Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide localization of the RNA polymerase III transcription machinery in human cells

ABSTRACT: To characterize the pol III transcriptome in actively growing human cells, we used MR90hTeIrt lung fibroblasts, which are immortal but not transformed, with an intact Rb pathway. After formaldehyde cross-linking, chromatin was extracted and submitted to immunoprecipitation. To identify actively transcribed RNAP-III transcription units, we used antibodies directed against the RPC4 subunit of RNAP-III. To identify promoter regions, we used antibodies directed against Bdp1, which is part of both Brf1-TFIIIB and Brf2-TFIIIB, and thus should mark all types of active RNAP-III promoters. Antibodies directed against Brf1 were used to mark type 1 and 2 promoters. For type 3 promoters, we engineered an IMR90hTert cell line expressing the SNAPc subunit SNAP45 tagged at its C-terminal end with the biotin acceptor domain as well as the biotin ligase BirA and performed M-bM-^@M-^\chromatin affinity purificationM-bM-^@M-^] (ChAP) with streptavidin beads. In each case, the precipitated material was then processed for deep sequencing. ChIP-enriched DNA or ChAP-enriched DNA from MR90hTeIrt cells were analyzed by Illumina high-throughput sequencing. This analysis includes ChIP data done with antibodies against : RPC4, Bfr1, Bdp1 and SNAP45. Two lanes were run for RPC4, Brf1 and SNAP45, and three for Bdp1. One lane was run with Input DNA for control.

ORGANISM(S): Homo sapiens

SUBMITTER: Viviane Praz

PROVIDER: E-GEOD-18184 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells.

Canella Donatella D Praz Viviane V Reina Jaime H JH Cousin Pascal P Hernandez Nouria N

Genome research 20100422 6

Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Po ...[more]

PMID: 20413673

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Project description:RNA polymerase (RNA Pol) III synthesizes the tRNAs, the 5S ribosomal RNA and a small number of untranslated RNAs. In vitro, it also transcribes short interspersed nuclear elements (SINEs). We investigated the distribution of RNA Pol III and its associated transcription factors on the genome of mouse embryonic stem (ES) cell using a highly specific tandem ChIP-Seq method. Only a subset of the annotated class-III genes was bound and thus transcribed. A few hundred SINEs were associated with the RNA Pol III transcription machinery. We observed that RNA Pol III and its transcription factors were present at thirty unannotated sites on the mouse genome, only one of which was conserved in human. An RNA was associated with more than 80% of these regions. More than 2200 regions bound by TFIIIC transcription factor were devoid of RNA Pol III. These sites are correlated with association of CTCF and the cohesin. Cohesin has been shown to occupy sites bound by CTCF and to contribute to DNA loop formation associated with gene repression or activation. This observation suggests that TFIIIC may play a role in chromosome organization in mouse. We also investigated the genome-wide distribution of the ubiquitous TFIIS variant, TCEA1. We found that, as in Saccharomyces cerevisiae, TFIIS is associated with class III genes and also with SINEs suggesting that TFIIS is a RNA Pol III transcription factor in mammals. We performed ChIP-seq experiment on mouse ES cells, in order to analyse the distribution of the RNA Pol III, with two of its subunits, RPC1 and RPC4, of the two distinct forms of the transcription factor TFIIIB, with BRF1 and BRF2, respectively subunit of TFIIIB-beta, and TFIIIB-alpha form, and three subunits of the transcription factor TFIIIC, TFIIIC90, TFIIIC110, TFIIIC220. We also analysed the distribution of the RNA Pol II elongation factor TCEA1. We used tagged proteins, in order to develop a highly specific and generic ChIP-seq protocol. A sequence encoding a 6 histidine-Flag-HA tag was inserted just after the last codon of the gene encoding proteins of the RNA Pol III machinery subunits, or just after the start codon for TCEA1, using the recombineering technology. Untagged ES cell line was used as negative control for data processing. Our dataset comprises of ten ChIP-seq samples, eight from tagged proteins, two from untagged cell line.

Dataset Information

Genome-wide localization of the RNA polymerase III transcription machinery in human cells

Publications

Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells.

Similar Datasets

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