A novel sequence in the type IV collagen promoter binds nuclear proteins from Engelbreth-Holm-Swarm tumor.
The production of extracellular matrix proteins is an important element of tumor formation, and alterations in matrix protein metabolism may be critical to the process of tumor metastasis. Abundant expression of type IV collagen, the major structural protein of the basement membrane, is characteristic of the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. In the present study, we evaluated mechanisms of transcriptional regulation of type IV collagen genes by analysing nuclear factors that bind to the promoter region. Gel mobility-shift assays indicated that specific proteins from EHS tumor bound the promoter and generated several unique shift patterns. The specific sequences to which these proteins bound were determined using DNAase I protection assays. DNA-binding proteins protected two regions from DNAase I digestion. The first region was similar to a GC box, the binding site for the transcription factor Sp1. The other footprint was a 30-bp region that contained the novel sequence motif, 'CCCTCCC' present in several other extracellular matrix promoters. Nuclear extracts isolated from tissues that variably express type IV collagen bound to this protected sequence with distinctly different shift patterns. Furthermore, in highly expressing tissues, unlabeled oligonucleotides containing the 'CCCTCCC' motif effectively inhibited nuclear protein binding with the entire promoter. Thus, it is likely that a novel protein or protein complex binds to these sequences. Furthermore, these sequences appear to be unique to the genes that encode basement membrane proteins, suggesting a specific role in their regulation.
Bruggeman
L A
LA
Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Burbelo
P D
PD
Yamada
Y
Y
Klotman
P E
PE
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003094:Collagen / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012513:Sarcoma, Experimental / Q000235:genetics*
D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D003094:Collagen; D003850:Deoxyribonuclease I