Role of histone modifications and early termination in pervasive transcription and antisense-mediated gene silencing in yeast

Manuele Castelnuovo,Jurgi Camblong,Françoise Stutz,Elisa Guffanti,Nicholas M Luscombe,Judith B Zaugg,Zhenyu Xu,Lars M Steinmetz,Andrea Maffioletti,Sandra Clauder-Münster

doi:10.1093/nar/gku100

Abstract

Most genomes, including yeast Saccharomyces cerevisiae, are pervasively transcribed producing numerous non-coding RNAs, many of which are unstable and eliminated by nuclear or cytoplasmic surveillance pathways. We previously showed that accumulation of PHO84 antisense RNA (asRNA), in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1. Here we investigate this process genome-wide and measure the whole transcriptome of various histone modification mutants in a Δrrp6 strain using tiling arrays. We confirm widespread occurrence of potentially antisense-dependent gene regulation and identify three functionally distinct classes of genes that accumulate asRNAs in the absence of Rrp6. These classes differ in whether the genes are silenced by the asRNA and whether the silencing is HDACs and histone methyl transferase-dependent. Among the distinguishing features of asRNAs with regulatory potential, we identify weak early termination by Nrd1/Nab3/Sen1, extension of the asRNA into the open reading frame promoter and dependence of the silencing capacity on Set1 and the HDACs Hda1 and Rpd3 particularly at promoters undergoing extensive chromatin remodelling. Finally, depending on the efficiency of Nrd1/Nab3/Sen1 early termination, asRNA levels are modulated and their capability of silencing is changed.

Highlights

The development of high-density tiling arrays and large-scale RNA sequencing approaches revealed that all eukaryotic genomes are pervasively transcribed and synthesize a myriad of non-coding RNAs
We previously showed that accumulation of PHO84 antisense RNA, in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1
To identify genes regulated by antisense ncRNAs accumulating in the absence of Rrp6 and to characterize the mechanisms by which antisense transcription affects gene expression, we performed tiling array expression profiling of strains lacking Rrp6 alone or in combination with disruptions of histone modification enzymes previously implicated in antisense-mediated regulation of PHO84: the H3K4 methyl transferase Set1 and the Hda1/2/3 HDAC complex subunit Hda2

Summary

Introduction

The development of high-density tiling arrays and large-scale RNA sequencing approaches revealed that all eukaryotic genomes are pervasively transcribed and synthesize a myriad of non-coding RNAs (ncRNAs). The yeast Saccharomyces cerevisiae produces a large number of long ncRNAs between 200 up to a few thousand bases in length, comprising both intergenic RNAs and transcripts antisense to coding open reading frames (ORFs). Antisense transcripts (asRNAs) have been divided into stable unannotated transcripts (SUTs) detected in wild-type (WT) cells and cryptic unstable transcripts (CUTs), which are rapidly degraded and detectable only in mutants of the nuclear exosome [2,3]. The degradation of CUTs by the 30–50 exonuclease Rrp is tightly linked to transcription termination by the Nrd1/Nab3/Sen (NNS) complex and polyadenylation by the TRAMP.

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