The functional specialization, healthy life, and proper disposal of cells in all metazoans require precise responses to developmental and environmental signals. These responses are largely mediated by enhancers, regulatory DNA elements that contain specific recognition sequences required for binding of transcription factors. An ideal model for investigating the actions of enhancers and transcription factors on transcriptional programs is Estrogen receptor (ER?), which is activated by binding of the sex hormone 17?-estradiol (E2) and interacts with ~7000 possible enhancers in the human genome. A major unanswered question is how this factor and the associated enhancers concurrently regulate global programs of estrogen-dependent gene activation and repression. While the key role of short-range enhancer-promoter interactions in transcriptional regulation is well established, the detailed influence of the three-dimensional chromosomal architecture on the putative estrogen-dependent regulatory enhancer network remains poorly understood. To uncover the links between chromosomal structure and regulation of gene transcriptional programs, I hypothesize that that E2/ER? and the putative hierarchical network of associated enhancers play a key role in dynamically altering the three-dimensional chromosomal architecture, and these alterations in turn contribute to the regulation of transcriptional programs. To investigate this hypothesis systematically, I propose to develop and apply bioinformatics pipelines for analyzing data from several powerful contemporary global genomic technologies, including 5C, 3D-DSL, Hi-C, GRO-seq, RNA-seq, and ATAC-seq. I also propose to build an accurate 3D model of chromosomal architecture and to document its dynamic dependence and influence on E2-regulatory enhancers in human Chr21. To this end, I will apply several computational strategies to infer ensembles of chromatin conformations from experimental contact maps, such as those provided by 3D-DSL experiments. By providing critical mechanistic insights into the regulation of transcriptional programs relevant to the endocrine system, the proposed research may help to advance the design of new therapeutic modalities to address diseases of development, metabolism, endocrinology, and aging. I so doing, I will not only become scholarly in central biological and molecular biological issues, but also I will become proficient in the use of recently- emerging advanced bioinformatics and statistical methods for the analysis of large experimental data sets. This NRSA Individual Postdoctoral Fellowship will therefore enable me to acquire invaluable knowledge, training, and experience, while making important contributions to biomedical science, critical to my goal of becoming a particularly effective independent investigator, dedicated to making transformative discoveries regarding fundamental questions in regulatory biology.
The mechanisms by which the dynamic constraints of three-dimensional chromosomal architecture combine with the activities of liganded nuclear receptors to influence global gene regulation programs are of central importance to contemporary endocrinology, but are as yet poorly understood. Relevant insights may ensue by using estrogen receptor as a model system to investigate how the putative hierarchical network of estrogen- dependent enhancers affects and is affected by chromosomal architecture in a cell-type specific manner. The mechanistic details obtained from such investigation will have particular significance for development and disease in the endocrine system, with profound implications on therapeutic approaches to inflammation, cancer, and metabolic disorders.