Determining the role of Tet1 in facultative intestinal stem cells

确定 Tet1 在兼性肠干细胞中的作用

• 批准号: 10678519
• 负责人: NICOLAS VAUGHAN JANTO
• 金额: $ 4.77万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678519
• 关键词: Ablation; Adult; Alleles; Apoptosis; Biological Assay; CASP3 gene; Cells; Chemotherapy and/or radiation; Chromatin; DNA; DNA Damage; Data; Data Analyses; Data Set; Dioxygenases; Diphtheria Toxin; Doxorubicin; Enzymes; Epithelial Cells; Epithelium; Event; Exposure to; Faculty; Gene Activation; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Global Change; Goals; Growth; Health; Histology; Homeostasis; In Situ Nick-End Labeling; Individual; Inflammation; Inflammatory Bowel Diseases; Injury; Intestines; Knock-out; Knockout Mice; LGR5 gene; Malignant Neoplasms; Maps; Measures; Modeling; Morphology; Mus; Natural regeneration; OLFM4 gene; Organoids; Pathogenicity; Population; Population Control; Positioning Attribute; Process; Proliferation Marker; Publishing; Radiation; Radiation therapy; Regenerative Medicine; Regulator Genes; Reporter; Research; Risk; Role; Shapes; Specific qualifier value; Stains; Testing; Therapeutic; Tissues; Training; Villus; Work; cancer therapy; cell injury; cell type; chemotherapy; chromatin modification; cytotoxic; demethylation; embryonic stem cell; epigenomics; epithelium regeneration; experimental study; gastrointestinal; genotoxicity; gut inflammation; insight; intestinal epithelium; intestinal injury; progenitor; radiation-induced injury; regenerative; response; seal; side effect; stem cell function; stem cell genes; stem cells; stemness; tissue regeneration; transcriptome sequencing; transcriptomics; unpublished works

PROJECT SUMMARY Active intestinal stem cells (aISCs) are tasked with maintaining the highly proliferative intestinal epithelium, a tissue with absorptive, secretory, and barrier functions that are critical to homeostasis and survival. Dysregulation of aISCs can lead to inflammation and cancer, while loss of aISCs due to cytotoxic, pathogenic, or radiation- induced injury can threaten epithelial integrity and pose a severe health risk. aISC injury is a common side effect of chemotherapeutic or radiation treatment of cancer. In response to aISC injury, the intestinal epithelium has the remarkable capacity to regenerate its stem cell pool through dedifferentiation of mature epithelial cells and their progenitors, which function as facultative ISCs (fISCs). However, little is understood about the genetic mechanisms that permit this high level of cellular plasticity. One way cells are able to simultaneously modulate the expression of many genes is through chromatin modification. Ten-eleven translocation methylcytosine dioxygenases (TET enzymes) are involved in DNA demethylation through the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), a process associated with gene activation. TET enzymes have well- known functions in maintaining stem cell-specific gene expression in mouse embryonic stem cells, and published data from our lab demonstrate that Tet1 is enriched in adult mouse ISCs and intestinal epithelial progenitors. My preliminary data show that the loss of Tet1 results in aberrant lineage specification, suggesting a central role for Tet1 in changing cell identity. The central hypothesis of this proposal is that Tet1 facilitates ISC identity transitions through activation of cell type specific gene expression. I will test this hypothesis with the following specific aims: Aim 1 will establish the genetic requirement for Tet1 during fISC dedifferentiation following aISC injury using both targeted aISC ablation and cytotoxic injury models. Post-injury regeneration will be assessed by histology and organoid growth assays. Aim 2A will characterize Tet1’s gene regulatory mechanisms during differentiation through genomic, epigenomic, and transcriptomic analysis of inducible Tet1 knockout (Tet1iKO) mice. Aim 2B will use the same approaches to describe Tet1’s gene regulatory mechanisms during dedifferentiation in Tet1iKO mice that have suffered ISC injury. This project will determine how Tet1 facilitates changes in cell identity and provide me with training towards my goal of obtaining a research-focused faculty position studying chromatin regulatory mechanisms of maintaining stem cell function and establishing cell identity. This work will advance the understanding of regenerative mechanisms and provide insight into intestinal epithelial responses to damage and injury.

项目摘要 活性肠干细胞（aISCs）的任务是维持高度增殖的肠上皮细胞， 具有吸收、分泌和屏障功能的组织，这些功能对体内平衡和生存至关重要。失调 aISCs的减少可导致炎症和癌症，而由于细胞毒性、致病性或辐射引起的aISCs的丧失- 诱发的损伤可威胁上皮完整性并造成严重的健康风险。aISC损伤是常见的副作用 化疗或放射治疗癌症。在对aISC损伤的反应中， 通过成熟上皮细胞的去分化再生其干细胞库的显著能力， 它们的祖细胞，其功能为兼性ISC（fISC）。然而，人们对遗传学知之甚少。 这种机制允许这种高水平的细胞可塑性。细胞能够同时调节 许多基因的表达是通过染色质修饰实现的。10 - 11易位甲基胞嘧啶 双加氧酶（泰特酶）通过5-甲基胞嘧啶的转化参与DNA去甲基化 (5mC)5-羟甲基胞嘧啶（5hmC），这是一个与基因激活有关的过程。泰特酶具有良好的- 在小鼠胚胎干细胞中维持干细胞特异性基因表达的已知功能， 我们实验室的数据表明Tet1在成年小鼠ISCs和肠上皮祖细胞中富集。我 初步数据显示Tet1的缺失导致异常的谱系特化，提示Tet1在人类遗传学中的核心作用。 Tet1改变细胞身份。这个建议的中心假设是Tet1促进ISC身份 通过激活细胞类型特异性基因表达的转变。我将测试这个假设与 以下具体目标：目标1将确定fISC脱分化期间Tet1的遗传要求 在aISC损伤后，使用靶向aISC消融和细胞毒性损伤模型。受伤后的再生将 通过组织学和类器官生长测定进行评估。目的2A将描述Tet1的基因调控 通过诱导Tet1的基因组、表观基因组和转录组学分析， 敲除（Tet1iKO）小鼠。目的2B将使用相同的方法来描述Tet1的基因调控机制 在患有ISC损伤的Tet1iKO小鼠中的去分化期间。该项目将确定Tet1 促进细胞身份的变化，并为我提供培训，以实现我获得以研究为重点的目标。 教师职位，研究维持干细胞功能和建立细胞的染色质调节机制 身份这项工作将推进对再生机制的理解，并为肠道疾病的治疗提供新的见解。 上皮对损伤和损伤的反应。