Figure 5.

A-utrophin transcript and a promoter A construct are both up-regulated during myogenic differentiation in vitro. (A) Quantitative RNase protection analysis of the endogenous utrophin transcripts was undertaken in C2C12 myoblasts as previously described (16), before (B) and 1, 4 and 7 days after starting differentiation by changing the culture medium to DMEM with 5% horse serum. Yeast RNA (Y) and mouse heart RNA (H) were included as negative and positive controls for the assay. A specific protected band for the A-utrophin transcript (205 bp) was detected in all C2C12 RNA samples. The amount of A-utrophin mRNA relative to total RNA increases by >2-fold during differentiation. These data indicate that promoter A drives the increase in total utrophin expression seen during myogenesis. (B) Proliferating and differentiating C2C12 cells were transfected with a construct in which 1.3 kb of utrophin promoter A drives luciferase. There was a 2-fold increase in luciferase activity on differentiation of the cells. This effect was not seen when the cells were transfected with the core promoter element only (0.3 kb) or an identical 1.3 kb fragment with a single point mutation in the conserved upstream E-box. Grey columns represent proliferating and white columns represent differentiating C2C12 cell lines.