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

项目摘要/摘要 前列腺癌的临床表现和进展因人而异。开发改进的临床 干预，我们需要更好地了解导致不同前列腺癌的分子机制 子类型。被称为增强子的远端调控元件的活动受特定细胞类型的结合 转录因子(TF)在调节细胞命运的决定中起关键作用。活性增强子调节定位的基因 通过形成染色质环在很大的基因组距离上。前列腺癌中活性增强剂的鉴定 亚型，我们和其他人进行了染色质免疫沉淀测序(芯片序列)使用 H3K27ac组蛋白标记抗体及其在前列腺癌中的12,000个差异富集峰的鉴定 子类型。然而，H3K27ac芯片序列峰(&gt；1kb)的大小太大，无法确定增强子区域 TF绑定的地方。识别前列腺癌增强剂的靶基因，全基因组染色体 进行构象捕获分析(Hi-C)，发现增强子附近有染色质环。然而，我们 由于Hi-C数据的分辨率有限，无法识别在给定细胞中激活的所有增强子的靶基因。 阐明与前列腺癌亚型相关的分子机制，更好地识别前列腺癌 迫切需要肿瘤亚型特异性增强子及其靶基因。在此，我们建议开发 经济有效的方法可以测量1)增强剂的活性和2)涉及染色质环在 单核小体分辨的前列腺癌亚型。在目标1中，我们将开发一种识别活动的方法 单核小体分辨的前列腺癌亚型中的增强剂。在初步研究中，我们表明 核小体占有率和甲基体测序(Nome-seq)可以定义Tf结合的核小体缺失 在单分子分辨率的增强子中的区域(&lt；200bp)。识别前列腺癌亚型特异性 与这些区域结合的增强子和转录因子，我们将首先整合芯片序列和开放染色质数据集 并确定所有在前列腺癌亚型中活跃的增强子。接下来，使用设计用于测量 识别的增强子的活性，我们将在前列腺癌细胞中进行靶向Nome-seq以识别 与前列腺癌亚型相关的增强剂和转录因子。在目标2中，我们将开发一种识别目标的方法 在单核小体分辨率下前列腺癌亚型中活跃的增强子基因。最近，Micro-C正在 报道了在单核小体分辨率下定位增强子和启动子之间的染色质环。我们会 首先使用匹配的DNA-seq识别前列腺癌亚型之间差异表达的基因 以及由TCGA和Orien联盟生成的RNA-seq数据集。接下来，我们将设计出能够 测量已识别基因的启动子和前列腺中活跃的增强子之间的染色质环路 在前列腺癌细胞中进行肿瘤亚型和靶向Micro-C。我们的综合多元经济分析和 聚焦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 前列腺癌亚型的结构，并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.