Multiprotein complex formation at the beta myosin heavy chain distal muscle CAT element correlates with slow muscle expression but not mechanical overload responsiveness.
To examine the role of the beta-myosin heavy chain (betaMyHC) 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 beta293WT 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 betaMyHC MCAT element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the betaMyHC 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 beta293 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.
Vyas
D R
DR
Department of Biochemistry, School of Medicine, School of Veterinary Medicine, and the Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211, USA.
McCarthy
J J
JJ
Tsika
G L
GL
Tsika
R W
RW
D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D001696:Biomechanical Phenomena; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008822:Mice, Transgenic; D046912:Multiprotein Complexes; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D018995:Myosin Heavy Chains / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C499330:MAX protein, human; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; C067850:Myc associated factor X; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D000091262:TEA Domain Transcription Factors; C057182:TEAD1 protein, human; D014157:Transcription Factors; C068422:Max protein, mouse; D015500:Chloramphenicol O-Acetyltransferase; D011065:Poly(ADP-ribose) Polymerases; D018995:Myosin Heavy Chains