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J Biol Chem, Vol. 274, Issue 43, 30832-30842, October 22, 1999

Nuclear Protein Binding at the -Myosin Heavy Chain A/T-rich Element Is Enriched following Increased Skeletal Muscle Activity

Dharmesh R. Vyas, John J. McCarthy, and Richard W. Tsika^§¶

From the  Department of Biomedical Sciences, School of Veterinary Medicine, the ^§ Department of Biochemistry, School of Medicine, and the ^¶ Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, Missouri 65211

In adult mouse skeletal muscle, -myosin heavy chain (MyHC) gene expression is primarily restricted to slow-type I fibers but can be induced in fast-type II fibers by mechanical overload (MOV). Our previous transgenic analyses have delimited an 89-base pair (bp) MOV-responsive region (293 to 205), and shown that mutation of the MCAT and C-rich elements within this region did not abolish MyHC transgene induction by MOV. In this study we describe an A/T-rich element (A/T-rich; 269 5'-GGAGATATTTTT-3' 258) located within this 89-bp region that, only under MOV conditions, revealed enriched binding as characterized by electrophoretic mobility shift assays and dimethyl sulfate and diethyl pyrocarbonate interference footprinting. Direct, competition, and supershift electrophoretic mobility shift assays revealed highly enriched specific binding activity at the A/T-rich element that was antigenically distinct from GATA-4, MEF2A-D, SRF, and Oct-1, nuclear proteins that were previously shown to bind A/T-rich elements. In vitro translated GATA-4, MEF2C, SRF, and Oct-1 bound to consensus GATA, MEF2, SRE, and Oct-1 elements, respectively, but not to the A/T-rich element. Two-dimensional UV cross-linking of the bromodeoxyuridine-substituted A/T-rich element with mechanically overloaded plantaris (MOV-P) nuclear extract detected two proteins (44 and 48 kDa). Our results indicate that the A/T-rich element may function in vivo as a MyHC MOV-inducible element during hypertrophy of adult skeletal muscle by binding two distinct proteins identified only in MOV-P nuclear extract.

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