Figure 2.

Evolutionarily conserved residues of ScSub1 ssDBD are crucial for its DNA binding capacity in vivo. (A) Analysis of Sub1-6HA levels in wt (Sub1-6HA), sub1Δ, and ssDBD mutants (sub1-K45A, sub1-Y66A) cells by Western blot. Levels of Pgk1 were used as a control of loaded proteins. (B) Yeast strains with the indicated genotypes were spotted onto solid media plates (1:10 serial dilutions) and grown at 28 °C or 37 °C for 2–3 days. spt4Δ cells were used as a control for cell growth and thermo-sensitivity defects. (C) ChIP analyses of Sub1-HA in the wt, sub1∆, and ssDBD mutants (sub1-K45A, sub1-Y66A and sub1-FRN54-56AGG). Sub1 occupancy at the 5′ region of two constitutively expressed genes, PMA1 and PYK1, and the induced gene, IMD2, were examined by qPCR, and quantifications were graphed (see Materials and Methods). The numbers on the Y-axis represent the occupancy of Sub1 at the promoters in mutant cells relative to wt cells, in which occupancy is considered 100%. (E) ChIP analysis of wt and sub1∆CT cells expressing Sub1-6HA, as in (D). In this case, four constitutively transcribed genes were examined: PGK1, PMA1, PYK1, and YEF3, as well as IMD2. (E) Left: co-immunoprecipitation assays (Co IP) to estimate Sub1-HA levels in wt and sub1ΔCT cells; whole cell extracts (WCE) were prepared from strains expressing Sub1-6HA, and either 1 mg or 10 mg of WCE were used to immunoprecipitate Sub1 from wt or sub1ΔCT cells, respectively. Immunoprecipitated (α-HA) or inputs (IN)—0.1 mg and 1 mg, respectively—were loaded onto a 12% SDS-PAGE gel and immunoblotted with anti-HA. Right: chemiluminescence of immunoreactive bands from Western blots were quantified and graphed. (F) Ratio ChIP/protein levels with values obtained in D and E, respectively. In all cases, the mean and standard deviation values were calculated from at least three independent experiments. Statistically significant levels are shown where ns = non-significant, ** = p < 0.01, *** = p < 0.001.