Point mutations and small substitution mutations in three different upstream elements inhibit the activity of the...

Point mutations and small substitution mutations were introduced in three different promoter segments of the mouse alpha 2(I) collagen gene that are binding sites for specific DNA-binding proteins. The first of these promoter elements contains a CCAAT motif between -80 and -84 and binds a novel factor consisting of two different components which are both needed for DNA binding (Hatamochi, A., Golumbek, P.T., Van Schaftingen, E., and de Crombrugghe, B. (1988) J. Biol. Chem. 263, 5940-5947). Four different point mutations in the CCAAT motif, which strongly inhibit binding of this heterodimeric CCAAT-binding factor, decrease the activity of the promoter at least 8-fold when assayed by DNA transfection of NIH 3T3 fibroblasts. The second cis-acting element is located around -250. Two mutations in this element, one a 3-base pair (bp), another a 4-bp substitution mutation, decrease the activity of the promoter in DNA transfection experiments by about 8- and 12-fold, respectively. The third element is located between -315 and -295 and binds nuclear factor 1 (NF1). Both 1- and 2-bp mutations which strongly inhibit binding of NF1 to this site decrease the activity of the promoter 8-10-fold in DNA transfection assays whereas another mutation in this site shows less inhibition in the activity of the promoter. Transfer of this NF1-binding site 5' to the SV40 early promoter increases the activity of this promoter more than 10-fold. These results indicate that the integrity of each of these three cis-acting elements is required for optimal activity of the alpha 2(I) collagen promoter and suggest a model whereby the factors which bind to these sequences cooperate in the formation of a transcription initiation complex.