ABSTRACT/SUMMARY - Project 3 Topological mapping of chromatin architectures for hormone-independent gene transcription Long-range chromatin interactions between ER/AR-bound enhancers and promoters are necessary for coordinated gene regulation in breast and prostate cancer cells. These interactions occur via the formation of 3D chromatin architecture that brings enhancers and transcription factor complexes into close contact with target genes. To decode this complex regulation, we and other investigators have previously used Hi-C to map topologically associated domains (TADs) in different cell types. In a further study, we have identified a cancer- specific TAD on chromosome 17q23 that can be partitioned into an ER-regulated transcription hub. Concordant up-regulation of its target genes is found to be associated with short disease-free survival in a subgroup of ER-positive breast cancer patients, irrespective of their anti-hormone treatments. Emerging evidence has also shown AR-specific TADs are present in the prostate cancer cell genome. Therefore, we hypothesize that 1) frequent hormone (i.e., estrogen or androgen) stimulation leads to the formation of ER/AR-related TADs that dynamically regulate transcription of multiple genes for aberrant proliferation of breast and prostate cancer cells and 2) in the presence of antagonists, a subset of these chromatin domains, herein termed transition TADs, continue to be exploited through chromatin redeployment for hormone- independent transcription. Whereas the majority of ER/AR-related TADs are functionally suppressed by antagonists, transition TADs may partially escape this blockade for constitutive regulation of gene transcription. To test these hypotheses, we will use a modified Hi-C method, called tethered conformation capture (TCC), to investigate dynamic changes of TAD structures in hormone-sensitive and -resistant cancer cell lines exposed to agonists or antagonists (Aim 1). ChIP-seq of repressive, active, and gene-body histone marks and CTCF insulator will also be conducted in this cell line panel. MNase-seq and MBDCap-seq datasets will be acquired to map euchromatinized and heterochromatinized TADs. To integrate omics-seq data, we will develop a computational model, PRAM3D, which applies a Poisson Random effect Architecture Model (PRAM) to recapitulate 3D chromatin architectures (Aim 2). A Bayesian hierarchical model will predict putative transition TADs that concordantly regulate hormone-independent transcription of target genes. Furthermore, we will use a nucleosome density method to classify transition TAD subdomains into different regulatory categories, i.e., active, repressive, or bivalent transcription hubs. CRISPR/Cas9 genome-editing of critical chromatin regions may functionally disassemble spatiotemporal organization of these TAD-associated hubs (Aim 3). Proliferation and invasion/migration assays will determine whether this genome editing partially re-sensitizes cancer cells to anti-hormone treatments. We will also interrogate mechanistic contribution of histone modifications and other epigenetic modulators for the establishment of transition TAD structures. In silico expression profiling and single-cell RNA seq will be conducted in primary tumors of TCGA cohorts and in cancer cell subpopulations, respectively, and determine whether concordant regulation of TAD-associated target genes is intrinsic predictors of hormone resistance in breast or prostate cancer.

PROJECT NARRATIVE - Project 3 We plan to use omics approaches to investigate functional transition of hormone-sensitive into hormone- resistant transcription regulation during cancer progression. Concordant gene signatures identified in primary tumors may predict treatment response to anti-hormone therapies. The proposed study is expected to identify druggable targets that show promises to mitigate the advanced development of breast and prostate cancers.