The objectives of this competitive renewal are to understand the regulation and function of the retinoic acid receptors RARalpha, beta, and gamma, and the high affinity cellular retinoic acid binding proteins CRABPI and CRABPII. Two model systems will be used: a) the differentiation of murine teratocarcinoma and embryonic stem (ES) cells induced by RA, 4-oxoretinol, and/or by removal of the cytokine leukemia inhibitory factor (LIF); and b) transgenic mice. In the ES differentiation system, the applicant will delineate the DNA elements which regulate expression of the RARbeta, CRABPI and CRABPII genes by RA and 4-oxoretinol and by the removal of LIF. She will also use an ES:neuronal cell differentiation system to characterize the neural enhancer of the CRABPI gene which she has shown to be active in regulating the appropriate neural expression of the lacZ gene in transgenic mice. Gel shift assays, DNAse I footprinting, and expression cloning will be used to identify and clone the protein factors which interact with these regulatory elements in the RARbeta, CRABPI and CRABPII genes. She will also identify and characterize relevant biological target genes of RARbeta through the use of differential hybridization and subtractive hybridization approaches, using wild type vs. RARbeta -/- knockout lines. Since she has recently shown that the RARbeta -/- lines have lost their response to the growth inhibitory actions of RA, these experiments should provide critical insights into the mechanism of cell growth arrest by RA. To investigate the functions of CRABPI, wild type ES vs. CRABPI -/- ES knockout lines will be used. In the transgenic animals, delineate the sequences which control CRABPII mRNA expression in embryogenesis and in adult skin and CRAPBII proteins in specific tissues (basal vs. suprabasal cells of the skin) and at specific times during development using the LAP or the tet inducible expression vectors will be delineated.
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