Muscle cell differentiation is inhibited by the helix-loop-helix protein Id3

Id3 (originally named HLH462) belongs to the Id family of the helix-loop-helix transcription factors. Members of the Id family do not contain basic DNA binding regions adjacent to the helix-loop-helix dimerization domain and are, therefore, hypothesized to act as negative regulators of other helix-loop-helix proteins by preventing the formation of functional dimers. We have investigated the potential role of Id3 in the control of muscle cell differentiation. Id3 mRNA is expressed at a high level in proliferating myoblasts and is down-regulated following induction of differentiation. We show that stable overexpression of Id3 mRNA inhibits differentiation of the Sol 8 muscle cell line. Both the HLH and COOH-terminal domains of Id3 are necessary and sufficient for its dominant-negative activity in muscle cells. DNA-binding activity present in nuclear extracts prepared from Id3-overexpressing cells was significantly reduced when compared to the wild-type or vector-transfected cells. Finally, we show by in situ hybridization that the Id3 mRNA is co-expressed with the myogenic regulatory factor myogenin in somites and developing muscle during embryogenesis, although unlike the myogenic regulatory factors, Id3 is also expressed in many other locations in the embryo. These data support a model in which Id3 negatively regulates muscle differentiation by inhibiting the DNA-binding activities of the myogenic regulatory factors.