Cellular Retinoic Acid Binding Proteins: Functional Studies

细胞视黄酸结合蛋白：功能研究

• 批准号: 7730984
• 负责人: LORRAINE J GUDAS
• 金额: $ 34.91万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-15 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7730984
• 关键词: 3T3 Cells; Acids; Acute Promyelocytic Leukemia; All-Trans-Retinol; Alleles; Antibodies; Biological Assay; Carcinoma; Cell Differentiation process; Cell Line; Cell Proliferation; Chemicals; Chromatin; Complex; DNA; Development; Differentiation Therapy; Direct Repeats; Disease; ES Cell Line; EZH2 gene; Effectiveness; Elements; Embryo; Enzymes; Event; Fibroblasts; Funding; Gene Expression; Gene Targeting; Genes; Goals; Grant; HRAS gene; Health; Histone Deacetylase; Histone H3; Human; Immunoprecipitation; Individual; Knock-out; Knowledge; Laboratories; Lysine; Malignant Neoplasms; Measures; Mediating; Molecular; Mus; Neuroblastoma; Normal Cell; Nuclear Receptors; Null Lymphocytes; Oncogenic; Pathway interactions; Pharmaceutical Preparations; Play; Polycomb; Polymerase Chain Reaction; Prevention; Process; Protein Binding; Proteins; RXR; Regulation; Regulatory Element; Research; Research Project Grants; Resistance; Retinoic Acid Receptor; Retinoic Acid Response Element; Retinoid Receptor; Retinoids; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Stem cells; Steroid Receptors; System; TNFRSF10B gene; Technology; Teratocarcinoma; Therapeutic; Time; Transcription Coactivator; Transcription Regulatory Protein; Transcriptional Activation; Transcriptional Regulation; Transferase; Tretinoin; Vitamin A; Vitamins; base; cancer cell; cancer pharmacology; cancer prevention; cancer therapy; cell type; cellular retinoic acid binding protein; chromatin immunoprecipitation; embryonic stem cell; genome-wide; homologous recombination; improved; leukemia; medical schools; neoplastic cell; overexpression; public health relevance; receptor; research study; response; retinoic acid 4-hydroxylase; self-renewal; stem; stem cell differentiation; tool; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Alterations in the expression of the genes that control stem cell differentiation and self-renewal often contribute to tumorigenesis. Our laboratory has studied the mechanisms by which retinoids (vitamin A (retinol) and its derivatives and metabolites), control gene expression in both normal stem cells and in tumors. Over this past grant period we have developed evidence that: a) the polycomb group (PcG) protein(s) play a major role in repressing retinoid signaling in embryonic stem (ES) cells; b) these polycomb mediated inhibitory signals are altered in tumor cells; and c) specific retinoid receptors act in concert with transcriptional coactivators in a gene specific context. Our immediate goal in the next grant period is to delineate the mechanisms by which the polycomb group mediated transcriptional repressive pathways and the retinoid transcriptional signaling pathways intersect. Our Specific Aims for the next grant period: 1) We will elucidate the mechanisms by which the polycomb proteins inhibit retinoid transcriptional activation and retinoic acid induced cell differentiation. This will be accomplished by a combination of ChIP (chromatin immunoprecipitation) assays; overexpression or reduced expression of key transcriptional regulatory proteins such as pCIP (SRC3), EZH2, SUZ12, and JMJD3 in F9 and ES Wt and RAR1, 2, and 3 null cells; and deletion of DNA regulatory elements such as the Hoxa1 RARE, followed by analysis of PcG protein binding. We also propose a comparison of 3T3 cells with H-Ras transformed 3T3 cells to assess how an oncogenic protein perturbs retinoid signaling. 2) We plan to define the domains of the specific retinoic acid receptors (RARs) 1, 2, and 3 (i.e. the receptors for all-trans retinoic acid (RA) and other retinoids) and the coactivators for these receptors that control the transcriptional events which result in cell differentiation (and, thus, aid in inhibiting tumor cell proliferation). 3) Finally, as part of our goal of understanding retinoid regulation of stem cell differentiation, we will undertake a genome-wide search for primary target genes of the RARs through the use of ChIP-on-chip (tiling) arrays or ChIP-seq technology. This approach will allow us to obtain key information about the genes transcriptionally activated as ES cells differentiate along different pathways. For these proposed experiments we will utilize (a) murine ES and teratocarcinoma stem cell lines with both alleles of each of the individual RARs "knocked out" by homologous recombination; (b) ES cell lines with some of the "downstream" RAR target genes "knocked out"; and (c) mice that have each of the individual RARs knocked out by homologous recombination. This proposed research will increase our knowledge of the molecular mechanisms by which signaling by retinoids is accomplished, provide new knowledge that will be useful for improvement of 'differentiation therapy' for cancer, delineate the mechanisms by which retinoids signal so that we will understand why some tumors are resistant to retinoid therapy, and allow us to manipulate the expression or function of polycomb repressive proteins to increase the effectiveness of retinoid based therapies and differentiation strategies for stem cells. PUBLIC HEALTH RELEVANCE: Cancer is a dangerous and dreaded disease. This research project will allow us to understand how normal mouse stem cells differentiate or mature in response to vitamin A, a vitamin required for health and without which we would all die. Our new research findings will allow us to improve therapies for cancer treatment, including new therapies in which chemicals related to vitamin A "force" cancer cells to differentiate and become more like normal cells ("differentiation therapy" for cancer).

