Contribution of transcription factor binding site motif variants to condition-specific gene expression patterns in budding yeast

Abstract

It is now experimentally well known that variant sequences of a cis transcription factor binding site motif can contribute to differential regulation of genes. We characterize the relationship between motif variants and gene expression by analyzing expression microarray data and binding site predictions. To accomplish this, we statistically detect motif variants with effects that differ among environments. Such environmental specificity may be due to either affinity differences between variants or, more likely, differential interactions of TFs bound to these variants with cofactors, and with differential presence of cofactors across environments. We examine conservation of functional variants across four Saccharomyces species, and find that about a third of transcription factors have target genes that are differentially expressed in a condition-specific manner that is correlated with the nucleotide at variant motif positions. We find good correspondence between our results and some cases in the experimental literature (Reb1, Sum1, Mcm1, and Rap1). These results and growing consensus in the literature indicates that motif variants may often be functionally distinct, that this may be observed in genomic data, and that variants play an important role in condition-specific gene regulation.

Figure 1. The expression profile distance between genes with the same binding site motif variant (BSMV) is smaller than the distance between genes with a different BSMV for functionally variant positions, but not for other positions with BSMVs.

VDRE distances are based on ranked expression profiles for 211 S98 Affymetrix microarrays, and all within-BSMV (grey) or between BSMV (blue) distances are grouped together either from all functionally variant binding site positions (first two bars) or all other positions with BSMVs (third and fourth bar).

Figure 2. Examples of binding site motif variants (BSMVs) associated with condition-specific gene expression.

Mean expression values (Affymetrix; y axis) of genes with each of two BSMVs are plotted on each graph (standard error of mean shown), although more BSMVs may be present at that position. The means are ordered across conditions (x axis) according to the difference in mean expression between the two BSMVs (black dashes). (A) Mcm1, involved in cell-type-specific transcription and pheromone response, has functional variants at position 4 of its binding motif. Genes with “T” at position 4 of the Mcm1 binding site (red) are induced relative to genes with “A” BSMVs (green) after DNA damage with MMS. While undergoing desiccation and rehydration, genes with “A” BSMVs are induced in comparison to genes with “T” BSMVs. (B) Sum1, a regulator of sporulation-specific genes, has functional variants at position 8 of its binding motif. Genes with “T” (red) at position 8 of the Sum1 binding site have higher expression than genes with “A” BSMVs (green) during rich media growth in lab or IFH1 myc-tagged strains or glucose pulse after starvation. In sporulation, genes with “A” BSMVs are expressed higher than genes with “T” BSMVs. (C) The effect of the functional variant at position 8 of Sum1 on target genes remains the same when also considering target genes under more complex regulatory control (multiple primary binding sites).

Figure 3. Conserved expression patterns associated with functional binding site motif variants (BSMVs).

The y-axis of each plot is the mean expression (Y6.4kv6 arrays) standard error of mean shown) of the stress condition relative to the non-stress condition and the x-axis is experimental treatment, ordered by the difference between the means of genes with each BSMV (black dashes). The function of variant nucleotides at position 9 of the Reb1 binding motif is conserved in (A) Saccharomyces cerevisiae, (B) S. paradoxus, and (C) S. mikatae. In all three species, genes associated with the “G” BSMV (orange) are more highly expressed than genes associated with the “A” BSMV (green) in starvation conditions (glycerol). The function of variant nucleotides at position 10 of the Rap1 binding motif is conserved in (d) S. cerevisiae, (e) S. paradoxus, (f) S. mikatae, and (g) S. kudriavzevii. In all four species, genes associated with the “C” BSMV (blue) are more highly expressed than genes associated with the “T” BSMV (red) in starvation conditions (glycerol), and the opposite relationship is apparent during nitrogen starvation. The expression differences between the BSMVs are significantly condition-specific in panels a-f (p<0.005).

Figure 4. Pairwise expression profile distances (VDRE) between genes that have different types of binding sites in common.

With “one primary” binding site in common, target genes have only a single primary binding site (posterior probability >0.7), and pairwise comparisons are between target genes that have a binding site with the same TF identity. With “primary (in the presence of multiple primary sites)” binding sites in common, target genes may have multiple primary binding sites, and pairwise comparisons are between target genes that have a binding site with the same TF identity. With “secondary” binding sites in common, pairwise comparisons are between target genes that have a secondary binding site (posterior probability <0.7 and >0.2) with the same TF identity. With “random” binding sites in common, pairwise comparisons are between random pairs of genes. Standard error bars are indicated.

Figure 5. Distribution of the number of within and between variant comparisons between gene expression profiles of positions with binding site motif variants (BSMVs).

For each motif position, the lower of either the number of within-BSMV comparisons or between BSMV expression comparisons was counted. The blue line and blue bars represent the distribution of all counts, while the orange line and orange dots represent the distribution of only the positions that are functionally variable. Triangles indicate the median of the two distributions. The distribution suggests that there are a reasonable number of comparisons available for most positions.

Figure 6. Variable and highly variable binding site motif positions are evolutionarily constrained.

The relative evolutionary rate of binding site motif positions that are variable (>1 bit of information) and highly variable (≤bit of information) evolve more slowly than putatively neutral sites: third codon positons, introns, and intergenic regions. First and second positions, which are more functionally constrained, are also shown. Rates were calcualted from a whole-genome alignment of Saccharomyces sensu stricto species using emperical Bayesian estimation.