The primary mediators of transcriptional regulation are the cis-regulatory elements (CREs), viz., promoters and enhancers, and the trans-acting factors (TFs) that bind to the CREs. First, the landscape of distinct sequence elements that regulate the spatio-temporal activity profiles of genes is far from complete. For example, several (or alternate) CREs can in a context-specific fashion regulate transcription of one gene. Second, mutations that occur in the coding sequences of TFs, or those occurring in CREs that determine TF binding sites, may change the identity of the cognate TF or alter the affinity with which a site is bound, respectively. This in turn introduces a change in the logic of the transcriptional regulatory circuits harboring these modifications and leads to adaptations in the form of novel gene expression patterns, or robust responses to internal or external signals. CREs and trans-acting factors thus provide an extensive platform for regulatory innovation; the extent of which is only beginning to be appreciated. In this thesis, we discuss three yet-unexplored avenues of regulatory innovation and provide novel insights into each program.

Cis-regulatory rewiring mediated by CREs: A co-regulated module of genes (“regulon”) can have evolutionarily conserved expression and yet have diverged upstream regulators across species, such as the ribosomal regulon which is regulated by the transcription factor (TF) TBF1 in C. albicans, instead of RAP1 in S. cerevisiae. Only a handful of such rewiring events have been established, and the prevalence or conditions conducive to such events are not well known. Here, we develop a novel probabilistic scoring method to comprehensively screen for rewiring within regulons across 23 yeast species. Our analysis recapitulates known events, and suggests TF candidates for certain processes reported to be under distinct regulatory controls in S. cerevisiae and C. albicans, for which the implied regulators are not known. Independent functional analyses of rewiring TF pairs revealed greater functional interactions, common upstream regulators and shared biological processes between them. Our study reveals that cis-rewiring is pervasive; and generated a high-confidence resource of specific events.

Interaction-mediated regulatory rewiring in TFs: Similar to evolutionary changes in the sequence of CREs, changes within coding regions of TFs can allow for altered protein-protein interaction capabilities and function, through motif and domain turnover across evolution. For example, FTZ, has switched from a homeotic TF in ancestral insect species, to being involved in segmentation in the Drosophila genus by the loss of a YPWM motif, and the gain of a LXXLL motif. Elucidating the occurrence of, and mechanisms underlying these switches in TF function is critical to our understanding of evolution. To this end, we developed an approach to detect protein interaction regulatory rewiring across 1200 TFs in 12 related arthropod species. Simulation studies show that the accuracy of event detection is approximately ~80-85%. We recapitulate the known FTZ rewiring event; and find several members of “enhancer of split” complex represented amongst top events, consistent with previous knowledge that the latter has undergone lineage specific losses and duplications across arthropod evolution. Overall, this work establishes that interaction-rewiring is quite prevalent in arthropod development, and provides a high-confidence list of such candidates.

Orphan CGI alternative promoter potential: CGIs are regions with a relatively high frequency of CpG sites. CGIs that occur within gene promoters are historically well studied. Yet, about 50% of all CGIs lie outside of promoter regions (called orphan CGIs), and not much is understood about their biological significance. We show through extensive analysis of the methylome and transcriptome in 34 tissues, that in many cases of highly expressed genes with methylated-promoters, transcription is initiated by a distal orphan CGI located several tens of kb away that functions as an alternative promoter. We found strong evidence of transcription initiation at the upstream CGI and a lack thereof at the methylated proximal promoter itself. CGI-initiated transcripts are associated with signals of stable elongation and splicing that extend into the gene body, as evidenced by tissue-specific RNA-seq and other DNA-encoded splice signals. Overall, our study describes an unreported mechanism of transcription of methylated proximal promoter genes in a tissue-specific fashion.