ABSTRACT Enhancers are cis-regulatory DNA sequences that increase the transcription of its cognate target genes in response to developmental and regulatory signaling at linear distances of kilo- to mega bases from their gene targets directing critical transcriptional programs. Mechanistically, considerable data support the model that interactions between enhancer and promoter are achieved by looping. Chromosome Conformation Capture Analysis (3C) and its derivative techniques provide information about 3D genomic architecture of enhancer- promoter interactions and suggest that the long-range nature of enhancer functions tightly connects their regulation to chromatin architecture. Genome folding into spatial domains facilitates transcriptional regulation consistent with the observation that promoters and enhancers within the same domain preferentially interact with each other. However, despite recent insights into 3D chromosomal organization and topologically associated contact domains, the mechanisms and disease risk implications underlying cognate promoter choice by an activated enhancer remain incompletely understood. To explore the mechanism underlying enhancer-promoter choice, we will employ gene editing by CRISPR/Cas9 to specifically delete or mutate a promoter of the estrogen- regulated gene. Intriguingly, the mutation of the cognate promoter causes its activating enhancer to “switch” its target gene promoter choice, resulting in activation of the alternative gene target. We term this previously- unappreciated phenomenon as “enhancer release and retargeting (ERR).” To systematically investigate the ERR phenomenon and establish it as a major disease risk mechanism, we propose to develop and apply bioinformatic pipelines to analyze data from several large cohorts of datasets from genomics and normal human population such as GWAS and GTEx. Therefore, we can computationally identify putative ERR events that may be associated with human disease including endocrine diseases in which the neighboring alternative genes, rather than the gene hosting the mutant promoter, are disease-causing. Further, we plan to experimentally corroborate this by identifying the putative ERR gene pairs in systemic approach by using CRISPR-KRAB mediated inhibition of promoters. We will investigate the ERR cases of disease significance in detail with appropriate powerful contemporary global genomic technologies, including a variety NGS methods (e.g. 4C, GRO-seq, RNA-seq, ATAC-seq, etc.), single-cell technologies (e.g. scRNA sequencing and live imaging), and the requisite bioinformatic pipelines for analyzing data to explore the mechanism underlying ERR phenomenon and enhancer- promoter choice. By gaining a comprehensive understanding of ERR, including the identification of promoter CTCF binding as a key determinant of promoter choice, the proposed research may represent an important step in understanding enhancer function and enhancer-promoter choice in mammalian cells as ...