Howe, F. S. | bioRxiv Science

bioRxiv2017

Antisense transcription-dependent chromatin signature modulates sense transcription and transcript dynamics

Antisense transcription is widespread in genomes. Despite large differences in gene size and architecture, we find that yeast and human genes share a unique, antisense transcription-associated chromatin signature. We asked whether this signature is related to a biological function for antisense transcription. Using quantitative RNA-FISH, we observed changes in sense transcript distributions in nuclei and cytoplasm as antisense transcript levels were altered. To determine the mechanistic differences underlying these distributions, we developed a mathematical framework describing transcription from initiation to transcript degradation. At GAL1, high levels of antisense transcription alters sense transcription dynamics, reducing rates of transcript production and processing, while increasing transcript stability, which is also a genome-wide association. Establishing the antisense transcription-associated chromatin signature through disruption of the Set3C histone deacetylase activity is sufficient to similarly change these rates even in the absence of antisense transcription. Thus, antisense transcription alters sense transcription dynamics in a chromatin-dependent manner.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC=\"FIGDIR/small/187237_fig8.gif\" ALT=\"Figure 8\">\nView larger version (52K):\norg.highwire.dtl.DTLVardef@4f872corg.highwire.dtl.DTLVardef@1339662org.highwire.dtl.DTLVardef@1d62cc2org.highwire.dtl.DTLVardef@148f24_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract\n\nC_FIG In this work, Brown et al. provide a mechanistic understanding of the effect of antisense transcription on the production and fate of sense transcripts. Antisense transcription buffers genes against the action of the Set3 lysine deacetylase, thus altering rates of transcript production, processing and stability. O_LIConserved antisense transcription-dependent chromatin architecture near promoters\nC_LIO_LIAntisense transcription alters sense transcription dynamics and transcript stability\nC_LIO_LIAntisense transcription functions in a chromatin-dependent manner\nC_LIO_LIIncreased acetylation by set3{Delta} mimics high antisense transcriptional dynamics\nC_LI

systems biology↗

bioRxiv2017

CRISPRi is not strand-specific and redefines the transcriptional landscape

CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, binding of sgRNA/dCas9 creates an environment that is permissive for transcription initiation and termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9 binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.

genetics↗