Identifying epigenetic states of TADs and targeting using epigenome editing

识别 TAD 的表观遗传状态并使用表观基因组编辑进行靶向

• 批准号: 10372076
• 负责人: Suhn Kyong Rhie
• 金额: $ 20.63万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2024-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372076
• 关键词: 3-Dimensional; Address; Biological; Biological Assay; CRISPR interference; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cells; Cellular Assay; ChIP-seq; Chromatin; Chromatin Structure; DNA Modification Methylases; Data; Data Set; Databases; Development; Diploidy; Disease; EZH2 gene; Encyclopedia of DNA Elements; Epigenetic Process; Epithelial Cells; Etiology; Frequencies; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomic Segment; Genomics; Guide RNA; Health; Hi-C; Histones; Human; Human Cell Line; Human Genome; Lead; Malignant Neoplasms; Malignant neoplasm of prostate; Maps; Measures; Medical; Molecular; Nucleic Acid Regulatory Sequences; Process; Proliferating; Prostate; Protocols documentation; Regulatory Element; Repression; Research; Research Personnel; Role; Specificity; Structure; System; Techniques; Technology; Testing; The Cancer Genome Atlas; Therapeutic; Tissues; Transcriptional Regulation; Variant; bioinformatics tool; cell type; chromatin immunoprecipitation; chromosome conformation capture; design; epigenome; epigenome editing; epigenomics; experimental study; genome-wide; histone modification; human disease; improved; insight; nervous system disorder; next generation sequencing; novel; prostate cancer cell; therapeutic development; tool; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY/ABSTRACT The formation of improper 3-dimensional (3D) chromatin structures and states can lead to many types of human disease. 3D epigenomic datasets were generated in many different cell-types, using genome-wide chromosome conformation capture-derivative techniques (e.g. Hi-C) and chromatin immunoprecipitation assays with sequencing (ChIP-seq). However, there are many unanswered questions about the role of chromatin interactions in cell-type specific gene regulation. Genomic regions that physically interact with each other with high frequency are called topologically associating domains (TADs). Our preliminary results found common TADs that share boundaries among cell types (aka invariant TADs). Interestingly, a subset of the TADs is heavily enriched with histone modifications, suggesting that the size of the TADs may be tightly associated with epigenetic states. We identified hundreds of H3K27me3-enriched (repressed), H3K9me3- enriched (heterochromatic) and H3K36me3-enriched (active) TADs in multiple human cell lines and primary cells, and we also found common TADs that changed epigenetic states among cell types. To elucidate the epigenomic mechanisms by which TADs are cell-type specific or invariant, and to develop tools that can alter TADs, we propose to use this two-pronged approach. In Aim 1, we will identify common TADs that have different chromatin states among cell types, using 3D epigenome and transcriptome data generated in >50 cell types by large consortia (e.g. Roadmap of Epigenomics, Encyclopedia of DNA Elements, PsychENCODE, 4DNucleome) and other epigenomic studies. We will classify large-scale structural features (TADs) into invariant or cell type-specific TADs, comparing the size of TADs. We will also identify epigenetic states of TADs in different cell and tissue types, integrating Hi-C, ChIP-seq, and RNA-seq datasets. In the process of carrying out this aim, we will develop databases and bioinformatics tools that facilitate researchers to identify epigenetic states of common TADs. In Aim 2, we will develop technologies to alter epigenetic states of the TADs using targeted epigenome editing. As preliminary data, we have selected candidate common TADs that are enriched with histone marks and have changed chromatin states between normal and prostate cancer. We also demonstrated that targeted epigenetic editing using the CRISPRi system enabled long-term repression of target genes. Using CRISPRi and gRNAs targeting boundaries of the H3K36me3-enriched TADs and active regulatory elements within the TADs, we will edit the epigenetic states of the selected TADs. Moreover, we will test if changing epigenetic states can alter chromatin structures or expression levels of multiple genes that are located in the TADs, using ChIP-seq, capture Hi-C and RNA-seq. We designed each of the aims can be performed, independently of the others. Our proposed studies will not only provide new insights into transcriptional regulation in 3D human epigenome but also further the development of therapeutic tools for targeted epigenome editing.

项目总结/摘要 不适当的3维（3D）染色质结构和状态的形成可导致许多类型的染色体畸变。 人类疾病3D表观基因组数据集在许多不同的细胞类型中生成，使用全基因组 染色体构象捕获衍生技术（如Hi-C）和染色质免疫沉淀 测序分析（ChIP-seq）。然而，关于《公约》的作用， 细胞类型特异性基因调控中的染色质相互作用。基因组区域与每一个 其他具有高频率的被称为拓扑关联域（TADs）。我们的初步结果发现 在细胞类型之间共享边界的常见TAD（也称为不变TAD）。有趣的是， TADs大量富含组蛋白修饰，这表明TADs的大小可能是紧密相关的。 与表观遗传状态有关我们发现了数百个H3 K27 me 3富集（抑制），H3 K9 me 3- 在多种人细胞系和原代细胞系中富集的（异染色质）和H3 K36 me 3富集的（活性）TAD 我们还发现了改变细胞类型之间表观遗传状态的常见TADs。阐明本 TADs是细胞类型特异性或不变的表观基因组机制，并开发可以改变TADs的工具。 我们建议采用这种双管齐下的方法。在目标1中，我们将确定具有以下特征的常见TAD： 不同细胞类型之间的不同染色质状态，使用在>50个细胞中生成的3D表观基因组和转录组数据 大财团的类型（例如表观基因组学路线图，DNA元件百科全书，PsychENCODE， 4DNucleome）和其他表观基因组学研究。我们将大规模结构特征（TADs）分为 不变量或细胞类型特定的TAD，比较TAD的大小。我们还将确定表观遗传状态， 不同细胞和组织类型的TADs，整合Hi-C，ChIP-seq和RNA-seq数据集。过程中 为了实现这一目标，我们将开发数据库和生物信息学工具，以便于研究人员识别 常见TADs的表观遗传状态。在目标2中，我们将开发技术来改变 使用靶向表观基因组编辑的TADs。作为初步数据，我们选择了候选的常见TAD， 富含组蛋白标记，并且改变了正常和前列腺癌之间的染色质状态。我们 还表明，使用CRISPRi系统的靶向表观遗传编辑能够长期抑制 靶基因使用CRISPRi和gRNA靶向H3 K36 me 3富集的TADs的边界， 在TADs内的调控元件，我们将编辑所选TADs的表观遗传状态。而且还要 测试改变表观遗传状态是否可以改变染色质结构或多个基因的表达水平， 位于TADs中，使用ChIP-seq，捕获Hi-C和RNA-seq。我们设计的每一个目标都可以 是独立于其他人的。我们提出的研究不仅将提供新的见解， 在3D人类表观基因组中的转录调控，而且还进一步开发了治疗工具， 靶向表观基因组编辑。