Contribution of specific cis-acting elements to activity of the mouse pro-alpha 2(I) collagen enhancer

Intronic transcriptional regulatory regions are found in several collagen genes. We demonstrate here the importance of two distinct cis-acting elements for the activity of a transcriptional enhancer located in the first intron of the mouse pro-alpha 2(I) collagen gene. Enhancer subfragments were tested for their ability to stimulate a linked promoter following transient transfection into NIH/3T3 fibroblasts. A 92-base pair subfragment retained significant enhancer activity. DNase I footprinting identified a binding site for a nuclear factor (designated CIBF-I) within this subfragment. Electrophoretic mobility-shift experiments demonstrated that an oligonucleotide containing 18 base pairs from the protected region was sufficient for specific binding of CIBF-I and suggested that CIBF-I may be related to a protein(s) that binds to the rat c-mos enhancer. A second cis-acting element, identified by electrophoretic mobility-shift and methylation interference experiments, bound affinity-purified Sp1 and an Sp1-like protein present in crude nuclear extracts. Small deletion mutations in either the CIBF-I or Sp1 site abolished factor binding in vitro and reduced enhancer activity in vivo. Our results represent the first identification of specific cis-acting elements required for full activity of the pro-alpha 2(I) collagen enhancer.