Nuclear protein binding at the beta-myosin heavy chain A/T-rich element is enriched following increased skeletal muscle activity.
In adult mouse skeletal muscle, beta-myosin heavy chain (betaMyHC) 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 betaMyHC transgene induction by MOV. In this study we describe an A/T-rich element (betaA/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 betaA/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 betaA/T-rich element. Two-dimensional UV cross-linking of the bromodeoxyuridine-substituted betaA/T-rich element with mechanically overloaded plantaris (MOV-P) nuclear extract detected two proteins (44 and 48 kDa). Our results indicate that the betaA/T-rich element may function in vivo as a betaMyHC MOV-inducible element during hypertrophy of adult skeletal muscle by binding two distinct proteins identified only in MOV-P nuclear extract.
Vyas
D R
DR
Department of Biomedical Sciences, School of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
McCarthy
J J
JJ
Tsika
R W
RW
D020856:AT Rich Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000502:physiology*; D018995:Myosin Heavy Chains / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014158:Transcription, Genetic; D020107:Troponin T / Q000235:genetics; D016474:Weight-Bearing
D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D020107:Troponin T; D018995:Myosin Heavy Chains