Analysis of intestinal genes regulated by the transcription factor CDX2

转录因子CDX2调控的肠道基因分析

基本信息

批准号：
8102916
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
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8102916
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 Homeostasis 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-wide genome-wide analysis improved in vivo insight intestinal crypt intestinal epithelium intestinal villi novel public health relevance response transcription factor transcription factor CDX2

项目摘要

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. PUBLIC HEALTH RELEVANCE: Common disorders of the gastrointestinal (GI) tract, including inflammation, malabsorption, ulcers and cancer, cause considerable suffering and death. Although these conditions are fundamentally related to mechanisms of normal gene expression and cell behavior, there is limited appreciation of their molecular underpinnings. The CDX2 protein, which is found almost exclusively in the intestine, is a pivotal regulator in normal and disease states. Investigation of its regulatory functions will improve understanding and, ultimately, treatment of many common GI diseases.

描述（由申请人提供）：对负责肠道特异性基因调节和细胞分化的分子机制的欣赏是有限的。一个重要的调节剂转录因子CDX2仅限于肠上皮，在整个隐层villus单元中表达，并需要体内分化肠道特异性柱状上皮。了解CDX2的转录目标和机制的知识是不完整的，对其作为主转录调节器的功能的理解也是如此。我们已经使用了全基因组染色质免疫沉淀（CHIP）来识别结肠上皮细胞中CDX2占用率的置信度。 CDX2结合区域是高度保守的，并且显示了一些其他序列特异性的DNA结合蛋白的基序的明显聚类，以前涉及肠道基因调节。因此，我们的研究正确地识别了许多CDX2靶标，并在肠道特异性基因调节中揭示了3个特定的候选伴侣转录因子。我们将扩展初步数据和见解，以测试有关CDX2功能和分子机制的特定假设。 AIM 1寻求确定〜1,100个CDX2结合位点中的哪个是肠细胞中的真正的顺式元素。我们将确定对表达因子和强迫CDX2表达的细胞中CDX2耗竭反应的转录本。我们还将在功能报告测定中测试推定的顺式元素，并批判性地检查CDX2靶基因是否反映了主调节器的预期。出乎意料的是，我们发现CDX2通常占据DNA非常接近TCF蛋白的结合位点，即规范Wnt途径的转录效应子。 Wnt信号在许多组织中传播，但在肠内稳态中至关重要。我们将检验以下新的假设，即CDX2在全局WNT响应中赋予肠道特异性。我们还发现，核受体HNF4）在CDX2激活的基因和CDX2似乎抑制的基因附近的GATA蛋白附近的显着共占者。 AIM 2采用了几种方法来检验HNF4）和GATA因子与CDX2结合以激活和沉默基因的假设。最后，在孤立的小鼠肠道隐窝和绒毛馏分上的全基因组芯片意味着CDX2控制了这两个功能室内的不同基因。在AIM 3中，我们将检验该假设并解决潜在机制。我们将描述CDX2伴侣蛋白，并询问是否需要CDX2结合来产生隐窝和绒毛特异性的染色质结构域，或者相反，是否需要CDX2响应其他因素对此类域的响应。为此，我们已经确定了可以调节肠道CDX2水平的信息性染色质标记和生成的小鼠的全基因组分析的可行性。这些研究代表了阐明肠道特异性基因调节机制的详细而全面的方法。公共卫生相关性：胃肠道（GI）的常见疾病，包括炎症，吸收不良，溃疡和癌症，会造成巨大的痛苦和死亡。尽管这些疾病与正常基因表达和细胞行为的机制从根本上相关，但对它们的分子基础的认识有限。 CDX2蛋白几乎完全在肠道中发现，是正常和疾病状态的关键调节剂。对其调节功能的研究将改善对许多常见GI疾病的理解，并最终对治疗。