenetic state of genes covered by the tiling array in control and mutant p53-depleted U251 cells we focused on changes in active histone marks, H3K4me3 and H3K9Ac, and RNA polymerase II recruitment and processivity. H3K4me3 and H3K9Ac marks are enriched in active promoter regions and the phosphorylation of serine 5 (S5-P) and serine 2 (S2-P) in the CTD of RNA polymerase II have been described to define initiated and elongating complexes, respectively. We performed the ChIP-chip experiments for H3K4me3, H3K9Ac, RNA polymerase II (S5-P) and RNA polymerase II (S2-P) from U251 cells transfected with p53-specific siRNA or control siRNA (2 biological replicates each). To analyze binding of mutant p53 to the genes covered by the tiling array we performed mutant p53 ChIP-chip experiments in 4 biological replicates of. In addition, we analyzed the distribution of SP1 and ETS1 binding sites in 3 biological replicates.

Dataset Information

Heptad-specific phosphorylation of RNA polymerase II CTD

Publications

Heptad-Specific Phosphorylation of RNA Polymerase II CTD.

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