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J. Biol. Chem., Vol. 276, Issue 2, 1173-1184, January 12, 2001
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From the § Department of Biochemistry, School of
Medicine, the To examine the role of the
Multiprotein Complex Formation at the
Myosin Heavy Chain
Distal Muscle CAT Element Correlates with Slow Muscle Expression
but Not Mechanical Overload Responsiveness*
,,, and§¶
Department of Biomedical Sciences, School
of Veterinary Medicine, and the ¶ Dalton Cardiovascular Research
Center, University of Missouri, Columbia, Missouri 65211
-myosin heavy chain
(MyHC) distal muscle CAT (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 with
wild type transgene 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 MyHC MCAT element that was highly enriched only when
using either MOV plantaris or control soleus nuclear extract.
Scanning mutagenesis of the 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
plantaris or control soleus nuclear extracts were antigenically 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 complex is tethered to the DNA via TEF-1. Protein-protein
interaction assays revealed interactions between nominal TEF-1, PARP,
and Max. Our studies show that for transgene 293 the distal MCAT element is not required for MOV responsiveness but suggest that a
multiprotein complex likely comprised of nominal TEF-1, PARP, and Max
forms at this element to contribute to basal slow fiber expression.
*
This work was supported by National Institutes of Health
Grants R01-AR41464 and R01-AR47197 (to R. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Missouri-Columbia, Dept. of Veterinary Biomedical Sciences and
Department of Biochemistry, 1600 E. Rollins Ave., W112 VET Medicine
Bldg., Columbia, MO 65211. Tel.: 573-884-4547; Fax:
573-884-6890; E-mail: tsikar@missouri.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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