Molecular cloning of gene sequences transcriptionally regulated by retinoic acid and dibutyryl cyclic AMP in cultured mouse teratocarcinoma cells.

F9 mouse teratocarcinoma stem cells differentiate into primitive endoderm cells in response to retinoic acid (RA). A cDNA library, synthesized from RA and dibutyryl cyclic AMP-treated F9 teratocarcinoma cells, has been screened for gene sequences regulated by RA. By hybridization-selection and in vitro translation, the pcJ6, pcJ31, and pcF117 homologous mRNAs encode polypeptides of 40,000; 35,000-37,000; and 24,000 Da respectively. The pcI56 and pcI5 homologous mRNAs encode laminin B and collagen IV (S-Y. Wang and L. J. Gudas, 1983, Proc. Natl. Acad. Sci. USA 80, 5880-5884). Prior to RA addition, these gene sequences correspond to low-abundance mRNAs (less than 0.05% of total cellular mRNAs). Within 24 hr after the addition of RA (5 X 10(-7) M) to F9 cells, the expression of these sequences increases dramatically, and at 72 hr after drug addition, a 5- to 30-fold induction of these genes can be attained. Addition of lower concentrations of RA results in a smaller increase in the expression of these genes. If the F9 cells are treated with both RA (5 X 10(-7) M) and dibutyryl cyclic AMP (but2cAMP), the levels of mRNA specific for these five inducible genes are further increased by approximately 30- to 110-fold over those in the stem cells; but2cAMP alone does not induce the expression of these genes. The expression of J6 and J31, but not I56 (laminin) and 15 (collagen IV), is also regulated by RA in the P19 teratocarcinoma stem cell line, which differentiates into a fibroblastic cell type in response to RA. In vitro transcription experiments demonstrate that laminin and collagen IV are transcriptionally regulated by RA; but2cAMP also enhances the rate of transcription of these genes in F9 cells.