Multiprotein complex formation at the beta myosin heavy chain distal muscle CAT element correlates with slow muscle expression but not mechanical overload responsiveness

To examine the role of the P-myosin heavy chain (beta MyHC) distal muscle C AT (MCAT) element in muscle fiber type-specific expression and mechanical o verload (MOV) responsiveness, we conducted transgenic and in vitro experime nts. In adult transgenic mice, mutation of the distal MCAT element led to s ignificant reductions in chloramphenicol acetyltransferase (CAT) specific a ctivity measured in control soleus and plantaris muscles when compared with wild type transgene beta 293WT but did not abolish MOV-induced CAT specifi c activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the beta MyHC MCA T element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the beta MyHC dista l MCAT element revealed that only the nucleotides comprising the core MCAT element were essential for LMC formation. The proteins within the LMC when using either MOV plantaris or control soleus nuclear extracts were antigeni cally related to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP -ribose) polymerase (PARP), and Max, Only in vitro translated TEF-1 protein bound to the distal MCAT element, suggesting that this multiprotein comple x is tethered to the DNA via TEF-1. Protein-protein interaction assays reve aled interactions between nominal TEF-1, PARP, and Max. Our studies show th at for transgene beta 293 the distal MCAT element is not required for MOV r esponsiveness but suggest that a multiprotein complex likely comprised of n ominal TEF-1, PARP, and Max forms at this element to contribute to basal sl ow fiber expression.