Disruption of the coordinate expression of muscle genes in a transfected BC₃H1 myoblast cell line producing a low level of the adenovirus E1A transforming protein

Abstract

Mouse BC3H1 myoblasts were stably transfected with the adenovirus 5 E1A gene. One clonal line, BC3E7, was found to differ in some important respects from those previously reported for E1A-transformed myoblasts. In contrast to BC3H1 cells which differentiate when confluent in medium containing 0.5% fetal calf serum (FCS), BC3E7 cells failed to elongate and align, to express acetylcholine receptor and creatine kinase, and to down-regulate expression of beta- and gamma-actins and tropomyosin isoform (TM) 1. However, increased synthesis of TMs 2, 3, and 4, and myosin light chain 1 associated with differentiation in BC3H1 still occurred in BC3E7 cells, and most surprisingly, alpha-actin was produced at a significant level in both proliferating and confluent BC3E7 cells. Interestingly, myogenin was expressed in confluent BC3E7 cells in 0.5% FCS, but not in 20%. The level of E1A expression in BC3E7 cells was found to be very low by analysis of mRNA, by immunoprecipitation of E1A protein, and by the ability of BC3E7 cells to complement the E1A-deficient adenovirus mutant dl312. These results suggest that different levels of E1A may be needed to repress different promoters and that E1A does not block myogenic differentiation by repressing myogenin expression, but represses each muscle gene independently.

Ability of adenovirus 5 E1A proteins to suppress differentiation of BC3H1 myoblasts correlates with their binding to a 300 kDa cellular protein.

“…From recent studies on BC3E7 cells, an ElA-transfected BC3H1 line that expresses ElA at a very low level, we concluded that ElA represses myogenic differentiation by repressing transcription of muscle-specific genes directly, rather than as a result of repressing expression of myogenin, a putative myogenic factor in these cells (Mymryk et al, 1992). All of the work discussed here suggests that by binding to p300, ElA proteins are able to repress transcription specifically from cellular genes associated with differentiation.…”

“…Graham, McMaster University. Transfected cell lines were selected and maintained as described previously (Mymryk et al, 1992 was washed six times in 0.75 ml of the same buffer with centrifugation, and immunoprecipitates were analyzed by electrophoresis on sodium dodecyl sulfate (SDS)-8.5% polyacrylamide gels followed by fluorography.…”

“…Cells Stably Transfected with ElA Mutants To verify that the results obtained with ElA mutants by viral infection could also be obtained by stable transfection, we transfected BC3H1 cells with plasmids containing E1A mutants dl101, dl1107, and d11151 (none in a d1520 background), together with the gene for neomycin resistance as described (Mymryk et al, 1992). (Figure 3).…”