During differentiation of C2C12 myoblasts in vitro, expression of 1(VI) collagen mRNA was transiently stimulated severalfold. Promoter assays on cells transfected with chloramphenicol acetyltransferase (CAT) chimeric constructs have identified a region of the 1(VI) collagen promoter that increases CAT activity about 8-fold during differentiation. The region, which overlaps with transcription initiation sites, was shown to contain three protected segments (A, B, and C) in DNase I footprinting assays. The contact points between nuclear factors and the protected segments were determined by methylation interference assay and included the sequence GGGAGGG (GA box) in all segments. Experiments in which CAT constructs were cotransfected with double-stranded oligonucleotides containing the GA box suggested that this motif was necessary for induction. Transfections with deletion constructs of the natural promoter and with minipromoters made of three copies of A, B, or C showed that the elements have inducing activity and that elements C and, to a lower extent, B are stimulatory for basal transcription, whereas the contribution of A in this process is limited. Electrophoretic mobility shift assays with nuclear extracts from C2C12 cells indicated that the three GA box-containing elements bound several transcription factors, including Sp1. Comparison of the properties of the bands shifted under different experimental conditions (presence of 10 mM EDTA, heating of the nuclear extracts, addition of different concentrations of competitor oligonucleotides) established that A, B, and C probes form nine, eight and five main retarded complexes, respectively, and indicated that nuclear factors binding to C and B are subsets of proteins binding to A. UV cross-linking assays identified several peptides (seven with probe A, six with B, and five with C) in the range of 150-32 kDa. Comparison of the gel retardation pattern obtained with nuclear extracts from proliferating and differentiating cells revealed a particular increased intensity of two retarded bands. The data establish that multiple GA boxes mediate induction of the 1(VI) collagen promoter during myoblast differentiation and suggest the attractive hypothesis that the effect may be related to variations of expression of transcription factors binding to these motifs.

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