描述（由申请人提供）：控制干细胞分化和自我更新的基因表达的改变通常有助于肿瘤发生。我们的实验室研究了类维生素A（维生素A（视黄醇）及其衍生物和代谢物）控制正常干细胞和肿瘤中基因表达的机制。在过去的资助期内，我们已经发现了以下证据：a）多梳组（PcG）蛋白在胚胎干（ES）细胞中抑制类维生素A信号传导中起主要作用; B）这些多梳介导的抑制信号在肿瘤细胞中改变;和c）特定的类维生素A受体在基因特异性背景下与转录辅激活因子协同作用。我们在下一个资助期的直接目标是描述polycomb组介导的转录抑制途径和类维生素A转录信号通路交叉的机制。我们下一个资助期的具体目标：1）我们将阐明polycomb蛋白抑制类维生素A转录激活和维甲酸诱导的细胞分化的机制。这将通过ChIP（染色质免疫沉淀）试验的组合来实现;在F9和ES Wt和RAR 1、2和3无效细胞中过表达或降低关键转录调控蛋白（如pCIP（SRC 3）、EZH 2、SUZ 12和JM JD 3）的表达;删除DNA调控元件（如Hoxa 1 RARE），然后分析PcG蛋白结合。我们还提出了3 T3细胞与H-Ras转化的3 T3细胞的比较，以评估致癌蛋白如何干扰类维生素A信号传导。2)我们计划定义特异性视黄酸受体（RAR）1、2和3（即全反式视黄酸（RA）和其他类视黄酸的受体）的结构域以及这些受体的共激活因子，这些受体控制导致细胞分化的转录事件（因此有助于抑制肿瘤细胞增殖）。3)最后，作为我们理解类维生素A调节干细胞分化的目标的一部分，我们将通过使用ChIP-on-chip（平铺）阵列或ChIP-seq技术对RAR的主要靶基因进行全基因组搜索。这种方法将使我们能够获得有关胚胎干细胞沿着不同途径分化时转录激活基因的关键信息。对于这些提出的实验，我们将利用（a）通过同源重组“敲除”每个单个RAR的两个等位基因的鼠ES和畸胎瘤干细胞系;（B）“敲除”一些“下游”RAR靶基因的ES细胞系;和（c）通过同源重组敲除每个单个RAR的小鼠。这项拟议的研究将增加我们对类维生素A信号传导完成的分子机制的知识，提供有助于改善癌症“分化治疗”的新知识，描述类维生素A信号传导的机制，以便我们理解为什么一些肿瘤对类维生素A治疗有抗性，并允许我们操纵多梳抑制蛋白的表达或功能，以增加基于类视色素的治疗和干细胞分化策略的有效性。公共卫生相关性：癌症是一种危险和可怕的疾病。这个研究项目将使我们了解正常的小鼠干细胞如何分化或成熟，以响应维生素A，维生素A是健康所需的维生素，没有它我们都会死亡。我们的新研究发现将使我们能够改进癌症治疗的疗法，包括与维生素A相关的化学物质“迫使”癌细胞分化并变得更像正常细胞的新疗法（癌症的“分化疗法”）。