Transgenic MyoD Is Associated with Upregulation of Id2 mRNA During Induction of Differentiation at Low Cell Density in Balb 10(1)‐ and 10T1/2‐Derived Myogenic Cells

Myogenesis is the process of muscle development. MyoD is one of 4 muscle‐specific basic helix‐loop‐helix (bHLH) transcriptional factors in the myogenic regulatory factor (MRF) family. MyoD binds at distinct DNA sites during proliferation and differentiation of myogenic cells in culture. During proliferation only, MyoD binds to conserved regulatory regions in the genes for Id2 and Id3, transcriptional regulatory factors that are expressed in dividing cells but are repressed in differentiated cells. During differentiation, when myogenic cells stop proliferating, MyoD binds to and induces the expression of muscle‐specific genes. The function of MyoD binding at the Id2 and Id3 genes is not clear. We hypothesized that MyoD contributes to the transient upregulation of Id2 and Id3 during the transition from proliferation to differentiation. Such transient upregulation could prevent premature differentiation. To test our hypothesis, we are using pairs of congenic cell lines in which one line expresses MyoD from a stably‐transfected transgene, while the other line contains control transgenic DNA. We modeled the transition from growth to differentiation by inducing differentiation in subconfluent (40%) cells. RNA was extracted from cells over a time course of 48 hours from induction by switch to low serum from high serum medium. Using qRT‐PCR, we determined the accumulation of the mRNAs for MyoD, myogenin (a marker of MyoD activity and onset of differentiation), Id2, Id3, and RP58, a repressor of Id2 and Id3 transcription. With the Balb 10(1) cells, Id2 mRNA was transiently upregulated in the presence of MyoD at 12 hrs, preceding the upregulation of myogenin mRNA. Id3 and RP58 mRNA levels were more nearly similar +/− MyoD. With the 10T1/2 cells, Id2 was upregulated in the presence of MyoD (N=1), but with no indication of transience. The results with both MyoD+ Balb10(1) and 10T1/2 lines suggest that MyoD is up‐regulating expression of Id2. When 10T1/2 +/−MyoD cells were cultured to 100% confluence and then exposed to differentiation medium, MyoD did not appear to upregulate the Id2 gene. Taken together, these results suggest that Id2, but perhaps not Id3, is transcriptionally regulated by MyoD, and that the upregulation of Id2 in differentiation conditions is density dependent. Id2 was also upregulated when cells of the C2C12 myogenic cell line, derived from satellite cells of mouse hindlimb muscle, were induced to differentiate at low density (N=2). Therefore, we are currently identifying C2C12 MyoD shRNA knock‐down cell lines to use in similar experiments. Our project should contribute to understanding the functional significance of MyoD binding to the Id2 and Id3 genes and ultimately may help to further identify key factors in muscle associated diseases. Taken together, these results suggest that Id2, but perhaps not Id3, is transcriptionally regulated by MyoD, and that the upregulation of Id2 in differentiation conditions is density dependent. Id2 was also upregulated when cells of the C2C12 myogenic cell line, derived from satellite cells of mouse hindlimb muscle, were induced to differentiate at low density (N=2). Therefore, we are currently identifying C2C12 MyoD shRNA knock‐down cell lines to use in similar experiments. Our project should contribute to understanding the functional significance of MyoD binding to the Id2 and Id3 genes and ultimately may help to further identify key factors in muscle associated diseases. Support or Funding Information CSU MBRS RISE NIH via Grant #GM061331; CSU LSAMP‐BD NSF via Grant #HRD‐1363399; NSF MCB RUI‐99831‐40 and REU Supplement to Sandra B Sharp; Cal State LA Culminating Project Fund