Expression evolution in yeast genes of single-input modules is mainly due to changes in trans-acting factors

Both cis- and trans-regulatory mutations contribute to gene expression divergence within and between species. To estimate their relative contributions, we examined two yeast strains, BY4741 (a laboratory strain) and RM11-1a (a wild strain), for their gene expression divergence first by microarray. By incorporating the obtained array data with ChIP-chip data, we generated a set of single-regulator-regulated genes that showed expression divergence between BY4741 and RM11-1a. We randomly selected 50 of these genes for further study. We found substantial variations in the DNA sequences of the promoter regions of these genes and in their regulators between the two strains, which are potential sources of expression divergence. We then developed a step-by-step approach to assess the relative contributions of cis- and trans-variations to expression divergence by using pyrosequencing to quantify the mRNA levels of the two alleles of BY4741 and RM11-1a in co-cultures and in hybrid diploids. Among the 50 genes studied, 4 genes showed no significant expression difference when the two strains were grown in separate cultures or in the same culture (co-culture) and 6 genes showed significant expression difference in separate cultures but not in co-culture. The remaining 40 genes showed expression divergence between the two strains in co-culture. Among these 40 genes, pyrosequencing of diploids showed that 18 cases of expression divergence (45%=18/40) can be attributed to differences in trans-acting factors alone, 7 cases (17.5%) mainly to trans-variations, 8 cases (20%) to both cis- and trans-acting factors, 3 cases (7.5%) mainly to cis-variations, and 4 cases (10%) to cis-acting factors alone. In addition, we replaced the BY promoter by the RM promoter in each of 10 BY genes that were found from our microarray data to have expression divergence between BY and RM, and in each case our quantitative PCR analysis revealed the cis effect of the promoter replacement on gene expression. In summary, our study suggests that trans-acting factors play the major role in expression evolution between yeast strains, but the role of cis variation is also important. Keywords: comparative gene expression between strains Yeast strains were grown in YPAD and harvested at the mid-log phrase. Overnight yeast cultures were used to prepare the starting cultures with OD600=0.1 and were grown in YPAD media at 30oC with 250 rpm shaking. The yeast cells were harvested at the OD600=1.0. Experiment was repeated eight times. Dye-swapping was also performed in each set of experiments to eliminate dye bias (a total of 16 slides).