The authors set out to devise an approach that would allow them to detect nascent transcripts in unperturbed cells. They immunoprecipitated tagged RNA polymerase II (RNAPII) from Saccharomyces cerevisiae and then did deep sequencing of 40 nucleotides from the 3′ end of the co-purified RNA. By using the density of these sequences at each position, they could determine the density of RNAPII at each site. Several features suggested the detection of nascent transcripts, including the presence of introns and regions beyond the polyadenylation sites. The ability of this technique to detect transcripts irrespective of their stability allowed the authors to ask whether antisense and sense transcription is tightly coordinated. They found that, although transcription was often divergent, in most cases there was more sense transcription than antisense transcription.
So, what determines the direction of transcription? The authors saw a correlation between antisense transcription and earlier increased histone H4 acetylation, and therefore tested whether this was important for directionality. Deletion of a key subunit of the Rpd3 small (Rpd3S) H4 deacetylation complex increased unstable antisense transcription during initiation, and this effect also required the histone methyltransferase SET domain-containing 2 (Set2). Together with previous studies, this suggested that Rpd3S is recruited to sites of Set2-mediated methylation, where it suppresses antisense transcription initiation by mediating histone de-acetylation.
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