Genome-wide transcriptional response to genetic and environmental variations. (A) Four examples of sense–antisense transcript pairs (three anti-correlated and one positively correlated). Expression data are displayed along the chromosome (x axis) for the Watson (W, upper half) and the Crick (C, lower half) strands. Normalized signal intensities (higher in dark) are shown for 24 out of 48 segregants, randomly selected and ordered (y axis). Vertical red lines represent inferred transcript boundaries. Genome annotations are shown in the center: annotated ORFs (blue boxes), their mapped UTRs (dashed grey lines), SUTs (orange boxes) and transcript start sites (arrows). (B) Expression data along 15 kb of chromosome VIII across three replicates each for yeast grown in synthetic complete media with glucose (SDC), rich media with galactose (YPGal) and with ethanol (YPE); and three rows (summarizing nine replicates) for yeast grown in rich media with glucose (YPD; Xu et al, 2009).

Sense expression variability depends on TSS overlap. (A) Overlap of sense–antisense pairs. d₁ (x axis) is the distance (base pairs) of the 3′ end of the antisense SUT to the TSS of the sense ORF-T and is positive if the 3′ end of the antisense extends beyond the sense TSS. d₂ (y axis) is the distance (base pairs) of the 3′ end of the sense ORF-T to the TSS of the antisense SUT. The panel shows the scatterplot of d₁ and d₂ and the histograms their marginal distributions, with dashed grey lines marking the modes of the distribution. (B) Variability across segregants for genes grouped by type of sense–antisense overlap. Mean of the expression standard deviation across the 48 segregants for ORF-Ts without antisense SUT (blue), with antisense SUT not overlapping the TSS (pink) and with antisense SUT overlapping the TSS (red). Error bars indicate standard error of the mean. The number of ORF-Ts in each category is shown below the bar.

Model of antisense-mediated regulation. The sense gene (red dashed line, coding sequence as blue box) and the antisense SUT (green dashed line) typically extend beyond the TSS of each other. In the absence of sufficiently strong activating signals on the sense promoter, the antisense is expressed and inhibits sense expression (left T-shaped arrow). Upon activation of the sense promoter, the inhibition from the antisense SUT is relaxed, possibly through reciprocal inhibition of sense on antisense expression (right T-shaped arrow). This leads to threshold behaviour of gene regulation, where the gene is switched-off unless activation reaches a certain threshold.

Antisense expression associates with larger gene expression variability. (A–D) Expression variability of ORF-Ts with or without antisense. (A) Mean of the log₂ expression standard deviation across all segregants and (B) across environmental conditions, (C) mean of the expression divergence across five yeast species as provided by Tirosh et al (2006) and (D) mean of cell-to-cell protein expression variability (DM as provided by Newman et al (2006)) for ORF-Ts without antisense (blue) and ORF-Ts with antisense (red). Error bars indicate standard error of the mean. The number of ORF-Ts in each category is shown below the bar. (E) Q-Q plots of expression levels of ORF-Ts with and without antisense. The Q-Q plot compares two distributions by plotting for every quantile the two corresponding expression values against each other (expression level for ORF-Ts without antisense (x axis) against those for ORF-Ts with antisense (y axis)). Two data sets with the same distribution would align on the diagonal (grey line). ORF-Ts with antisense have lower expression levels (below the diagonal, bottom left) for the small quantiles, and similar expression levels for the large quantiles (top right). The shade around the curve (black line) represents bootstrap standard deviation (Materials and methods). (F, G) Smoothed histograms (distribution density estimates) of minimum (F) and maximum (G) expression values across the 48 segregants for ORF-Ts with antisense (red) and without (blue). Vertical line at x=0 indicates our threshold for calling a transcript expressed.

Antisense-mediated regulation of SUR7. (A) Expression maps around the SUR7–GAL80 locus. Expression data is displayed along the chromosome (x axis) for the Watson (W, upper half) and the Crick (C, lower half) strands. Normalized signal intensities (higher in dark) are shown for the profiled samples (y axis, bottom-up): wild-type strain grown in YPD (WT_YPD1-3), wild-type strain grown in YPGal (WT_YPGal1-3), mutant strain with Gal4 binding site knocked out grown in YPD (dAS_YPD1-3), mutant strain with Gal4 binding site knocked out grown in YPGal (dAS_YPGal1-3), all for three biological replicates and without α-factor stimulation. The next 12 samples (bottom up) follow the same order, but with α-factor stimulation. Vertical red lines represent inferred transcript boundaries. Nucleosome positions (green tracks, darker for more significant scores; Mavrich et al (2008)) and genome annotations are shown in the center: annotated ORFs (blue boxes), their mapped UTRs (dashed grey lines), SUTs (orange boxes) and transcript start sites (arrows). (B) Mean of the log₂ expression levels of SUR7 across three biological replicates for strains grown in YPGal, with (left) or without (right) α-factor stimulation for the wild-type strain (orange) or the antisense-disrupted strain (green). Values of individual samples are shown by open circles. (C) Mean of the log₂ expression levels of SUR7 across three biological replicates for strains under α-factor stimulation grown in YPD (purple) or YPGal (turquoise), for the wild-type (left) or the antisense-disrupted strain (right). Values of individual samples are shown by open circles. (D) Model for spreading of regulatory signals on the SUR7–GAL80 locus. SUR7 and GAL80 are two ORFs (blue boxes) in tandem configuration. The promoter of GAL80 is bidirectional and initiates SUT719, a transcript antisense to SUR7. Regulatory signals in response to galactose media (Gal4 transcription factor, green oval) upregulate expression of GAL80 as well as the antisense SUT719, thereby repressing SUR7.