Project Summary/Abstract The clinical behavior and progression of prostate tumors vary case by case. To develop improved clinical intervention, we need to better understand the molecular mechanisms leading to different prostate tumor subtypes. The activities of distal regulatory elements called enhancers, which are bound by cell-type specific transcription factors (TFs), are critical in regulating cell fate decisions. Active enhancers regulate genes located at great genomic distances by forming chromatin loops. To identify enhancers active in prostate tumor subtypes, we and others performed chromatin immunoprecipitation with sequencing (ChIP-seq) using the H3K27ac histone mark antibody and identified >12,000 peaks differentially enriched among prostate tumor subtypes. However, the size of H3K27ac ChIP-seq peaks (>1kb) is too large to determine enhancer regions where TFs bind. To identify target genes of prostate cancer enhancers, genome-wide chromosome conformation capture assay (Hi-C) was performed, revealing chromatin loops near enhancers. However, we could not identify target genes of all enhancers active in a given cell due to the limited resolution of Hi-C data. To elucidate molecular mechanisms linked to prostate tumor subtypes, better strategies to identify prostate tumor subtype-specific enhancers and their target genes are crucially needed. Herein, we propose to develop cost-effective methods that can measure the activities of 1) enhancers and 2) involved chromatin loops in prostate tumor subtypes at single nucleosome resolution. In Aim 1, we will develop a method to identify active enhancers in prostate tumor subtypes at single nucleosome resolution. In preliminary studies, we showed that Nucleosome Occupancy and Methylome sequencing (NOMe-seq) can define TF-bound nucleosome-depleted regions (<200bp) in enhancers at single molecule resolution. To identify prostate tumor subtype-specific enhancers and TFs that bind to these regions, we will first integrate ChIP-seq and open chromatin datasets and identify all enhancers active in prostate tumor subtypes. Next, using probes designed to measure the activities of identified enhancers, we will perform targeted NOMe-seq in prostate cancer cells to identify enhancers and TFs linked to prostate tumor subtypes. In Aim 2, we will develop a method to identify target genes of enhancers active in prostate tumor subtypes at single nucleosome resolution. Recently, Micro-C is reported to map chromatin loops between enhancers and promoters at single nucleosome resolution. We will first identify genes that are differentially expressed among prostate tumor subtypes using matched DNA-seq and RNA-seq datasets generated by TCGA and ORIEN consortia. We will next design probes that can measure the chromatin looping between promoters of the identified genes and enhancers active in prostate tumor subtypes and perform targeted Micro-C in prostate cancer cells. Our integrative multi-omic analyses and focused deep s...