Retinoic acid (RA), a natural metabolite of retinol (vitamin A), is a powerful signalling molecule that influences the differentiation programs of many types of cells throughout life. RA also affects morphogenesis and pattern formation (the formation of structures such as the brain, face, limbs, etc. during embryogenesis). Moreover, RA can inhibit or reverse the process of malignant transformation in some cell types. How does RA accomplish this? Increasing evidence indicates that RA, acting through nuclear receptors (RARs and RXRs), regulates the expression of a number of different genes that encode transcription factors. We previously demonstrated that in F9 embryonic teratocarcinoma stem cells, RA treatment results in the rapid activation of the expression of the homeobox gene Hox 1.6 (formerly named ERA-1) and conversely, in the rapid decrease in the rate of transcription of the transcription factor REX-1. We have also identified an RA-sensitive enhancer several kilobases 3' of the Hox 1.6 gene through which the Hox 1.6 gene is directly activated by RA in F9 cells. These results demonstrate a molecular link between RA action and homeobox gene expression for the first time. We now want to determine 1) what regulatory proteins in addition to the RARs interact with this enhancer DNA, 2) whether this enhancer is the major enhancer that regulates Hox 1.6 expression in the embryo, and 3) if this enhancer is a """"""""master"""""""" enhancer that regulates the expression of other Hox genes in the Hox 1 cluster. Therefore, this RA-sensitive enhancer will be further characterized and dissected at a molecular level by using gel retardation assays, DNAse I footprinting, and in vivo footprinting techniques to identify potential regulatory proteins that bind to the enhancer DNA in F9 cells. Whether the enhancer regulates Hox 1.6 expression and the expression of other Hox genes in the Hox 1 cluster in the embryo will be addressed by the creation of appropriate transgenic mice that carry Hox 1.6/beta-galactosidase transgenes + the enhancer, and by knocking out both copies of enhancer DNA by homologous recombination. We also plan to identify the target genes regulated by the Hox 1.6 homeoprotein; this will be accomplished by employing cell lines that overexpress the Hox 1.6 gene vs. F9 wild type cells for differential screening of F9 cDNA libraries. We have some data suggesting that some of the Hox 1.6 target genes may be involved in the regulation of cell shape. We will use similar experimental approaches, utilizing both the F9 cell culture model differentiation system and transgenic animals, to determine how transcription of the REX-1 gene is inhibited by RA and what REX-1 genomic sequences impart cell type specific expression of REX-1 in embryos. The target genes of the REX-1 protein will be identified by using cell lines in which REX-1 expression is altered for """"""""differential screening"""""""" of libraries or by whole genome PCR. These studies of the regulation of the expression of Hox 1.6 and REX-1 by RA, combined with our investigations of the functions of the Hox 1.6 and REX-1 proteins, should provide us with significantly greater knowledge of the processes of cell differentiation, embryogenesis, and carcinogenesis.
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