Specific communication between enhancers and alternative promoters mediates finer regulation of transcription initiation

Enhancers are DNA regulatory elements that play an important role in the precise/nregulation of Pol II transcription initiation and, consequently, in the generation and/nmaintenance of patterns of gene expression. A defining characteristic of enhancers is their/ncapacity to regulate transcription from their target promoter, independent of the genomic/ndistance that separates them. Importantly, in order for enhancers to exert their regulatory/naction, they must first relocalize into close spatial proximity to their target promoter and/nform long-range interactions. Although these interactions have been demonstrated to/noccur, the mechanisms that allow their specific and efficient formation remains largely/nobscure. In this thesis, I focus on the development of tools and the expansion of our/nunderstanding with regard to this important topic./nIn the first chapter, I begin by providing a perspective of transcription initiation as/na hub of gene regulation. Then, I perform a concise overview of promoters and enhancers/nas DNA regulatory elements and share the most important functional and structural/ncharacteristics. I finalize the chapter by discussing the known regulatory mechanisms that/nallow the formation of specific interactions between enhancers and promoters to take place./nIn the second chapter, I present my findings on the development of imaging systems/nto permit the visualization of enhancer-promoter interactions in live cells. I begin by/ndescribing the limitations of current systems. Then, I discuss the importance of the/ndevelopment of novel, innovative systems that allow specific labelling of genomic loci./nFrom there, I present the possibilities and limitations of a developed dCas9/Aptamer/nsystem for the visualization of repetitive and non-repetitive loci. I also present my findings/non the development of a two-step integration system of operator arrays and share the/nprincipal technical limitations encountered in its development. I finish by discussing and/nproviding a critical perspective of relevant published work that has been produced since/nthese efforts were undertaken./nIn the third chapter, I begin by identifying and characterizing potential enhancers/nin the PCSK9 locus in hepatic derived cells. I identify three candidate enhancers with/nstrong transcriptional activity by reporter assays. Then, I demonstrate how the regulatory/nactivity of these candidate enhancers is specific to hepatic cells and their regulatory activity/ncan be modulated by two small molecules in a similar fashion to the endogenous PCSK9/ngene. From there, I dissect these DNA regulatory sequences to identify transcription factor/nbinding sites responsible for transcriptional activity by bioinformatic analysis and fine/nmutagenic studies. These results point towards the presence of three DNA regulatory/nelements required for PKCS9 expression./nIn the fourth and final chapter, I investigate two alternative promoters (P1 and P2)/nthat drive MYC expression and are regulated by enhancers. The two MYC promoters can/nbe differentially regulated across cell-types, and their selective usage is largely mediated by/ndistal regulatory sequences. Moreover, in colon carcinoma cells, Wnt-responsive enhancers/nprefer to upregulate transcription from the P1 promoter, using reporter assays in the/ncontext of the endogenous Wnt induction. In addition, multiple enhancer deletions using/nCRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, I explore how/npreferential activation between Wnt-responsive enhancers and the P1 promoter is/ninfluenced by the distinct core promoter elements that are present in the MYC promoters./nTaken together, these results provide new insight into how enhancers can specifically target/nalternative promoters and suggest that the presence of alternative promoters could create a/nmore efficient formation of long-range interactions and a more precise combinatorial/nregulation of transcription initiation.

Advisor: Darzacq, Xavier