Project description:RNA polymerase III transcribes many noncoding RNAs (e.g. tRNAs) important for translational capacity and other functions. Here, we localized RNA polymerase III, alternative TFIIIB complexes (BRF1/2) and TFIIIC in HeLa cells, determining the Pol III transcriptome, defining gene classes, and revealing ‘TFIIIC-only’ sites. Pol III localization in other transformed and primary cell lines revealed both novel and cell-type specific Pol III loci, and one occupied miRNA. Surprisingly, only a fraction of the in silico-predicted Pol III loci are occupied. Interestingly, many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer binding proteins such as ETS1 and STAT1. Remarkably, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. Taken together, active promoter and enhancer-like chromatin appears to gate Pol III accessibility to the genome. Use of ChIP-array to identify genomic regions bound by RNA Polymerase III machinery
Project description:We present a genome-wide map of RNA Polymerase III subunit localization in human THP-1 monocytes and THP-1 derived macrophages after 72 hr exposure to PMA, as well as profiles of POLR3G and POLR3GL occupancy in THP-1 monocytes after 4 hr exposure to Pol III drug inhibitor ML-60218; 27 uM
Project description:RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type–specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico–predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome. Use of ChIP-seq to identify genomic regions bound by RNA Polymerase III machinery in multiple cell types as well as RNA-seq in HeLa for gene expression analysis. See GSE20609 for whole human genome raw Pol III ChIP-array data. See link below for supplementary methods and analysis.
Project description:RNA polymerase (RNAP) is the key transcription machinery and its interaction with genomic DNA orchestrates gene expression in response to environmental cues. Dynamic interaction and localization between RNAP and nucleoid were observed before and after osmotic stress. Chromatin immunoprecipitation (ChIP) of RNAP β’ subunit together with chromatin profiling by ChIP-on-chip analysis demonstrated the dynamics of genome-wide RNAP-DNA interactions during osmotic stress.
Project description:SNAPc-dependent promoters are unique among cellular promoters in being very similar to each other, even though some of them recruit RNA polymerase II and other RNA polymerase III. We have examined all SNAPc-bound promoters present in the human genome. We find that there is a surprisingly small number of them, some 70 promoters. Among these, the large majority is bound by either RNA polymerase II or RNA polymerase III, as expected, but one gene hitherto considered an RNA polymerase III gene is also occupied by significant levels of RNA polymerase II, which synthesizes an RNA distinct from that synthesized by RNA polymerase III. Both RNA polymerase II and RNA polymerase III SNAPc- dependent promoters use a largely overlapping set of a few transcription activators, including GABP, a novel factor implicated in snRNA gene transcription.
Project description:tRNA genes are transcribed by RNA polymerase III (RNAPIII). During recent years it has become clear that RNAPIII activity is strictly regulated by the cell in response to environmental cues and the homeostatic status of the cell. However, the molecular mechanisms that control RNAPIII activity to regulate the amplitude of tDNA transcription in normally cycling cells are not well understood. Here, we show that tRNA levels fluctuate during the cell cycle and reveal the underlying molecular mechanism. The cyclin Clb5 recruits the cyclin dependent kinase Cdk1 to tRNA genes to boost tDNA transcription during S phase. At tDNA genes, Cdk1 promotes the recruitment of TFIIIC, stimulates the interaction between TFIIIB and TFIIIC, and increases the dynamics of RNA polymerase III in vivo. Furthermore, we identified Bdp1 as an important Cdk1 substrate in this process. Preventing Bdp1 phosphorylation prevented cell cycle-dependent recruitment of TFIIIC and abolished the cell cycle-induced increase in tDNA transcription. Our findings demonstrate that under optimal growth conditions Cdk1 gates tRNA synthesis in S phase by regulating the RNAPIII machinery, revealing a direct link between the cell cycle and RNAPIII activity.