Characterization of a cis-acting element required for efficient transcriptional activation of the collagen IV enhancer.
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.
Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
P D
PD
Burbelo
L A
LA
Bruggeman
G C
GC
Gabriel
P E
PE
Klotman
Y
Y
Yamada
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002872:Chromosome Deletion; D003094:Collagen / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D015183:Restriction Mapping; D013724:Teratoma; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D003094:Collagen; D003850:Deoxyribonuclease I