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 β- myosin heavy chain (βMyHC) promoter conferred mechanical overload (MOV) and non-weight-bearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene. Whether the same DNA regulatory element(s) direct βMyHC expression following MOV or NWB activity in vivo remains unknown. We now show that a 293-base pair βMyHC promoter fused to chloramphenicol acetyltransferase (β293) responds to MOV, but not NWB activity, indicating a segregation of these two diverse elements. Inclusion of the βMyHC negative regulatory element (-332 to -300; βNRE) within transgene β350 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 βNRE sense strand (dβNRE-S; -332 to -311). Supershift electrophoretic mobility shift assay revealed that the binding at the distal region of the βNRE 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 crosslinking and shift Southwestern blotting analyses detected two proteins (50 and 52 kDa) that bind to this element. These in vivo results demonstrate that segregated βMyHC promoter elements transcriptionally regulate βMyHC transgene expression in response to two diverse modes of neuromuscular activity.