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High-resolution mapping of transcribing Pol III by CRAC in the WT and C128 mutants

ABSTRACT: High-resolution mapping of transcribing Pol III by CRAC in the WT and C128 mutants

PROVIDER: PRJNA804707 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR17954814.fastq.gz	Fastqsanger.gz
	SRR17954815.fastq.gz	Fastqsanger.gz
	SRR17954816.fastq.gz	Fastqsanger.gz
	SRR17954817.fastq.gz	Fastqsanger.gz
	SRR17954818.fastq.gz	Fastqsanger.gz

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High-resolution mapping of transcribing Pol III by CRAC in the WT and C128 mutants

Project description:Here we report high-resolution analyses of transcribing RNA polymerase III (Pol III) in a wild-type (WT) strain and three C128 mutants. C128 is the second largest subunit of Pol III, which is the key subunit to recognize termination signal. Mutating key residues in C128 interferes with transcription termination in vitro and affect yeast viability as well, raising the question that whether these mutations provoke Pol III termination defects in vivo. Here we show that C128 mutants display termination defects at most PolIII-dependent genes, indicating that the most disruptive C128 mutations indeed impair Pol III transcription termination in vivo

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High-resolution mapping of transcribing RNAPIII by CRAC upon depletion of Nrd1 [Experiment 2]

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Project description:The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is associated with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here that antisense RNA Polymerase II (Pol II) transcription is critical to prime and maintain nucleosome depletion at Pol III loci and allow efficient Pol III recruitment upon re-initiation of growth from stationary phase. Antisense Pol II is recruited by the Pcr1 transcription factor, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.

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MAF1 Represses CDKN1A through a Pol III-Dependent Mechanism

Project description:MAF1 represses Pol III-mediated transcription by interfering with TFIIIB and Pol III. Herein, we found that MAF1 knockdown induced CDKN1A transcription and chromatin looping concurrently with Pol III recruitment. Simultaneous knockdown of MAF1 with Pol III or BRF1 (subunit of TFIIIB) diminished the activation and looping effect, which indicates that recruiting Pol III was required for activation of Pol II-mediated transcription and chromatin looping. ChIP analysis after MAF1 knockdown indicated enhanced binding of Pol III and BRF1, as well as of CFP1, p300, and PCAF, which are factors that mediate active histone marks, along with the binding of TBP and POLR2E to the CDKN1A promoter. Simultaneous knockdown with Pol III abolished these regulatory events. Similar results were obtained for GDF15. Our results reveal a novel mechanism by which MAF1 and Pol III regulate the activity of a protein-coding gene transcribed by Pol II.

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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.

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