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.