Three distinct mechanisms of long-distance modulation of gene expression in yeast

Abstract

Recent Hi-C measurements have revealed numerous intra- and inter-chromosomal interactions in various eukaryotic cells. To what extent these interactions regulate gene expression is not clear. This question is particularly intriguing in budding yeast because it has extensive long-distance chromosomal interactions but few cases of gene regulation over-a-distance. Here, we developed a medium-throughput assay to screen for functional long-distance interactions that affect the average expression level of a reporter gene as well as its cell-to-cell variability (noise). We ectopically inserted an insulated MET3 promoter (MET3pr) flanked by ~1kb invariable sequences into thousands of genomic loci, allowing it to make contacts with different parts of the genome, and assayed the MET3pr activity in single cells. Changes of MET3pr activity in this case necessarily involve mechanisms that function over a distance. MET3pr has similar activities at most locations. However, at some locations, they deviate from the norm and exhibit three distinct patterns including low expression / high noise, low expression / low noise, and high expression / low noise. We provided evidence that these three patterns of MET3pr expression are caused by Sir2-mediated silencing, transcriptional interference, and 3D clustering. The clustering also occurs in the native genome and enhances the transcription of endogenous Met4-targeted genes. Overall, our results demonstrate that a small fraction of long-distance chromosomal interactions can affect gene expression in yeast.

Heat Shock Factor 1 Drives Intergenic Association of Its Target Gene Loci Upon Heat Shock

“…As alluded above, a particularly striking aspect of our study is that constitutively active genes (PAU17 and FRA1), despite located in close linear proximity to HSP genes, do not coalesce with them. Such specificity contrasts with a recent report of methionineresponsive genes in yeast that engage in intrachromosomal clustering upon their induction as assessed by 3C, yet unlike what we observed here, unrelated neighboring genes also tended to interact (Du et al, 2017). More similar to the specificity and selectivity of Hsf1-target gene coalescence are observations that TNFα-responsive genes in human endothelial cells engage in intrachromosomal interactions upon cytokine stimulation (Papantonis et al, 2010), whereas an actively transcribed gene interposed between them, and located nearby to one of them, is excluded from such colocalization (Fanucchi et al, 2013).…”

3D clustering of co-regulated genes and its effect on gene expression

“…Du et al developed a medium-throughput assay to screen for functional long-distance interactions that affect the expression of a reporter gene in the budding yeast genome (Du et al 2017). An insulated MET3 promoter flanked by ∼1 kb invariable sequences was integrated into thousands of genomic loci, allowing it to make contacts with different parts of the genome.…”

“…For example, among the insertion sites tested, GAL1 reporters only interact when they are at allelic locations, or on non-homologous chromosomes but have equal distance to centromere (Mirkin et al 2013; Zhang and Bai 2016). Similarly, only a small fraction of Met4-targeted genes seems to cluster, and the MET3 reporter makes contacts with the cluster only when inserted into certain loci (Du et al 2017). These results indicate that the search for interaction partners is constrained to a nuclear sub-volume imposed by the chromosome context (Noordermeer et al 2011).…”

Enhancement of LacI binding in vivo

“…We used the instrumentation and data acquisition platform as described in a previous study (13). Briefly, cells were grown in synthetic complete (SCD) liquid media at 30°C to an OD 660 ∼0.2, washed, diluted to an OD 660 ∼0.1, and then transferred onto a coverslip with a SCD agarose pad.…”