! ! PROJECT ABSTRACT The central goal of this proposal is to shed new light on the roles of chromosomal architecture and phase separation in regulation of gene transcriptional programs by liganded nuclear receptors, and to use this project and the experimental skills acquired to transition to an academic career as an independent investigator studying the mechanisms underlying regulated gene transcriptional programs. My focus is on gene enhancers. I will examine a cohort of active MegaTrans-bound enhancers separated by vast genomic distances and use several approaches to investigate whether E2 induces rapid changes in chromosomal architecture, resulting in spatial proximity and chromosome-wide cooperativity of these enhancers. Two new ideas, the possible condensin- dependency of this inter-TAD enhancer association and cooperation and the potential role of phase separation events occurring between MegaTrans enhancers and RNP nuclear structures, including inter-chromatin granules (ICGs) will be specifically investigated. Finally, I propose to develop and utilize contemporary real time imaging and accompanying informatics approaches to uncover the functional alterations in chromosomal and subnuclear architectural structures induced by estrogen. Together, this approach can significantly alter our current concepts about regulated gene transcriptional programs.
A central question in gene regulation is to determine whether enhancers separated by vast spatial distances in a chromosome can exhibit functional interactions to synergistically increase the activation of the interacting enhancers. Here, I propose to use contemporary global genomic technologies that permit quantification of the transcription of all genes and regulatory elements genome-wide to identify a cohort of most robust, active enhancers (?first tier?) that, upon E2 stimulation, exhibit dynamic interactions and cooperative activation based on enhancer-mediated regulation of chromosomal architecture and phase separation events. Finally, I propose to develop contemporary real time imaging and accompanying informatics approaches to uncover the functional alterations in chromosomal and subnuclear architectural structures induced by estrogen.
