Figure 2: The role of Fun30^Fft3 in regulating RNAPII occupancy largely overlaps with that of Spt16.

(a) A spotting assay was used to assess the genetic interactions between Fun30^Fft3 and transcription elongation factors such as Spt16, Paf1 and Spt6. Serial dilutions (5-fold) of wild-type, fft3Δ, spt16-ts (spt16-1 and spt16-2), paf1Δ, spt6-1, fft3Δ spt16-ts (spt16-1 and spt16-2), fft3Δ paf1Δ and fft3Δ spt6-1 cells were spotted on rich medium and grown at the permissive (25 °C) and semi-permissive (30 °C) temperatures of spt16-ts cells. Cells grown at 25°C were incubated for an additional day to allow full growth. (b) Venn diagram depicting the overlap of genes showing decreased RNAPII occupancy at transcribing regions (log₂ fold-change≤−0.5) in fft3Δ versus spt16-1 cells. P values for the significance of list overlap were calculated according to hypergeometric distribution. (c) Boxplots of RNAPII occupancy changes at transcribing regions of fft3Δ, spt16-1, and fft3Δ spt16-1 cells for the overlapping genes identified in (b). The upper and lower whiskers extend from the upper and lower hinges to the highest and the lowest value that are within 1.5 × interquartile range (IQR), and the dots represent outliers. P values were calculated according to Tukey’s multiple comparison test. NS, P≥0.1; ***P≤0.001. (d) Venn diagram depicting the overlap of genes which show high Fun30^Fft3 occupancies at transcribing regions and those which show decreased RNAPII occupancies (log₂ fold-change≤−0.5) in both fft3Δ cells and spt16-1 cells (right) or only in fft3Δ cells (left). P values for the significance of list overlap were calculated according to hypergeometric distribution. The Fun30^Fft3 occupancy profiles in wild-type cells grown at 30 °C were subject to k-means clustering (k=3) to determine genes with high Fun30^Fft3 occupancy.