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， 快速产生多种功能细胞。这种上皮细胞还显示出显著的细胞可塑性， 包括成熟细胞在失去ISC后去分化为干细胞的能力。许多基因和 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， 重新连接细胞的可塑性，在很大程度上仍然未知。使用最先进的单细胞sc-Multiome分析， 揭示染色质可及性（scATAC-seq）和基因表达（scRNA-seq）平行，我们最近 产生了高质量的数据，比较了天然上皮细胞和ISC后再生的细胞， 消融术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 表达动态的基础细胞命运的变化，在肠再生，并确定关键表观遗传 该过程的调节剂和转录因子控制剂。结合这些分析，我们建议 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。 异常的染色质结构可导致基因失调，并有助于肿瘤发生，包括 结肠直肠癌DNA甲基化也是结直肠癌中最常见的表观遗传方式 和其他癌症一样。然而，DNA甲基化和DNA甲基化之间的相互作用的决定因素， 突变变化或其他表观遗传层尚未得到严格研究。同样缺少的是 DNA甲基化变化在肿瘤发生的早期发生， 细胞内DNA甲基化。技术限制一直是最重要的障碍， 分析，因为目前在单细胞水平表征DNA甲基化的方法受到低CpG 覆盖范围仅限于基因启动子和GpG岛，这妨碍了对 DNA甲基化改变，特别是在基因调控增强子处。我们建议推翻这些限制 通过开发一种表征DNA甲基化以及RNA表达的新方法， 单细胞水平。我们将联合收割机化学方法与温和的细胞核处理相结合， 基于我们的方法中的磁珠捕获，称为scTaMT-seq，这将提供一个强大的协议， 以尽可能高的分辨率以前所未有的细节分析DNA甲基化。这项技术将推动 了解表观基因组对各种生物过程的影响，特别是癌症。