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Abstract
Compared to other stages in the RNA polymerase II transcription cycle, the role of chromatin in transcription termination is poorly understood. We performed a genetic screen in Saccharomyces cerevisiae to identify histone mutants that exhibit transcriptional readthrough of terminators. Amino acid substitutions identified by the screen map to the nucleosome DNA entry-exit site. The strongest H3 mutants revealed widespread genomic changes, including increased sense-strand transcription upstream and downstream of genes, increased antisense transcription overlapping gene bodies, and reduced nucleosome occupancy particularly at the 3' ends of genes. Replacement of the native sequence downstream of a gene with a sequence that increases nucleosome occupancy in vivo reduced readthrough transcription and suppressed the effect of a DNA entry-exit site substitution. Our results suggest that nucleosomes can facilitate termination by serving as a barrier to transcription and highlight the importance of the DNA entry-exit site in broadly maintaining the integrity of the transcriptome.
Highlights
Packaging of the eukaryotic genome into chromatin presents a regulatory barrier to DNA templated processes
To investigate a role for chromatin structure in regulating transcription termination, we performed a genetic screen for amino acid substitutions in histones H3 and H4 that cause defective transcription termination of a reporter construct containing the 70 bp NNS-dependent termination element of the SNR47 gene (Carroll et al, 2004)
Using an unbiased genetic screen of a comprehensive histone mutant library (Nakanishi et al, 2008) and a well-established termination reporter (Carroll et al, 2004), we identified residues in the DNA entry-exit site of the nucleosome required for transcription termination in vivo
Summary
Packaging of the eukaryotic genome into chromatin presents a regulatory barrier to DNA templated processes. To faithfully express protein-coding genes as well as noncoding regions of the genome, RNA polymerase II (Pol II) employs a host of regulatory factors. Among these factors are enzymes that post-translationally modify histones with small chemical moieties (Lawrence et al, 2016), histone chaperones that maintain chromatin organization in the wake of Pol II (Hammond et al, 2017), and chromatin remodelers that can reposition, exchange, or remove histones from the DNA template (Clapier et al, 2017). The mechanisms by which these factors modify chromatin to facilitate or impede transcription initiation and elongation are the subject of much investigation. Little is understood about how chromatin structure affects transcription termination
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