The vimentin promoter as a tool to analyze the early events of retinoic acid-induced differentiation of cultured embryonal carcinoma cells

The vimentin gene encodes an intermediate filament protein expressed in the parietal endoderm, mesodermal, and early neural cells in vivo but by most in-vitro-cultured cells regardless of their embryonic origin. Here we show that the vimentin gene promoter is very active in F9 embryonal carcinoma cells and increases in activity during differentiation. Using a series of 5′- deletion mutants, we provide evidence that the regions of the promoter involved in F9 cell activity are different from those previously demonstrated to be active in differentiated cell lines. Furthermore, we show that in differentiating F9 cells the activities of two different regions of the promoter are significantly enhanced. A distal region (−1710/−957) appears to contain functional binding sites for the murine Hox-A5 homeoprotein as demonstrated by band shift and footprinting experiments. A proximal region (−140/−78) contains a 30-bp repetitive sequence found in other genes activated during differentiation of F9 cells. Using band shift assays and methylation interference, we present evidence that a sequence-specific single-stranded DNA-binding protein(s) specifically interacts with the minus strand of the 30-bp sequence.