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 TTATTAACA. Greater binding is observed at these sites by electrophoretic mobility shift assay when cells are cultured with RA and cyclic AMP analogs versus RA alone, and no binding is seen in extracts from RA-treated F9 RAR gamma-/- cells which lack RAR gamma mRNA and protein. Laminin B1 mRNA is not induced by RA in the RAR gamma-/- 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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U43597
To locate additional cis control regions that could regulate the transcription of the LAMB1 gene in F9 cells during the differentiation process, the murine LAMB1 gene was assayed for DNase I hypersensitive sites (DHSS)
In order to define the regulatory elements in the DHSS 2 region of the murine LAMB1 gene in greater detail, the series of LAMB1 promoter/CAT constructs indicated in Fig. 2A were transfected into F9 cells, and this transfection was followed by treatment of the cells with Retinoic acid (RA) for 24 h
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U43597. There was no augmentation of the RA response by cyclic AMP analogs using these LAMB1/CAT constructs in transient transfection assays (48).3 To locate additional cis control regions that could regulate the transcription of the LAMB1 gene in F9 cells during the differentiation process, the murine LAMB1 gene was assayed for DNase I hypersensitive sites (DHSS).
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