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.
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