Disordered sequences of transcription factors regulate genomic binding by integrating diverse sequence grammars and interaction types.

Intrinsically disordered regions (IDRs) guide transcription factors (TFs) to their genomic binding sites, raising the question of how structure-lacking regions encode for complex binding patterns. We investigated this using the TF Gln3, revealing sets of IDR-embedded determinants that direct Gln3 binding to respective groups of functionally related promoters, and enable tuning binding preferences between environmental conditions, phospho-mimicking mutations, and orthologs. Through targeted mutations, we defined the role of short linear motifs (SLiMs) and co-binding TFs (Hap2) in stabilizing Gln3 at respiration-chain promoters, while providing evidence that Gln3 binding at nitrogen-associated promoters is encoded by the IDR amino-acid composition, independent of SLiMs or co-binding TFs. Therefore, despite their apparent simplicity, TF IDRs can direct and regulate complex genomic binding patterns through a combination of SLiM-mediated and composition-encoded interactions.

Evolution of transcription factor binding through sequence variations and turnover of binding sites.

Variations in noncoding regulatory sequences play a central role in evolution. Interpreting such variations, however, remains difficult even in the context of defined attributes such as transcription factor (TF) binding sites. Here, we systematically link variations in cis-regulatory sequences to TF binding by profiling the allele-specific binding of 27 TFs expressed in a yeast hybrid, in which two related genomes are present within the same nucleus. TFs localize preferentially to sites containing their known consensus motifs but occupy only a small fraction of the motif-containing sites available within the genomes. Differential binding of TFs to the orthologous alleles was well explained by variations that alter motif sequence, whereas differences in chromatin accessibility between alleles were of little apparent effect. Motif variations that abolished binding when present in only one allele were still bound when present in both alleles, suggesting evolutionary compensation, with a potential role for sequence conservation at the motif's vicinity. At the level of the full promoter, we identify cases of binding-site turnover, in which binding sites are reciprocally gained and lost, yet most interspecific differences remained uncompensated. Our results show the flexibility of TFs to bind imprecise motifs and the fast evolution of TF binding sites between related species.

Independent evolution of transcript abundance and gene regulatory dynamics.

Changes in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells modulate gene expression in response to changing environments. Previous comparative studies focused on specific conditions, describing interspecies variation in expression levels, but providing limited information about variation across different conditions. To close this gap, we profiled mRNA levels of two related yeast species in hundreds of conditions and used coexpression analysis to distinguish variation in the dynamic pattern of gene expression from variation in expression levels. The majority of genes whose expression varied between the species maintained a conserved dynamic pattern. Cases of diverged dynamic pattern correspond to genes that were induced under distinct subsets of conditions in the two species. Profiling the interspecific hybrid allowed us to distinguish between genes with predominantly cis- or trans-regulatory variation. We find that trans-varying alleles are dominantly inherited, and that cis-variations are often complemented by variations in trans Based on these results, we suggest that gene expression diverges primarily through changes in expression levels, but does not alter the pattern by which these levels are dynamically regulated.

The architecture of binding cooperativity between densely bound transcription factors.

The binding of transcription factors (TFs) along genomes is restricted to a subset of sites containing their preferred motifs. TF-binding specificity is often attributed to the co-binding of interacting TFs; however, apart from specific examples, this model remains untested. Here, we define dependencies among budding yeast TFs that localize to overlapping promoters by profiling the genome-wide consequences of co-depleting multiple TFs. We describe unidirectional interactions, revealing Msn2 as a central factor allowing TF binding at its target promoters. By contrast, no case of mutual cooperation was observed. Particularly, Msn2 retained binding at its preferred promoters upon co-depletion of fourteen similarly bound TFs. Overall, the consequences of TF co-depletions were moderate, limited to a subset of promoters, and failed to explain the role of regions outside the DNA-binding domain in directing TF-binding preferences. Our results call for re-evaluating the role of cooperative interactions in directing TF-binding preferences.

Accumulation of cis- and trans-regulatory variations is associated with phenotypic divergence of a complex trait between yeast species.

Gene regulatory variations accumulate during evolution and alter gene expression. While the importance of expression variation in phenotypic evolution is well established, the molecular basis remains largely unknown. Here, we examine two closely related yeast species, Saccharomyces cerevisiae and Saccharomyces paradoxus, which show phenotypical differences in morphology and cell cycle progression when grown in the same environment. By profiling the cell cycle transcriptome and binding of key transcription factors (TFs) in the two species and their hybrid, we show that changes in expression levels and dynamics of oscillating genes are dominated by upstream trans-variations. We find that multiple cell cycle regulators show both cis- and trans-regulatory variations, which alters their expression in favor of the different cell cycle phenotypes. Moreover, we show that variations in the cell cycle TFs, Fkh1, and Fkh2 affect both the expression of target genes, and the binding specificity of an interacting TF, Ace2. Our study reveals how multiple variations accumulate and propagate through the gene regulatory network, alter TFs binding, contributing to phenotypic changes in cell cycle progression.