RESUMO
Genomes from yeast to humans are subject to pervasive transcription. A single round of pervasive transcription is sufficient to alter local chromatin conformation, nucleosome dynamics and gene expression, but is hard to distinguish from background signals. Size fractionated native elongating transcript sequencing (sfNET-Seq) was developed to precisely map nascent transcripts independent of expression levels. RNAPII-associated nascent transcripts are fractionation into different size ranges before library construction. When anchored to the transcription start sites (TSS) of annotated genes, the combined pattern of the output metagenes gives the expected reference pattern. Bioinformatic pattern matching to the reference pattern identified 9542 transcription units in Saccharomyces cerevisiae, of which 47% are coding and 53% are noncoding. In total, 3113 (33%) are unannotated noncoding transcription units. Anchoring all transcription units to the TSS or polyadenylation site (PAS) of annotated genes reveals distinctive architectures of linked pairs of divergent transcripts approximately 200nt apart. The Reb1 transcription factor is enriched 30nt downstream of the PAS only when an upstream (TSS -60nt with respect to PAS) noncoding transcription unit co-occurs with a downstream (TSS +150nt) coding transcription unit and acts to limit levels of upstream antisense transcripts. The potential for extensive transcriptional interference is evident from low abundance unannotated transcription units with variable TSS (median -240nt) initiating within a 500nt window upstream of, and transcribing over, the promoters of protein-coding genes. This study confirms a highly interleaved yeast genome with different types of transcription units altering the chromatin landscape in distinctive ways, with the potential to exert extensive regulatory control.
Assuntos
Saccharomyces cerevisiae , Transcrição Gênica , Humanos , Saccharomyces cerevisiae/genética , Cromatina , Fatores de Transcrição/genética , Regiões Promotoras GenéticasRESUMO
In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change.