Segregated regulatory elements direct β-myosin heavy chain expression in response to altered muscle activity

Our previous transgenic analyses revealed that a 600-base pair beta-myosin heavy chain (beta MyHC) promoter conferred mechanical overload (MOV) and non-weightbearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene. Whether the same DNA regulatory element(s) direct beta MyHC expression following MOV or NWB activity in vivo remains unknown. We now show that a 293-base pair beta MyHC promoter fused to chloramphenicol acetyltransferase (beta 293) responds to MOV, but not NWB activity, indicating a segregation of these two diverse elements. Inclusion of the beta MyHC negative regulatory element (-332 to -300; beta NRE) within transgene beta 350 repressed expression in all transgenic lines. Electrophoretic mobility shift assays showed highly enriched binding activity only in NWB soleus nuclear extracts that was specific to the distal region of the beta NRE sense strand (d beta NRE-S; -332 to -311). Supershift electrophoretic mobility shift assay revealed that the binding at the distal region of the beta NRE sense strand was antigenically distinct from cellular nucleic acid-binding protein and Y-box-binding factor 1, two proteins shown to bind this element, Two-dimensional UV cross-linking and shift Southwestern blotting analyses detected two proteins (50 and 52 kDa) that bind to this element. These in vivo results demonstrate that segregated beta MyHC promoter elements transcriptionally regulate beta MyHC transgene expression in response to two diverse modes of neuromuscular activity.