Analysis of intestinal genes regulated by the transcription factor CDX2

转录因子CDX2调控的肠道基因分析

• 批准号: 8717640
• 负责人: Ramesh A Shivdasani
• 金额: $ 35.7万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8717640
• 关键词: Abbreviations; Address; Binding; Binding Sites; Biological Assay; CDX2 gene; CDX2 protein; Caco-2 Cells; Cancer Etiology; Cell Differentiation process; Cell Line; Cells; Cessation of life; Chromatin; Columnar Epithelium; DNA; Data; Data Set; Databases; Defect; Digestion; Disease; Elements; Epithelial; Epithelial Cells; Family; Gastrointestinal Diseases; Gastrointestinal tract structure; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genome; Genomics; Heart; Homeobox; Human; Human Genome; Immunoprecipitation; In Situ Hybridization; Indium; Inflammation; Intestines; Investigation; Knowledge; Life; Ligation; Malabsorption Syndromes; Maps; Measures; Mediating; Metabolism; Modeling; Molecular; Mus; Nuclear Receptors; Pathway interactions; Physiological; Proteins; RNA; Regulation; Regulatory Element; Reporter; Research Proposals; Resolution; Role; Sequence-Specific DNA Binding Protein; Signal Transduction; Site; Specificity; System; TCF7L2 gene; Testing; Tissue-Specific Gene Expression; Tissues; Transcript; Ulcer; Undifferentiated; Villus; absorption; base; cell behavior; chromatin immunoprecipitation; chromatin modification; combinatorial; computerized tools; gastrointestinal epithelium; genome analysis; genome-wide; genome-wide analysis; improved; in vivo; insight; intestinal crypt; intestinal epithelium; intestinal homeostasis; intestinal villi; novel; public health relevance; response; transcription factor; transcription factor CDX2

DESCRIPTION (provided by applicant): Appreciation of the molecular mechanisms responsible for intestine-specific gene regulation and cell differentiation is limited. One important regulator, the transcription factor CDX2, is restricted to intestinal epithelium, where it is expressed throughout the crypt-villus unit and required in vivo for differentiation of gut- specific columnar epithelium. Knowledge of CDX2's transcriptional targets and mechanisms is incomplete, as is understanding of how it functions as a master transcriptional regulator. We have used whole-genome chromatin immunoprecipitation (ChIP) to identify, with high confidence, regions of CDX2 occupancy in colonic epithelial cells. CDX2-binding regions are highly conserved and show significant clustering of motifs for a handful of other sequence-specific DNA-binding proteins previously implicated in intestinal gene regulation. Our studies hence correctly identify numerous CDX2 targets and reveal 3 specific candidate partner transcription factors in intestine-specific gene regulation. We will extend the preliminary data and insights to test specific hypotheses on CDX2 function and molecular mechanisms. Aim 1 seeks to identify which among ~1,100 CDX2-binding sites are bona fide cis-elements in intestine cells. We will identify transcripts that respond to CDX2 depletion in cells that express the factor and to forced CDX2 expression in cells that don't. We will also test putative cis-elements in functional reporter assays and critically examine whether CDX2 target genes reflect the activities expected of a master regulator. Unexpectedly, we find that CDX2 commonly occupies DNA very close to binding sites for Tcf proteins, transcriptional effectors of the canonical Wnt pathway. Wnt signals are transmitted in many tissues but are critical in intestinal homeostasis. We will test the novel hypothesis that CDX2 imparts intestinal specificity within a global Wnt response. We also find significant co-occupancy of the nuclear receptor HNF4) near CDX2-activated genes and of GATA proteins near genes that CDX2 appears to repress. Aim 2 takes several approaches to test the hypothesis that HNF4) and GATA factors combine with CDX2 to activate and silence genes, respectively. Lastly, genome-wide ChIP on isolated mouse intestinal crypt and villus fractions implies that Cdx2 controls distinct genes within these two functional compartments. In Aim 3 we will test this hypothesis and address the underlying mechanisms. We will delineate Cdx2 partner proteins and ask if Cdx2 binding is needed to generate crypt- and villus-specific chromatin domains or, conversely, if Cdx2 responds to the creation of such domains by other factors. To this end, we have established the feasibility of whole-genome analysis of informative chromatin marks and generated mice in which intestinal Cdx2 levels can be modulated. These studies represent a detailed and comprehensive approach to elucidate mechanisms of intestine-specific gene regulation.

描述（由申请人提供）：对肠道特异性基因调控和细胞分化的分子机制的认识是有限的。转录因子 CDX2 是一种重要的调节因子，仅限于肠上皮，在整个隐窝绒毛单位中表达，并且是体内分化肠道特异性柱状上皮所必需的。对 CDX2 转录靶点和机制的了解并不完整，对其作为主转录调节因子如何发挥作用的了解也是如此。我们使用全基因组染色质免疫沉淀 (ChIP) 来高可信度地识别结肠上皮细胞中 CDX2 占据的区域。 CDX2 结合区高度保守，并且显示出先前与肠道基因调控有关的少数其他序列特异性 DNA 结合蛋白的显着基序聚集。因此，我们的研究正确识别了众多 CDX2 靶标，并揭示了肠道特异性基因调控中的 3 个特定候选伙伴转录因子。我们将扩展初步数据和见解，以测试有关 CDX2 功能和分子机制的具体假设。目标 1 旨在确定约 1,100 个 CDX2 结合位点中哪些是肠细胞中真正的顺式元件。我们将鉴定对表达该因子的细胞中 CDX2 耗尽做出反应的转录物，以及对不表达该因子的细胞中强制 CDX2 表达作出反应的转录物。我们还将在功能报告分析中测试假定的顺式元件，并严格检查 CDX2 靶基因是否反映了主调节因子的预期活动。出乎意料的是，我们发现 CDX2 通常占据非常靠近 Tcf 蛋白（经典 Wnt 途径的转录效应子）结合位点的 DNA。 Wnt 信号在许多组织中传递，但对于肠道稳态至关重要。我们将测试 CDX2 在整体 Wnt 反应中赋予肠道特异性的新假设。我们还发现 CDX2 激活基因附近的核受体 HNF4 和 CDX2 似乎抑制的基因附近的 GATA 蛋白显着共占据。目标 2 采用多种方法来检验 HNF4 和 GATA 因子与 CDX2 结合分别激活和沉默基因的假设。最后，对分离的小鼠肠隐窝和绒毛部分进行全基因组 ChIP 表明 Cdx2 控制这两个功能区室中的不同基因。在目标 3 中，我们将测试这个假设并解决潜在的机制。我们将描述 Cdx2 伴侣蛋白，并询问是否需要 Cdx2 结合来生成隐窝和绒毛特异性染色质结构域，或者相反，Cdx2 是否对其他因素产生的此类结构域做出反应。为此，我们建立了对信息性染色质标记进行全基因组分析的可行性，并生成了可以调节肠道 Cdx2 水平的小鼠。这些研究代表了阐明肠道特异性基因调控机制的详细而全面的方法。