Mapping regulatory elements and chromatin structures in prostate tumor subtypes at single nucleosome resolution

项目总结/摘要 前列腺肿瘤的临床表现和进展因情况而异。改善临床 因此，我们需要更好地了解导致不同前列腺肿瘤的分子机制。 亚型被称为增强子的远端调节元件的活性，其被细胞类型特异性结合。 转录因子（TF）在调节细胞命运决定中至关重要。活性增强子调节位于 通过形成染色质环在很大的基因组距离上。鉴定在前列腺肿瘤中有活性的增强剂 亚型，我们和其他人进行了染色质免疫沉淀测序（ChIP-seq），使用 H3 K27 ac组蛋白标记抗体，并在前列腺肿瘤中鉴定了> 12，000个差异富集的峰 亚型然而，H3 K27 ac ChIP-seq峰的大小（> 1 kb）太大而不能确定增强子区域 TF结合的地方。为了确定前列腺癌增强子的靶基因，全基因组染色体 进行构象捕获测定（Hi-C），显示增强子附近的染色质环。但我们 由于Hi-C数据的分辨率有限，不能鉴定给定细胞中所有活性增强子的靶基因。 为了阐明与前列腺肿瘤亚型相关的分子机制， 肿瘤亚型特异性增强子及其靶基因是非常需要的。在此，我们建议开发 具有成本效益的方法，可以测量1）增强子和2）参与染色质环的活性， 单个核小体分辨率下的前列腺肿瘤亚型。在目标1中，我们将开发一种方法， 增强子在前列腺肿瘤亚型在单个核小体分辨率。在初步研究中，我们发现， 核小体占据和甲基组测序（NOMe-seq）可以定义TF结合的核小体缺失 区域（<200 bp）。识别前列腺肿瘤亚型特异性 增强子和TF结合到这些区域，我们将首先整合ChIP-seq和开放染色质数据集 并鉴定所有在前列腺肿瘤亚型中有活性的增强子。接下来，使用设计用于测量 为了验证所鉴定的增强子的活性，我们将在前列腺癌细胞中进行靶向NOMe-seq，以鉴定 与前列腺肿瘤亚型相关的增强子和TF。在目标2中，我们将开发一种识别目标的方法 在单个核小体分辨率下在前列腺肿瘤亚型中有活性的增强子的基因。近日，微C在 据报道，以单个核小体分辨率绘制增强子和启动子之间的染色质环。我们将 首先使用匹配的DNA-seq鉴定在前列腺肿瘤亚型中差异表达的基因 以及TCGA和ORIEN联盟生成的RNA-seq数据集。我们接下来将设计出 测量所鉴定基因的启动子和前列腺中活性增强子之间的染色质环 肿瘤亚型，并在前列腺癌细胞中进行靶向Micro-C。我们的综合多组学分析和 集中的深度测序方法将使我们能够更好地评估增强子活性和染色质 前列腺肿瘤亚型的结构和阐明不同肿瘤亚型的分子机制。

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 sequencing method will allow us to better assess the enhancer activities and chromatin structures of prostate tumor subtypes and shed light on molecular mechanisms of the distinct tumor subtypes.