High resolution characterization of epigenetic dynamics in gastrointestinal cell plasticity

胃肠道细胞可塑性表观遗传动力学的高分辨率表征

• 批准号: 10576232
• 负责人: Unmesh Jadhav
• 金额: $ 12.38万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10576232
• 关键词: Ablation; Abnormal Cell; Address; Binding; Biological Assay; Biological Process; Boranes; Cancer Etiology; Cell Nucleus; Cells; Cellular Assay; Cessation of life; Charge; Chemicals; Chromatin; Chromatin Structure; Cluster Analysis; Colorectal; Colorectal Cancer; Complementary DNA; Computing Methodologies; DNA; DNA Damage; DNA Methylation; DNA Sequence Alteration; DNA amplification; DNA analysis; Data; Development; Developmental Gene; Disease; Disease Progression; Enhancers; Epigenetic Process; Epithelial Cells; Epithelium; Event; Evolution; Future; Gene Expression; Gene Expression Profile; Gene Mutation; Genes; Genetic; Genetic Transcription; Genome; Genomic approach; Intestines; Island; KRAS2 gene; Knowledge; Malignant Neoplasms; Maps; Measures; Mesenchymal; Messenger RNA; Methods; Methylation; Modality; Modification; Molecular; Mutation; Natural regeneration; Process; Proteins; Protocols documentation; RNA; Regulator Genes; Research; Resolution; Signal Transduction; Sorting; Stains; TP53 gene; Technology; Testing; Therapeutic Intervention; Time; Tissues; base; bisulfite; cell behavior; cell type; disease prognosis; drug development; early onset colorectal cancer; epigenomics; gastrointestinal; histone modification; innovation; intestinal epithelium; magnetic beads; multiple omics; new therapeutic target; novel; preservation; promoter; stem cells; therapy design; tissue regeneration; tissue repair; transcription factor; transcriptomic profiling; tumor; tumorigenesis

Project Summary Continuous differentiation of intestinal stem cells (ISCs) maintains the integrity of intestinal epithelium while producing diverse functional cells at rapid pace. This epithelium also displays remarkable cell plasticity, including the ability of mature cells to dedifferentiate into stem cells upon loss of ISCs. As many genetic and signaling drivers of this cell fate reversion are being discovered, cell intrinsic epigenetic controls that must be rewired for cell plasticity, remain largely unknown. Using state-of-the-art single-cell sc-Multiome assays that reveal chromatin accessibility (scATAC-seq) and gene expression (scRNA-seq) in parallel, we have recently produced high-quality data comparing native epithelial cells and cells undergoing regeneration upon ISC ablation. Using these data and innovative computational methods, we will chart the chromatin and gene expression dynamic underlying cell fate changes in intestinal regeneration, and identify key epigenetic modulators and transcription factor controllers of this process. In conjunction with these analyses, we propose to delineate promoter and enhancer relevant epigenetic controls (histone modifications) through the time course of regeneration. Aberrant chromatin structure can cause gene dysregulation and contribute to tumorigenesis, including colorectal cancer. DNA methylation is the most frequently modulated epigenetic modality in colorectal as well as many other cancers. However, determinants of the reciprocal interactions between DNA methylation and mutational changes or other epigenetic layers have not been studied critically. Also missing is the knowledge of how early in tumorigenesis does the DNA methylation change occur and how uniform are the changes in DNA methylation across cells. Technological limitations have been the most significant barrier for such analyses, as current methods to characterize DNA methylation at single-cell level suffer from low CpG coverage limited to gene promoters and GpG islands, which precludes understanding of the true breadth of DNA methylation change, particularly at gene regulatory enhancers. We propose to override these limitations through development of a novel assay for characterizing DNA methylation as well as RNA expression at single-cell level. We combine chemical methods geared to preserve DNA integrity and gentler nuclei handling based on magnetic bead capture in our method called scTaMT-seq, which will provide a robust protocol for analyzing DNA methylation in unprecedented detail at highest possible resolution. This technology will propel the understanding of epigenomic impact on various biological processes, particularly cancer.

项目摘要 肠干细胞(ISCs)的持续分化维持了肠上皮的完整性 快速产生不同功能的细胞。这种上皮细胞也表现出显著的细胞可塑性， 包括成熟细胞在失去ISCs后去分化为干细胞的能力。同样多的基因和 这种细胞命运逆转的信号驱动因素正在被发现，细胞内在的表观遗传控制 对于细胞可塑性的重新连接，在很大程度上仍然是未知的。使用最先进的单细胞sc-Multiome分析 同时揭示染色质可及性(scatac-seq)和基因表达(scrna-seq)，我们最近已经 制作了高质量的数据，比较了自然上皮细胞和在ISC上再生的细胞 消融。利用这些数据和创新的计算方法，我们将绘制染色质和基因图 肠道再生中潜在的细胞命运变化的表达动态，并确定关键的表观遗传学 这一过程的调节器和转录因子控制器。结合这些分析，我们建议 通过时间描述启动子和增强子相关的表观遗传控制(组蛋白修饰) 再生的过程。 染色质结构的异常可导致基因失调，并有助于肿瘤的发生，包括 结直肠癌。DNA甲基化也是结直肠癌最常见的表观遗传方式 和许多其他癌症一样。然而，DNA甲基化和DNA甲基化之间相互作用的决定因素 突变变化或其他表观遗传层还没有得到严格的研究。同样遗漏的是知识 在肿瘤发生的早期，DNA甲基化的变化是如何发生的，以及这些变化是如何均匀的 细胞间的DNA甲基化。技术限制一直是实现这一目标的最大障碍 作为目前在单细胞水平表征DNA甲基化的方法，分析受到低CpG的影响 报道仅限于基因启动子和GPG岛，这排除了对 DNA甲基化改变，特别是在基因调控增强子。我们建议取消这些限制 通过开发一种新的方法来表征DNA甲基化和RNA在 单细胞水平。我们结合了旨在保持DNA完整性的化学方法和更温和的核处理 基于我们称为scTaMT-seq的方法中的磁珠捕获，该方法将为 以尽可能高的分辨率以前所未有的详细程度分析DNA甲基化。这项技术将推动 理解表观基因组对各种生物过程的影响，尤其是癌症。