Antisense non-coding transcription represses the PHO5 model gene at the level of promoter chromatin structure

Abstract

Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis, and use of the CRISPRi system. Furthermore, we showed that enhanced elongation of PHO5 antisense leads to a more repressive chromatin conformation at the PHO5 gene promoter, which is more slowly remodeled upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated upon inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, the data in this work reveal an additional level in the complex regulatory mechanism of PHO5 gene expression by showing antisense transcription-mediated repression at the level of promoter chromatin structure remodeling.

Alternative TSS use is widespread inCryptococcusfungi in response to environmental cues and regulated genome-wide by the transcription factor Tur1

“…This change in transcription factor usage could be due to a change in chromatin structure due to the increase in TSS1 usage observed during this phase of growth. Indeed, modification of the transcriptional sense or antisense can alter the chromatin structure allowing TF binding as previously shown (18,74). It is important to say that the present model is probably an oversimplification of what happens during the ten hours between the exponential and stationary time points during which the switch is TSS usage can be observed.…”

Streamlining N-terminally anchored yeast surface display via structural insights into S. cerevisiae Pir proteins

“…S. cerevisiae strains carrying reporter plasmids were grown at 30 °C/180 rpm in chemically defined media without histidine (-his: 6.70 g/l Difco Yeast nitrogen base without amino acids, 20.0 g/l glucose, 1.6 g/l drop-out without histidine, and 20.0 g/l agar for solid media). To induce the PHO5 promoter, yeast cells were first grown in a repressive and then in a permissive medium, i.e., in -his medium supplemented with 1 g/l of KH 2 PO 4 and then in -his medium lacking all phosphate sources, respectively, as in Novačić, et al [ 43 ].…”

Boosting N-terminally anchored yeast surface display via structural insights intoS. cerevisiaePir proteins

“…S. cerevisiae strains carrying reporter plasmids were grown at 30 °C/180 rpm in chemically defined media without histidine (-his: 6.70 g/l Difco Yeast nitrogen base without amino acids, 20.0 g/l glucose, 1.6 g/l drop-out without histidine, and 20.0 g/l agar for solid media). To induce the PHO5 promoter, yeast cells were first grown in a repressive and then in a permissive medium, i.e., in -his medium supplemented with 1 g/l of KH2PO4 and then in -his medium lacking all phosphate sources, respectively, as in Novačić et al (2022).…”