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cell function; stem cell genes; stem cells; stemness; tissue regeneration; transcriptome sequencing; transcriptomics; unpublished works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.

项目总结 活性肠干细胞(AISCs)的任务是维持高度增殖的肠上皮，a 具有吸收、分泌和屏障功能的组织，这些功能对动态平衡和生存至关重要。调控失调 AISCs的缺失会导致炎症和癌症，而由于细胞毒性、致病性或辐射导致的AISCs的丧失- 诱发性损伤可威胁上皮细胞的完整性，并对健康构成严重威胁。AISC损伤是一种常见的副作用 癌症的化疗或放射治疗。作为对AISC损伤的反应，肠上皮有 通过成熟上皮细胞的去分化再生其干细胞库的非凡能力和 它们的祖细胞，功能是兼性的ISCs(FISCC)。然而，人们对基因的了解很少 允许这种高水平的细胞可塑性的机制。细胞能够同时调制的一种方式 许多基因的表达是通过染色质修饰来实现的。10-11易位甲基胞嘧啶 双加氧酶(Tet酶)通过5-甲基胞嘧啶的转化参与DNA去甲基化 (5mC)到5-羟甲基胞嘧啶(5hmC)，这是一个与基因激活相关的过程。Tet酶有很好的- 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，并发表 我们实验室的数据表明，Tet1在成年小鼠的ISCs和肠道上皮祖细胞中丰富。我的 初步数据显示，Tet1的缺失导致了异常的谱系指定，这表明Tet1在 Tet1在改变细胞身份中的作用。这一建议的中心假设是Tet1有助于ISC身份认同 通过激活细胞类型特定的基因表达进行过渡。我将用 以下具体目标：目标1将确定FEC去分化过程中Tet1的遗传要求 AISC损伤后，使用靶向AISC消融和细胞毒性损伤模型。损伤后的再生将 通过组织学和有机物生长分析进行评估。Aim 2A将表征Tet1‘S基因的调控 通过对诱导型Tet1的基因组、表观基因组和转录转录分析研究其分化过程中的机制 基因敲除(Tet1iKO)小鼠。Aim 2B将使用相同的方法来描述Te1‘S基因的调控机制 在遭受ISC损伤的Tet1iKO小鼠的去分化过程中。该项目将决定Tet1如何 促进细胞身份的改变，并为我提供培训，以实现我获得以研究为重点的 研究维持干细胞功能和建立细胞的染色质调节机制的教职 身份。这项工作将促进对再生机制的理解，并提供对肠道的洞察 上皮对损伤和损伤的反应。