Retinoic Acid Binding Proteins: Functional Studies

维甲酸[维生素A及其代谢物，如维甲酸(RA)]深刻影响细胞分化 在开发过程中。维甲酸还可以抑制恶变过程，目前正在 用于治疗人类肿瘤。有大量证据表明，RA受体RARC_、13和7，以及 细胞维甲酸结合蛋白CRABP-I和CRABP-II参与了维甲酸的许多作用。 我们的长期目标是了解Rare、13和.v以及CRABP-I和CRABP-I的调节和功能 CRABP-II。我们的假设是，不同的RAR中的每一个，R，13和_，控制着不同的转录 靶基因集合以及这些靶基因的激活随后导致扩增 红色信号与细胞分化和增殖停滞。由于RAR/3表达缺失是 肿瘤进展，了解RAR_靶基因的作用是实现这些目标的重要目标， 在此授权期内，分离了一些特定的RAR_和RAR7靶基因；消减杂交 并利用Gone表达芯片技术比较了F9野生型和F9的Gone表达 RAR_-/-或F9 RAR？V细胞，本实验室通过同源重组获得。据我们所知，这是 利用这种遗传方法首次鉴定了RAR13和_1特异性靶基因。在AIM(1)中，我们将 使用两个模型系统：a)胚胎干细胞和畸胎瘤细胞的诱导分化 RA或其他生物活性维甲酸；以及b)RARC_，13和_，基因敲除小鼠。我们将通过以下方式来描述机制(S) 这些rarf基因和稀有靶基因的启动子是通过使用 凝胶移位分析、DNase I足迹和启动子/报告结构的瞬时转染。我们会让男人 利用染色质免疫印迹(ChIP)分析鉴定与视黄醇反应结合的蛋白质 靶基因启动子的区域。我们还将描述这些靶基因在调节细胞周期中的功能。 维甲酸通过产生这些靶基因异常表达的F9细胞株的作用，使用 同源重组/基因打靶方法或四环素启动子激活和正义消失 表情。利用RAR基因敲除小鼠，我们将研究RAR_1基因的时空表达模式。 和RAR/靶基因在胚胎发育和成年小鼠不同组织中的表达。在AIM(2)中，我们将确定 WT ES与CRABP-I/-系和转基因动物比较研究CRABP-I和CRABP-II的功能 其中CRABP-I和CRABP-II基因异常过表达。这些实验应该会导致 关于维甲酸的作用和介导维甲酸效应的受体的关键新信息。这 需要知识来理解复杂的过程，如发育中的图案形成、细胞 增殖控制、分化、致畸和肿瘤发生。

Retinoids [vitamin A and its metabolites such as retinoic acid (RA)] profoundly influence cell differentiation during development. Retinoids can also inhibit the process of malignant transformation, and are currently being used in the treatment of human tumors. There is much evidence that the RA receptors RARc_, 13,and 7, and the cellular RA binding proteins CRABP-I and CRABP-II are involved in mediating many of the effects of retinoids. Our long-term goals are to understand the regulation and functions of RARe, 13,and.v, and the CRABP-I and CRABP-II. Our hypothesis is that each of the different RARs, R, 13,and _,,controls the transcription of different sets of target genes and that activation of these target genes subsequently results in the amplification of the rednoid signal and cell differentiation and proliferation arrest. Since loss of RAR/3 expression is a feature of tumor progression, understanding the roles of RAR_ target genes is an important goal To achieve these goals, during this grant period _ e isolated a number of specific RAR_ and RAR7 target genes; subtractive hybridization and gone expression microarray techniques were employed to compare gone expression in F9 wild type vs. F9 RAR_ -/- or F9 RAR¿ V cells, generated in our lab by homologous recombination. To our knowledge, this is the first identification of RAR13 and _,specific target genes using such a genetic approach. In AIM (1), we wiil employ two model systems: a) the differentiation of teratocarcinoma and embryonic stem (ES) cells induced by RA or other bioactive retinoids; and b) RARc_, 13,and _,knockout mice. We will delineate the mechanism(s) by which RAR specificity is achieved by characterizing the promoters of these RARf_ and RARe, target genes using gel shift assays, DNAse I footprinting, and transient transfections of promoter/reporter constructs. We wilt men utilize chromatin immunopreclpitation (CHIP) assays to identify the proteins bound to the retinoid responsive regions of the target gene promoters. We will also delineate the functions of these target genes in mediating the effects of retinoids by generating F9 cell lines in which these target genes are aberrantly expressed, using homologous recombmation/gene targeting approaches or tetracycline promoter activated andsense gone expression. Using the RAR knockout mice, we will examine the spatio4emporal expression patterns of RAR_ and RAR,/target genes during embryogenesis and in different tissues of adult mice. In AIM (2) we will ascertain the functions of CRABP-I and CRABP-II by analyzing WT ES vs. CRABP-I /- lines and transgenic animals in which the CRABP-I and CRABP-II genes are aberrantly overexpressed. These experiments should result in critical new information about the actions of retinoids and the receptors which mediate retinoid effects. This knowledge is required to understand complex processes such as pattern formation in development, cell proliferation control, differentiation, teratogenesis, and tumorigenesis.