Divergence in species and regulatory role of β-myosin heavy chain proximal promoter muscle-CAT elements

We examined the functional role of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB) regulation of a wild-type 293-base pair β-myosin heavy chain (βMyHC) transgene. Electrophoretic mobility shift assays (EMSA) revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding at the human distal MCAT element when using NWB soleus vs. control soleus nuclear extract. Compared with the wild-type transgene, expression assays revealed that distal MCAT element mutation decreased basal transgene expression, which was decreased further in response to NWB. EMSA analysis of the human proximal MCAT (pMCAT) element revealed low levels of NTEF-1 binding that did not differ between control and NWB extract, whereas the rat pMCAT element displayed robust NTEF-1 binding that decreased when using NWB soleus extracts. Differences in binding between human and rat pMCAT elements were consistent whether using rat or mouse nuclear extract or in vitro synthesized human TEF-1 proteins. Our results provide the first evidence that 1) different binding properties and likely regulatory functions are served by the human and rat pMCAT elements, and 2) previously unrecognized βMyHC proximal promoter elements contribute to NWB regulation.