Genomic strategies to identify novel gene regulatory mechanisms by ligand-activated transcriptional enhancers

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PROJECT SUMMARY Eukaryotic cells interact with cellular environment through transcription regulatory programs activated by various endocrine ligands. Gene regulation by estrogen receptor alpha (ER), a class of nuclear hormone receptor, has been an important paradigm in understanding ligand-induced transcriptional programs. In response to the ligand -estradiol (E2), ER binds predominantly to the transcriptional enhancers, which are regulatory DNA sequence that control the gene expression from distant genomic loci. E2 stimulation also led to rapid reorganization of three-dimensional chromatin architecture and enhancer cooperativity. Biochemical and genomic studies in the past decade have identified many coregulators of ER, and also have largely resolved the composition of ER- bound enhancer complex. However, multidisciplinary studies over the past few years have challenged the conventional models in ligand-induced activation of transcriptional enhancers and the mechanism of enhancer- promoter communication. Live-cell imaging studies have revealed that in response to acute hormonal signaling, ER form distinct compartments inside the cell nucleus that concentrate transcription machinery at robust E2 activated transcriptional enhancers. Compositionally, these “nuclear receptor condensates” are ribonucleoprotein complexes composed of not only proteins, but also non-coding enhancer RNA (eRNA) that contribute to the physical properties and activity. However, the contribution of other RNA species in ligand- induced enhancer condensate assembly and chromatin architecture is not yet understood. The proposed study will focus on identifying new RNA coregulators of ER that facilitate ribonucleoprotein condensate assembly and regulated enhancer cooperativity. The central objective of AIM I will be to identify and characterize new lncRNAs that contribute to enhancer complex assembly and gene regulation using biochemical, genomics and genome engineering. The focus of AIM II will be to understand the prevalence and functional role of ligand-induced spatial enhancer cooperativity. Preliminary studies have identified few protein candidates that might be playing a mechanistic role in this long-distance enhancer cooperativity. The contribution of these proteins, and the lncRNAs identified from the screening strategy implemented in AIM I, in long distance enhancer cooperativity will also be examined. These studies promise to unravel new RNA regulators of enhancer assembly and will pave the way towards a new understanding of ribonucleoprotein interactome in the regulation of various endocrine signaling programs. Importantly, completion of this proposal will generate the data and resources necessary to design a larger study focusing on the in vivo relevance and disease condition contributed by these new molecular players by altering hormonal signaling programs.