Project 3 Topological mapping of chromatin architectures for hormone-independent gene transcription

摘要/摘要--项目3 激素非依赖性基因转录染色质结构的拓扑图 ER/AR结合增强子和启动子之间的染色质长程相互作用是 乳腺癌和前列腺癌细胞中的协调基因调控。这些相互作用通过形成 3D染色质结构，使增强子和转录因子复合体与 靶基因。为了破译这一复杂的规则，我们和其他研究人员之前曾使用Hi-C映射 不同细胞类型中的拓扑相关结构域(TADS)。在进一步的研究中，我们发现了一种癌症-- 染色体17q23上特定的TAD，可被划分为ER调控的转录中心。 研究发现，其靶基因的一致性上调与患者较短的无病生存期有关。 ER阳性乳腺癌患者的亚群，与他们的抗激素治疗无关。新兴 证据还表明，前列腺癌细胞基因组中存在AR特异性TADs。因此，我们 假设1)频繁的激素(即雌激素或雄激素)刺激导致 ER/AR相关TADs动态调控多基因转录促进肿瘤细胞异常增殖 乳腺和前列腺癌细胞以及2)在拮抗剂存在的情况下，这些染色质结构域的子集， 在这里被称为过渡性TADS，通过染色质重新分配激素继续被利用。 独立转录。而大多数与ER/AR相关的TAD在功能上受到抑制 拮抗剂、过渡性TADS可能部分地逃脱了这一障碍，对基因转录进行结构性调控。 为了验证这些假设，我们将使用一种改进的Hi-C方法，称为拴系构象捕获(TCC)，以 激素敏感和耐药癌细胞株TAD结构的动态变化 至激动剂或拮抗剂(目标1)。抑制性、活性和基因体组蛋白标志物和CTCF的芯片序列 绝缘体也将在该单元线面板中导通。将获取MNase-seq和MBDCap-seq数据集 绘制常染色化和异染色化TADS图谱。为了整合组学-序列数据，我们将开发一种 计算模型PRAM3D，它将泊松随机效果体系结构模型(PRAM)应用于 概述3D染色质结构(目标2)。贝叶斯分层模型将预测假定的过渡 TADS，协调调节目标基因的激素非依赖性转录。此外，我们将使用 核小体密度法将转换TAD亚区划分为不同的调控类别，即， 活跃的、抑制的或二价转录中心。CRISPR/Cas9关键染色质区域的基因组编辑 可以在功能上分解这些与TAD相关的中枢的时空组织(目标3)。扩散 侵袭/迁移分析将确定这种基因组编辑是否会部分地使癌细胞对 抗激素治疗。我们还将审问组蛋白修饰和其他 表观遗传调节剂对过渡TAD结构的建立。在电子计算机表达谱分析和 单细胞RNA序列将在TCGA队列的原发肿瘤和癌细胞亚群中进行， 并确定TAD相关靶基因的一致性调节是否是内在的 乳腺癌或前列腺癌激素抵抗的预测因子。

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.