Murine laminin B1 gene regulation during the retinoic acid- and dibutyryl cyclic AMP-induced differentiation of embryonic F9 teratocarcinoma stem cells

Abstract

Retinoic acid (RA) and cyclic AMP analogs cause the differentiation of F9 embryonic teratocarcinoma stem cells into parietal endoderm, an epithelial cell of the early mouse embryo. Laminin B1 is induced in this differentiation process, but is not transcriptionally activated until 24-48 h after RA addition and is not maximally induced until approximately 72 h. Cyclic AMP analogs enhance this transcriptional activation. Although several DNase I hypersensitive sites (DHSS) were observed in the LAMB1 5'-flanking DNA, one of the sites, DHSS2, was detected only after 72 h of RA treatment. Transient transfections have demonstrated that the DHSS2 region functions as a 'late- acting RA-inducible enhancer,' and motifs in this enhancer contain the homeobox protein-binding site TTATYAACA. Greater binding is observed at these sites by electrophoretic mobility shift assay when cells are cultured with HA and cyclic AMP analogs versus RA alone, and no binding is seen in extracts from RA-treated F9 RARγ(-/-) cells which lack RARγ mRNA and protein. Laminin B1 mRNA is not induced by RA in the RARγ(-/-) cells (Boylan, J. F., Lohnes, D., Taneja, R., Chambon, P., and Gudas, L. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9601-9605). Our data show that these DNA regulatory elements contribute to the transcriptional activation of the LAMB1 gene during the later stages of the differentiation process.