A unique nucleosome arrangement, maintained actively by chromatin remodelers facilitates transcription of yeast tRNA genes

Abstract

BackgroundRNA polymerase (pol) III transcribes a unique class of genes with intra-genic promoters and high transcriptional activity. The major contributors to the pol III transcriptome, tRNAs genes are found scattered on all chromosomes of yeast. A prototype tDNA of <150 bp length, is generally considered nucleosome-free while some pol III-transcribed genes have been shown to have nucleosome-positioning properties.ResultsUsing high resolution ChIP-chip and ChIP-seq methods, we found several unique features associated with nucleosome profiles on all tRNA genes of budding yeast, not seen on nucleosome-dense counterparts in fission yeast and resting human CD4+ T cells. The nucleosome-free region (NFR) on all but three yeast tDNAs is found bordered by an upstream (US) nucleosome strongly positioned at −140 bp position and a downstream (DS) nucleosome at variable positions with respect to the gene terminator. Perturbation in this nucleosomal arrangement interferes with the tRNA production. Three different chromatin remodelers generate and maintain the NFR by targeting different gene regions. Isw1 localizes to the gene body and makes it nucleosome-depleted, Isw2 maintains periodicity in the upstream nucleosomal array, while RSC targets the downstream nucleosome. Direct communication of pol III with RSC serves as a stress-sensory mechanism for these genes. In its absence, the downstream nucleosome moves towards the gene terminator. Levels of tRNAs from different families are found to vary considerably as different pol III levels are seen even on isogenes within a family. Pol III levels show negative correlation with the nucleosome occupancies on different genes.ConclusionsBudding yeast tRNA genes maintain an open chromatin structure, which is not due to sequence-directed nucleosome positioning or high transcription activity of genes. Unlike 5′ NFR on pol II-transcribed genes, the tDNA NFR, which facilitates tDNA transcription, results from action of chromatin remodeler Isw1, aided by Isw2 and RSC. The RSC-regulated nucleosome dynamics at the 3′ gene-end serves as a novel regulatory mechanism for pol III transcription in vivo, probably by controlling terminator-dependent facilitated recycling of pol III. Salient features of yeast tDNA chromatin structure reported in this study can explain the basis of the novel non-transcriptional roles ascribed to tDNAs.

TORC1-dependent sumoylation of Rpc82 promotes RNA polymerase III assembly and activity

“…Meta-analysis of this dataset showed that SUMO overlaps the transcription unit of tRNA genes, forming a peak right at the transcription start site (TSS; Fig. 2B), resembling previously published meta-analyses of the chromatin localization of RNAPIII and its nascentome (14,15). These high-throughput data were validated by ChIP followed by quantitative PCR (ChIP-qPCR) using primers specific for a panel of RNAPIIItranscribed genes (Fig.…”

Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation

“…It is known that RNAP III-transcribed genes are free of nucleosomes during repression. 46 The much longer RNAP II-transcribed genes have developed intricate ways to regulate the activity of nucleosomes that occupy their lengths. The TFIIIB/C-tRNA gene complexes occupy a DNA length similar to nucleosomes, although perhaps more stably so.…”

tRNA biology in the omics era: Stress signalling dynamics and cancer progression

“…One of the most unexpected findings of ChIP-seq profiling in human cells was the large number of annotated loci devoid of Pol III [20,22,24,30], a situation that strictly differs from the uniform binding pattern observed in yeast [14][15][16][17]. In immortalised human fibroblasts (IMR90hTert) grown under multiple conditions, Pol III was absent from $32% of annotated loci [30], suggesting that a large fraction of Pol III genes is in a permanently repressed state.…”

“…Genome-wide analyses to identify in vivo Pol III binding sites were first carried out in S. cerevisiae by the mean of chromatin immunoprecipitation (ChIP). With this approach, DNA is cross-linked to Pol III or its general transcription factors (GTFs), immunoprecipitated, and subjected to microarray {ChIP-chip [14][15][16]} or next generation sequencing {ChIP-seq [17]} analysis. These experiments revealed Pol III binding at the vast majority of known targets and an overall correlation with transcription as determined by measurement of the corresponding RNA levels.…”