Muscle-specific transcription factors in fibroblasts expressing the alpha-striated tropomyosin 3 ' untranslated region

The alpha-striated tropomyosin 3' untranslated region (TM UTR) promotes dif ferentiation of fibroblasts into cells resembling skeletal muscle. To inves tigate the mechanism of this observation, RNA harvested from transfected pr imary fibroblasts was used for semiquantitative RT-PCR with primers specifi c for muscle transcription factors, showing that myoD and myogenin transcri pts are detected in these cells, but that differentiation after TM UTR expr ession is independent of a detectable increase in these transcripts. Double immunofluorescent staining with antibodies to myoD family members and to t itin confirms that muscle differentiation in TM UTR-transfected fibroblasts is independent of production of any transcription factor in this family. I n contrast, the muscle transcription factor myocyte enhancer factor 2 (mef- 2) is strongly expressed after transfection of fibroblasts with the TM UTR, The increase in mef-2 protein is due to an increase in the steady-state le vel of its mRNA, as shown by Northern analysis. The expression of p21 ordin arily observed in skeletal myogenesis before the expression of muscle-speci fic proteins is not seen in fibroblasts induced to differentiate by the TM UTR, These results demonstrate that posttranscriptional regulation of myoD family members is seen in fibroblasts, and that the TM UTR induces muscle d ifferentiation independent of the myoD transcription factors and without ex pressing proteins characteristic of terminal withdrawal from the cell cycle . Finally, an increase in the steady-state level of mef-2 transcripts appea rs in the proximal pathway of myogenic activation in response to expression of the TM UTR. These results imply that fibroblasts can utilize an additio nal differentiation route upon TM UTR expression resulting in mature muscle other than that requiring myoD family members, (C) 1999 Academic Press.