Wednesday, May 7, 2025
5/7/2025
Enhancer Connectomes in Regulation of Gene Expression - ABSTRACT Mammalian cells exhibit a precise gene regulation process, during which enhancers mediate rapid gene activation programs in response to different signals and ligands. While many principles of chromosomal mechanisms underlying enhancer functions have been deduced in the 39 years since their initial discovery, striking gaps in our knowledge remain. These include actions of a series of previously unknown, but required, enhancer-recruited complexes; contributions of physicochemical properties of enhancer condensates formed on the estrogen receptor (ER) in signaling-dependent regulation; and the potentially important roles of interactions with subnuclear architectural structures. Further, because global genomic methods determine epigenomic events at only a single point in time, another striking gap in our knowledge is understanding the temporal principles underlying regulation of regulated transcriptional programs. Under physiological conditions, many signals present as a continuum from acute to chronic stimulation; however, the mechanistic and functional distinctions between acute and chronic enhancer activation remain poorly understood. Here, we take advantage of our knowledge of gene transcriptional control by nuclear receptors, exemplified by estrogen 17β-estradiol (E2)- dependent activation of transcriptional programs to address these basic questions. The central challenge in this competitive renewal is to provide a unified molecular logic that mechanistically links a series of required, but overlooked, components of signal-dependent enhancer activation strategies that underlie acute and chronic regulated gene expression programs. We will initiate a comprehensive investigation of new dynamic enhancer complexes and their interactions with the subnuclear architectural structures critically regulating transcription. These include the function of promoter antisense transcripts in promoter pause-release regulation; investigation of required contributions of Ku70 homodimers reading the TopoisomerseI:DNA covalent intermediate to assemble a new complex required for promoter pausing and enhancer activation events. We will investigate whether short sequences and specific amino acids in the ERN-terminal IDR mediate assembly of key RNP condensate components required for robust activation, including an unexpected role of KAP1 in these activation events. We will establish real time, multicolor imaging to uncover the dynamics of activated enhancer interactions, enhancer bursting events and relationship of activation to interactions with subnuclear architectural structures. We will explore the hypothesis that the most robust eRNA:protein enhancer condensates form a homotypic enhancer network resulting in cooperative activation of a subset of similarly activated homotypic enhancers separated by multiple TADs or even located on other chromosomes, properties are lost following chronic signal/ligand activation. These approaches will be complemented by alternative approaches for defining the dynamics of interactions between enhancers and subnuclear architectural structures. Our goal is to provide novel insights into regulated enhancer and promoter activation mechanisms.
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