TRANSCRIPTIONAL ACTIVATION OF THE ALPHA-1(VI) COLLAGEN GENE DURING MYOBLAST DIFFERENTIATION IS MEDIATED BY MULTIPLE GA BOXES

During differentiation of C2C12 myoblasts in vitro, expression of alph a 1(VI) collagen mRNA was transiently stimulated severalfold. Promoter assays on cells transfected with chloramphenicol acetyltransferase (C AT) chimeric constructs have identified a region of the alpha 1(VI) co llagen promoter that increases CAT activity about 8-fold during differ entiation. The region, which overlaps with transcription initiation si tes, was shown to contain three protected segments (A,B, and C) in DNa se I footprinting assays. The contact points between nuclear factors a nd the protected segments were determined by methylation interference assay and included the sequence GGGAGGG (GA box) in all segments. Expe riments in which CAT constructs were cotransfected with double-strande d oligonucleotides containing the GA box suggested that this motif was necessary for induction. Transfections with deletion constructs of th e natural promoter and with minipromoters made of three copies of A, B , or C showed that the elements have inducing activity and that elemen ts C and, to a lower extent, B are stimulatory for basal transcription , whereas the contribution of A in this process is limited. Electropho retic mobility shift assays with nuclear extracts from C2C12 cells ind icated that the three GA box-containing elements bound several transcr iption factors, including Spl. Comparison of the properties of the ban ds shifted under different experimental conditions (presence of 10 mM EDTA, heating of the nuclear extracts, addition of different concentra tions of competitor oligonucleotides) established that A, B, and C pro bes form nine, eight and five main retarded complexes, respectively, a nd indicated that nuclear factors binding to C and B are subsets of pr oteins binding to A. UV crosslinking assays identified several peptide s (seven with probe A, six with B, and five with C) in the range of 15 0-32 kDa. Comparison of the gel retardation pattern obtained with nucl ear extracts from proliferating and differentiating cells revealed a p articular increased intensity of two retarded bands. The data establis h that multiple GA boxes mediate induction of the alpha 1(VI) collagen promoter during myoblast differentiation and suggest the attractive h ypothesis that the effect may be related to variations of expression o f transcription factors binding to these motifs.