The intent of this proposal is to study the regulation of the genes for basement membrane proteins such as laminin by the vitamin A derivative retinoic acid (RA). F9 mouse teratocarcinoma stem cells differentiate into parietal endoderm, an embryonic epithelia, in response to RA. This laboratory has recently isolated cDNA clones for the basement membrane constituents laminin B1, laminin B2, and collagen IV (alpha 1), and these basement membrane protein genes are expressed at high levels in F9 parietal endoderm cells, but not in F9 stem cells. Preliminary evidence suggests that these RA-associated increases in basement membrane gene specific mRNA expression are the result of increases in gene transcription. To understand how RA regulates the expression of four of these parietal endoderm cell specific genes, laminin B1, laminin B2, J6, and J31, cDNAs which cover the entire four mRNAs and genomic clones which cover the 5' flanking regions of the genes must be isolated. To delineate the DNA sequences necessary for RA- dependent transcriptional regulation, varying amounts of 5' flanking DNA from each of the gene will be fused to an indicator gene, chloramphenicol acetyl transferase (CAT); teratocarcinoma cells plus or minus RA will be transfected with these fusion genes, and transient expression of the CAT gene will be measured by enzyme assay. Cell lines stably transfected with these CAT expression vectors may also be analyzed. Other portions of these four genes (eg. introns) may also be examined to determine if intragenic sequences are required for RA responsiveness. A similar approach utilizing CAT analysis will be employed to delineate the DNA sequences necessary for the cyclicAMP- associated enhancement of the RA-induced increase in transcription of these genes. finally, both in vivo competition assays and in vitro DNA-binding assays will be performed in order to identify nuclear proteins which bind specifically to DNA sequences (""""""""RA responsive elements"""""""") which are involved in the regulation of laminin gene expression by RA. Vitamin A (retinol) is an essential nutrient, required for the proper growth and differentiation of many types of cells. Vitamin A regulates the differentiation of epithelial cells, and the production of extracellular matrix by these cells. Retinoids can also act as late stage inhibitors of the process of malignant transformation. Therefore, it is crucial that a better understanding of the mechanism by which retinoids regulate cell growth and differentiation be developed.
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