The 3' untranslated region (UTR) of alpha-striated tropomyosin promotes differentiation of fibroblasts into cells resembling striated muscle. These cells fuse, become multinucleate, elongate, and express striated muscle proteins in a pattern reminiscent of mature muscle. RNA harvested from transfected chick embryo fibroblasts was used for semiquantitative RT-PCR with primers specific for muscle transcription factors, showing that differentiation after transfection with the UTR is independent of a quantitative increase in transcription of myoD or myogenin. Vector-, mock-, and un-transfected fibroblasts, and osteoblasts express similar levels of these myogenic transcription factors as UTR-transfected fibroblasts, yet only UTR-transfected cells express muscle-specific proteins. Double immunofluorescent staining of UTR-transfected fibroblasts with antibodies to the muscle transcription factors of the myoD family and to the muscle-specific protein titin confirms that muscle differentiation is independent of translation of any muscle transcription factor in this family. In contrast, strong nuclear staining for the muscle transcription factor Mef-2 is detected after transfection of fibroblasts with the UTR, but not with vector alone. By RT-PCR, the expression of the cardiac transcription factors Nkx 2.5 and GATA-4 are abundantly detected in avian heart, but are not found in UTR-transfected fibroblasts. The expression of p21 ordinarily observed in skeletal myogenesis before the expression of striated muscle proteins is not seen in fibroblasts induced to differentiate by the UTR. These results demonstrate that the UTR of alpha-striated tropomyosin induces muscle differentiation in fibroblasts independent of the myoD family of muscle transcription factors or the cardiac transcription factors GATA-4 or Nkx 2.5, and without expressing proteins characteristic of terminal withdrawal from the cell cycle. Mef-2 is the only muscle-specific transcription factor whose activation accompanies this differentiation event. This implies that fibroblasts have access to additional differentiation routes that culminate in mature muscle than has been previously appreciated.
