Role of the transcription factor Cdx2 in intestinal stem cells

转录因子Cdx2在肠干细胞中的作用

• 批准号: 8524730
• 负责人: Adrianna K San Roman
• 金额: $ 2.96万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8524730
• 关键词: Address; Adenomatous Polyposis Coli; Adult; Animals; Binding; Biological Models; CDX2 gene; CDX2 protein; Cancer Etiology; Cancerous; Cell Differentiation process; Cell Proliferation; Cell physiology; Cells; Cessation of life; ChIP-seq; Chromatin; Colorectal Cancer; DNA Binding; DNA-Binding Proteins; Data; Defect; Detection; Development; Disease; Enhancers; Enterocytes; Epidermal Growth Factor; Epithelium; Future; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Models; Genetic Transcription; Genomics; Homeobox; Homeostasis; Human; Human Cell Line; Immunohistochemistry; Indium; Intestinal Cancer; Intestinal Neoplasms; Intestines; Knowledge; Location; Maintenance; Mediating; Messenger RNA; Mitotic; Modeling; Molecular Conformation; Mus; Natural regeneration; Organoids; Pathogenesis; Pathway interactions; Play; Population; Process; Property; Regenerative Medicine; Regulation; Research; Role; Signal Pathway; Signal Transduction; Small Intestines; Staging; Stem cells; Structure of intestinal gland; System; Testing; Time; Tissue Differentiation; Tissues; Transcript; Transcriptional Regulation; United States; Villus; Work; adenoma; base; cancer cell differentiation; cell type; chromatin immunoprecipitation; colon cancer cell line; genome-wide; homeodomain; improved; in vivo; insight; intestinal epithelium; intestinal villi; mortality; mutant; notch protein; overexpression; progenitor; programs; promoter; public health relevance; regenerative therapy; self-renewal; stem; stem cell population; transcription factor; transcriptome sequencing; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Intestinal stem cells (ISCs) replenish the intestinal epithelium every few days and represent a general model for tissue differentiation and stem cell properties. Differentiation of ISCs and their immediate progeny into mature cells requires precise control of genes that are expressed in each stage. Although mature intestinal cells could always be obtained in large numbers to study gene regulation, ISCs were, until recently, difficult to isolate. Discovery of the ISC marker Lgr5 has made it possible to isolate and characterize this cell population, which is also important to regenerative medicine and the study of intestinal cancers. Homeostasis of the intestinal epithelium is sustained by rapidly dividing Lgr5+ ISCs. One crucial factor for gene activation in differentiated intestinal cells is the transcription factr Cdx2 (caudal type homeobox 2), however its role in ISCs was previously unknown. My analysis of Cdx2-/- intestines reveals that ISCs lacking Cdx2 are significantly impaired in replication and in the long term unable to give rise to mature cell types. Thus, Cdx2 seems to serve distinct roles in ISCs and their terminally differentiated progeny and this difference is reflected in my detection of Cdx2 binding at distinct enhancer and promoter elements in Lgr5+ ISCs and mature intestinal villus cells in vivo. This proposal builds on these preliminary data to address the specific role of Cdx2 in ISCs. In Aim 1 I will test the hypothesis that Cdx2 directly regulates key signaling pathways required for ISC proliferation. To reveal Cdx2's direct transcriptional targets in ISCs in vivo, I will integrate data from Cdx2 chromatin immunoprecipitation (ChIP-seq) in ISCs with global mRNA transcript profiling (RNA-seq) of wild-type and Cdx2-/- mouse ISCs. As my preliminary data point to epidermal growth factor (EGF) signaling as a strong direct Cdx2 target pathway, I will use mouse intestinal organoid cultures to additionally test the specific hypothesis that Cdx2 controls ISC proliferation through EGF signaling. In Aim 2 I will determine whether Cdx2 loss, which limits ISC proliferation, also limits the formation of tumors (adenomas) that originate specifically in ISCs. Although Lgr5+ ISCs are the likely cell of origin for intestinl tumors, including human colorectal cancer, the role of Cdx2 in the disease is confusing. To test the hypothesis that tumor formation is significantly compromised in the absence of Cdx2 function, I will conditionally remove both Cdx2 and Apc (a negative regulator of the tumor-promoting Wnt signaling pathway) in Lgr5+ ISCs. This strategy will address the controversy about Cdx2 functions in intestinal tumors in a genetic model system. Together, Aims 1 and 2 have the potential for significant impact on understanding of ISC regulation, which can be applied to promising stem cell-based regenerative therapies as well as improved understanding of colorectal cancer, the second leading cause of cancer mortality in the United States.

描述（由申请人提供）：肠道干细胞（ISC）每隔几天补充肠上皮，代表组织分化和干细胞特性的一般模型。 ISC及其直接后代对成熟细胞的分化需要精确控制每个阶段表达的基因。尽管始终可以大量获得成熟的肠细胞来研究基因调节，但直到最近，ISC还是很难分离。 ISC标记LGR5的发现使分离和表征该细胞种群成为可能，这对于再生医学和肠癌的研究也很重要。肠上皮的稳态通过迅速分裂的LGR5+ ISC维持。分化肠细胞中基因激活的一个关键因素是转录FACTR CDX2（尾型同源蛋白蛋白蛋白酶2），但是其在ISC中的作用以前尚不清楚。我对CDX2 - / - 肠道的分析表明，缺乏CDX2的ISC在复制方面受到了显着损害，并且从长远来看无法引起成熟的细胞类型。因此，CDX2似乎在ISC中起着不同的作用，并且它们的终端分化后代，并且这种差异反映在我检测到LGR5+ ISCS和成熟的肠绒毛细胞中不同增强子和启动子元素处的CDX2结合。该建议基于这些初步数据，以解决CDX2在ISC中的特定作用。在AIM 1中，我将测试CDX2直接调节密钥的假设 ISC增殖需要的信号通路。为了揭示在体内ISC中CDX2的直接转录靶标，我将在ISC中整合具有野生型和CDX2 - / - 小鼠ISC的ISC中CDX2染色质免疫沉淀（CHIP-SEQ）的数据。作为我的初步数据指向表皮生长因子（EGF）信号传导作为强大的直接CDX2目标途径，我将使用小鼠肠道器官培养物来替代特定的假设，即CDX2通过EGF信号传导CDX2控制ISC增殖。在AIM 2中，我将确定CDX2损失限制了ISC增殖，还限制了特有源自ISC的肿瘤（腺瘤）的形成。尽管LGR5+ ISC可能是包括人结肠直肠癌在内的肠肿瘤的起源细胞，但CDX2在疾病中的作用令人困惑。为了测试在没有CDX2功能的情况下肿瘤形成显着损害的假设，我将在LGR5+ ISC中有条件地删除CDX2和APC（促进肿瘤Wnt信号通路的负调节剂）。该策略将解决有关遗传模型系统中肠道肿瘤中CDX2功能的争议。总之，目标1和2具有对理解ISC调控的重大影响，这可以应用于有前途的干细胞再生疗法以及对大肠癌的理解，这是美国癌症死亡率的第二大主要原因。