Spt6 Regulates Intragenic and Antisense Transcription, Nucleosome Positioning, and Histone Modifications Genome-Wide in Fission Yeast

Abstract

Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the roles of Spt6, we performed genome-wide analyses of transcription, chromatin structure, and histone modifications in a Schizosaccharomyces pombe spt6 mutant. Our results demonstrate dramatic changes to transcription and chromatin structure in the mutant, including elevated antisense transcripts at >70% of all genes and general loss of the ؉1 nucleosome. Furthermore, Spt6 is required for marks associated with active transcription, including trimethylation of histone H3 on lysine 4, previously observed in humans but not Saccharomyces cerevisiae, and lysine 36. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones. Studies over the last few years have revealed that transcription across eukaryotic genomes is much more widespread and complex than previously believed (1). Although it was once thought that transcription occurs primarily across protein-coding regions, genome-wide studies have now shown that transcription is also prevalent in intergenic regions and on antisense strands, in organisms ranging from yeast to humans (2, 3). Although roles for a small amount of this transcription have been established, for most, we have little understanding of its biological functions. Furthermore, while some factors have been shown to control the level of noncoding and antisense transcripts, many questions remain regarding the regulation of their synthesis and stability.One factor that plays a prominent role in the genome-wide control of transcription is Spt6. Originally identified in Saccharomyces cerevisiae (4, 5), Spt6 is conserved throughout eukaryotes and also has homology to the prokaryotic activator Tex (6). Spt6 interacts directly with several important factors, including RNA polymerase II (RNAPII) (7-11), histones (12, 13), and the transcription factor Iws1/Spn1 (7,14,15), suggesting that it is multifunctional. Recent studies in mammalian cells show that Spt6 also interacts directly with other chromatin related factors, including H3K27 demethylases (16, 17). Several gene-specific studies have demonstrated roles for Spt6 in transcription initiation (18)(19)(20), elongation (21, 22), and termination (23, 24). In addition, Spt6 is required for H3K36 methylation (25-28) and regulates nucleosome positioning and occupancy, particularly over highly expressed genes (12,19,29). Finally, Spt6 can assemble nucleosomes in vitro in an ATP-independent fashion (12). These results suggest that Spt6 acts as a histone chaperone by restoring nucleosomes in the wake of RNAPII transcription (30,31).In vivo, Spt6 is critical for normal growth and transcription. It is either essential or nearly essential for viability in all organisms tested, and viable spt6 mutations cause severe defects. In S. cerevisiae spt6 mutants,...

A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin

“…This resulted in comprehensive maps of HA-tagged H3, which were synthesized before the genetic switch, and T7-tagged H3, which was produced after the genetic switch. Comparison with MNase-seq data (DeGennaro et al 2013) revealed that ChIP-exo identified nucleosome peaks at a similar resolution (Supplemental Fig. S1A).…”

Native elongating transcript sequencing reveals global anti-correlation between sense and antisense nascent transcription in fission yeast

“…In addition, it is required for H3K4me3 and H3K36me3 (DeGennaro et al 2013). As genes with asXUTs display reduced levels of both histone modifications, we investigated whether asXUTs' expression is controlled by Spt6.…”

“…To test this, we analyzed published RNA-seq data from WT and spt6-1 cells, shifted for 2 h at 37°C (DeGennaro et al 2013). We observed significant overexpression for 871 XUTs upon heat inactivation of Spt6 (Supplemental Fig.…”

“…Spt6 is a conserved histone chaperone that was shown to modulate intragenic and antisense transcription in fission yeast (DeGennaro et al 2013). In addition, it is required for H3K4me3 and H3K36me3 (DeGennaro et al 2013).…”

“…Previous transcriptomics analyses in S. pombe have identified hundreds of aslncRNAs, thereby providing several important and pioneer characterizations of antisense transcription in this organism (Dutrow et al 2008;Wilhelm et al 2008;Ni et al 2010;Rhind et al 2011;DeGennaro et al 2013;Eser et al 2016). Some of these aslncRNAs respond to sexual differentiation (Bitton et al 2011) or to environmental stress (Leong et al 2014).…”