Retinoic Acid Binding Proteins: Functional Studies

类维生素A [维生素A及其代谢产物，如视黄酸（RA）]深刻影响细胞分化 在发展过程中。维甲酸也可以抑制恶性转化的过程，目前正在 用于治疗人类肿瘤。有很多证据表明RA受体RARc_、13和7以及RARc_、13和7的表达与RA受体的表达有关。 细胞RA结合蛋白CRABP-I和CRABP-II参与介导类维生素A的许多作用。 我们的长期目标是了解RARe，13，和. v的调节和功能，以及CRABP-I和 CRABP-II。我们的假设是，每个不同的RAR，R，13，和_，控制不同的转录。 这些靶基因的激活随后导致靶基因的扩增。 类红信号和细胞分化和增殖停滞。由于RAR/3表达缺失是 肿瘤进展中，了解RAR_靶基因的作用是一个重要的目标。为了实现这些目标， 在此期间，我们分离了一些特定的RAR_和RAR 7靶基因;消减杂交 和gone表达微阵列技术被用来比较F9野生型与F9 RAR_ -/-或F9 RAR <$V细胞，在我们实验室通过同源重组产生。据我们所知，这是 使用这种遗传方法首次鉴定RAR 13和_特异性靶基因。在AIM（1）中，我们将 采用两种模型系统：a）由以下诱导的畸胎瘤和胚胎干（ES）细胞的分化： RA或其它生物活性类维生素A;和B）RARc_、13和_敲除小鼠。我们将通过以下方式描述机制： 所述RAR特异性是通过使用以下方法表征这些RARf和RARe靶基因的启动子来实现的： 凝胶迁移分析、DNA酶I足迹法和启动子/报告基因构建体的瞬时转染。我们让男人枯萎 利用染色质免疫沉淀（CHIP）分析来鉴定与类维生素A反应性结合的蛋白质 靶基因启动子区域。我们还将描述这些靶基因在介导细胞凋亡中的功能。 通过产生其中这些靶基因异常表达的F9细胞系，使用 同源重组/基因打靶方法或四环素启动子激活和正义基因缺失 表情利用RAR基因敲除小鼠，我们将检测RAR基因的时空表达模式。 和RAR/靶基因在胚胎发生期间和成年小鼠的不同组织中。在AIM（2）中，我们将确定 通过分析WT ES与CRABP-I /-系和转基因动物， 其中CRABP-1和CRABP-2基因异常过表达。这些实验应该会导致 关于类维生素A和介导类维生素A作用的受体的作用的重要新信息。这 知识是理解复杂过程所必需的，如发育中的模式形成、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.