AN ENHANCER FOR TRANSCRIPTION OF COLLAGEN-IV GENES IS ACTIVATED BY F9CELL-DIFFERENTIATION

Expression of collagen IV genes is developmentally regulated and cell type-specific. To identify transcriptional control elements for the mo use alpha2(IV) collagen gene, several promoter constructs were transie ntly transfected into mouse PYS-2 (parietal yolk sac) cells. Within th e 5.5-kb (kilobase) upstream and 8.5-kb downstream sequences from the transcription start site, we have identified several regulatory active regions. Here, we report characterization of the most proximal 0.3-kb enhancer found at 4.5-kb upstream of the alpha2(IV) collagen gene. Th is enhancer is transcriptionally active in cells that make collagen IV such as PYS-2 cells and differentiated F9 cells, but has little if an y activity in cells that do not make collagen IV including NIH 3T3 cel ls and undifferentiated F9 embryonal carcinoma stem cells. This enhanc er, linked to the herpes simplex virus TK gene promoter, confers a cel l type-specific and differentiation-induced expression on the TK gene as well. Mutational and 5' and 3' deletion analysis demonstrate that t his enhancer activity requires two identical response elements (GAACAA T) present in the 0.3-kb enhancer sequence. In gel shift assay, the GA ACAAT element forms a complex that is specific for cells that make col lagen IV.