Identification, cloning and characterization of MRF4: A new muscle regulatory factor
Abstract
Muscle development proceeds by the commitment of precursor cells to determined myoblast lineages. Myoblasts subsequently differentiate into fused, multinucleate myofibers that express muscle-specific genes such as those encoding the contractile proteins. To understand further the molecular mechanisms that regulate muscle development, a myogenic transcription factor, which has been named MRF4, has been identified and cloned. Strikingly, transfection of fibroblasts with MRF4 cDNA expression vectors alone is sufficient to convert the cells to stable muscle lineages. These MRF4-converted myogenic cells can be maintained as proliferating, determined myoblasts or can be induced to differentiate into myofibers. MRF4 is a member of a structurally-related, myogenic muscle regulatory factor gene family that also includes MyoD1, myogenin and Myf-5. However, MRF4 displays activities different from those of the other factors, consistent with MRF4 having a distinct role in myogenic development. MRF4-transfected cells express each of the other muscle regulatory factor genes, but transfection with MyoD1, myogenin or Myf-5 does not trans activate the endogenous MRF4 locus, suggesting that MRF4 may have an early role in the myogenic cascade. In addition, MRF4 is the predominant muscle regulatory factor transcript in adult skeletal muscle implying that it may regulate the establishment and maintenance of adult musculature. MRF4-converted myoblast cells are inhibited from differentiating into myofibers by added serum growth factors indicating regulation of muscle regulatory factor activity by growth factor-mediated pathways. The MRF4 protein contains a basic/helix-loop-helix (bHLH) motif, proposed to enable DNA-binding and protein oligomerization, that also has been observed in many other transcriptional regulators of diverse function defining a bHLH protein superfamily. MRF4 fusion proteins bind to contractile protein gene enhancer sequences revealing that MRF4 likely is involved in the terminal differentiation of muscle cells. Specific polyclonal antisera against each of the four muscle regulatory factors also were generated. Immunoprecipitation and immunolocalization studies with affinity-purified antibodies demonstrated that MRF4 is a serine/threonine phosphoprotein localized to the nucleus of the cell. These data are consistent with MRF4 being a muscle-specific myogenic transcription factor that may regulate both determination and differentiation events during skeletal myogenesis.
Degree
Ph.D.
Advisors
Konieczny, Purdue University.
Subject Area
Molecular biology
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