RETINOIC ACID--ROLE IN DIFFERENTIATION

DESCRIPTION: Retinoids are a group of natural and synthetic signaling molecule related to retinol (vitamin A). Retinoids such as retinoic acid (RA) can influence the growth and differentiation of many types of cells both during development and in the adult. Currently, retinoids are used clinically in both cancer prevention and treatment. The major goals of this proposal are to elucidate the molecular mechanisms by which retinoids regulate gene expression in target cells and to determine some of the functions of retinoid-regulated genes in development. Over this past grant period the P. I. has shown that RA transcriptionally activates the homeobox (Hox) genes Hoxa1 and Hoxb1 in both cultured embryonic stem (ES) cells and in transgenic animals through conserved RA-inducible 3' enhancers, termed RAIDR5s, which contain DR5 RAREs (RXR:RAR binding sites) and other regulatory elements such as CE1 and CE2. RARg is required for Hoxa1 mRNA induction by RA in these embryonic cells. Transgenic animal analysis has also shown that the CE2 element is a Hoxa1 somite/mesenchymal cell enhancer in embryos. Knockout of the Hoxa1 RAIDR5 enhancer in ES cells leads to reduced RA inducibility of several Hox genes of the Hoxa chromosomal cluster, indicating that this RAIDR5 is important in initiating retinoid effects on Hox genes. The P. I. has also identified severa putative Hoxa1 protein target genes, including Ran and "clone 62," a novel gen induced in Hoxa1 overexpressing cells. Over the next grant period the P. I. plans to knockout the RAIDR5 in animals and to analyze the phenotype which results. Moreover, the RAIDR5 enhancer will be relocated to a more 5' position in the Hoxa chromosomal cluster by homologous recombination to test its function in a more 5' location. It is also proposed to clone the protein which binds to the C2 element (somite/mesenchymal enhancer) of the RAIDR5. The putative Hoxa1 protein target genes Ran and clone 62 will be further characterized, and Hoxa2 and Hoxb1 target genes will be identified by differential screening/subtractive hybridization. The REX-1 gene, a gene transcriptionally repressed by RA, will be analyzed in both ES cells and transgenic animals to elucidate the mechanism by which RA reduces REX-1 expression via octamer and Rox-1 elements in the REX-1 promoter and to delineate the REX-1 DNA elements which impart cell type specific expression of REX-1 in embryos.