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Identification of master switch genes and MyoD
- The molecular basis of transdifferentiation

Identification of master switch genes

A number of genes exist that exemplify the definition of a master switch gene. For example, MyoD is thought to be the master switch gene for myogenesis while peroxisome proliferating activating receptor- γ (PPARγ) and CCAAT-enhancer binding protein- α (C/EBPα) are both involved in adipogenesis. The role of these transcription factors have largely been determined by in vitro studies. Pancreatic transcription factors such as Pancreatic-duodenal homeobox gene (Pdx1), Neurogenin3 (Ngn3), Hairy and Enhancer-of-split-1 (Hes1) and Pancreas transcription factor-1a/p48 (PTF1-p48) can also act as master switch genes. Each of these examples is discussed in more detail below.

Identification of MyoD

The master switch gene for muscle, MyoD, was found by a rather unusual experimental approach. It had been known for some time that treatment of the mouse fibroblast cell line 10T1/2 with the hypomethylating agent 5-azacytidine is sufficient to produce three different mesenchymal lineagesmyocyte, chondrocyte or adipocyte clones [4, 5]. All three phenotypes persist after withdrawal of 5- azacytidine, suggesting a stable switch has occurred. To identify which gene(s) was responsible for the switch from a fibroblast to a muscle phenotype, subtracted cDNA probes were initially prepared by isolating cDNA clones that are present in the 5-azacytidine-treated 10T1/2 cells and the mouse myogenic cell line C2C12 but not in the undifferentiated 10T1/2 cells. The myoblast-specific cDNA probes were then positively selected by taking the remaining cDNA probes from either 5- azacytidine-treated or C2C12 myoblasts and screening a myoblast cDNA library. One cDNA was identified which, when transfected into 10T1/2 cells, induced stable myoblast expression [6]. The cDNA was called MyoD. It is now known that at least four myogenic regulatory transcription factors (MRFs) are important for skeletal muscle commitment and myotube formation - Myf5, MyoD, myogenin and MRF4 - and all are basic helix-loop-helix (bHLH) nuclear proteins [7]. To determine whether MyoD was indeed the master switch gene for muscle, the transcription factor was introduced into primary fibroblasts, adipocytes, smooth muscle cells, baby hamster kidney cells, and hepatocytes. Upon MyoD expression, the cells started to adopt the muscle phenotype (myotube formation and fusion) and muscle cell markers myosin heavy chain (MHC) and myosin light chain (MLC2) became expressed. These results suggest that MyoD is indeed the master switch gene for muscle [8]. It is worth noting that in these examples of cell type conversions, germ layer boundaries are crossed; fibroblasts, adipocytes, smooth muscle and kidney cells are mesodermal in origin, but hepatocytes are endodermal. However, not all cells forced to express MyoD caused the muscle differentiation programme to be activated. Expression of MyoD in CV1 (an African green monkey kidney-derived cell) or HeLa (human cervical carcinoma) cells failed to activate differentiated muscle markers [8]. For melanoma and neuroblastoma cells the authors found co-expression of the parental melanocyte or neural markers with the muscle markers. This contrasts with adipocytes, in which the co-expression of adipocyte markers and muscle markers in the transdifferentiated myocytes was absent. This observation is possibly a result of myogenesis inhibiting differentiation pathways that are very close to the muscle lineage (e.g. the adipocyte lineage), but not those that are further away from the normal developmental pathway.

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