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J. Biol. Chem., Vol. 266, Issue 33, 22297-22302, 11, 1991
PD Burbelo, LA Bruggeman, GC Gabriel, PE Klotman and Y Yamada
Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Two regulatory regions in the murine collagen IV enhancer were identified. Transient transfection assays delimited a 210-base pair fragment within the first intron of the alpha 1(IV) collagen gene that had significant transcriptional enhancer activity. DNase I protection and gel mobility shift confirmed that two regions, designated footprints A and B, within this fragment bound nuclear factors. Gel shift studies suggested that the CCTTATCTCTGATGG motif (A-34) in the footprint A region was important for specific nuclear factor binding. Mutations in the A-34 motif abolished factor binding as detected by gel shift and resulted in a significant decrease in enhancer activity in transient transfection assays of F9 teratocarcinoma cells. Two putative transcription factors of Mr = 37,000 and Mr = 94,000, which interact with the A-34 motif, were purified from Engelbreth-Holm-Swarm tumor tissue using DEAE-Sephacel, heparin-Sepharose, salmon sperm DNA- Sepharose, and specific A-34 oligonucleotide affinity chromatography. Southwestern analysis revealed that both of these factors were capable of binding the A-34 oligonucleotide directly and did not require additional subunits for binding. These data suggest that positively acting transcription factor(s) interact with the A-34 site in the enhancer and are required for efficient transcription of the alpha 1 and alpha 2(IV) collagen chain genes.
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