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Genome-wide analysis of Gtf3c1, RNAP3 occupancy and H3K4me1, H3K27ac chromatin modifications in primary mouse cortical neurons

ABSTRACT: SINEs are a highly abundant class of retrotransposons that were initially considered a non-functional “junk” DNA due to their non coding, repetitive nature. SINEs frequently possess an internal RNAPIII promoter, which recruits the RNAPIII transcriptional machinery, including the general nuclear complex TFIIIC. We recently discovered that in response to neuronal depolarisation, SINEs may represent a novel class of neuronal enhancers and reasoned that RNAPIII and TFIIIC binding may provide a means of identifying enhancer SINEs genome-wide. To this end, we first performed ChIPseq analysis on resting and depolarised mouse cortical neurons, using an antibody that targets the DNA binding subunit of TFIIIC (Gtf3c1), RNA Polymerase III and the enhancer markers H3K4me1 and H3K27ac .

ORGANISM(S): Mus musculus

PROVIDER: GSE75191 | GEO | 2017/12/05

SECONDARY ACCESSION(S): PRJNA302756

REPOSITORIES: GEO

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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.

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Genome-wide analysis of Gtf3c1, RNAP3 occupancy and H3K4me1, H3K27ac chromatin modifications in primary mouse cortical neurons

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