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Papers In Press, published online ahead of print September 28, 2000
J. Biol. Chem, 10.1074/jbc.M007750200
Submitted on August 24, 2000
Revised on September 26, 2000
Accepted on September 28, 2000

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

Dharmesh R. Vyas, John J. McCarthy, Gretchen L. Tsika, and Richard W. Tsika

Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211

Corresponding Author: tsikar{at}missouri.edu

To examine the role of the beta -Myosin Heavy Chain (beta MyHC) distal MCAT element in muscle fiber-type specific expression and mechanical overload (MOV)-responsiveness, we conducted transgenic and in vitro experiments. In adult transgenic mice, mutation of the distal MCAT element led to significant reductions in chloramphenicol acetyltransferase (CAT) specific activity measured in control soleus and plantaris muscles when compared to wild type transgene beta 293wt, but did not abolish MOV-induced CAT specific activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the beta MyHC distal MCAT element that was highly enriched only when using either MOV-Plantaris (MOV-P) or control soleus (CS) nuclear extract. Scanning mutagenesis of the beta MyHC distal 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-P or CS nuclear extracts were antigenically related to nominal TEF-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 complex is tethered to the DNA via TEF-1. Protein-protein interaction assays revealed interactions between NTEF-1, PARP, and Max. Our studies show that for transgene beta 293, the distal MCAT element is not required for MOV-responsiveness, but suggest that a multiprotein complex likely comprised of NTEF-1, PARP and Max forms at this element to contribute to basal slow fiber expression.


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