Ectopic expression of either Pax3 or Pax7 in C2C12 cells led to the induction of Id1, Id2, and Id3, specifically when these cells were cultured under low serum conditions. Although these genes are both expressed in proliferating C2C12 cells grown in 10% fetal bovine serum, their expression is significantly attenuated when these cells are cultured in low serum conditions (2% horse serum) for 3 d. In striking contrast, in C2C12 cells programmed to express either exogenous Pax3 or Pax7, expression of these genes is maintained in low serum-containing medium. Although the Id genes have previously been linked to cellular proliferation in at least some cellular contexts (Benezra et al., 1990
), in satellite cells Id2 and Id3 are expressed in both quiescent and proliferating cells, indicating that their expression can be uncoupled from the cell cycle in this cell type. shRNA-mediated knockdown of Pax7 expression in purified satellite cells leads to a significant loss in the expression of both Id2 and Id3 in activated satellite cells in vitro, suggesting that Pax7 is necessary to maintain high level expression of these Id family members in satellite cells. Because there are conserved paired domain and homeodomain binding sites located upstream of the mouse, rat, human, orangutan, dog, and horse Id3 genes, which are necessary for efficient induction of an Id3-luciferase reporter by either Pax3 or Pax7, we propose that Id3 is a direct transcriptional target of Pax3/7. Consistent with this notion, we have documented that Pax3 and Pax7 bind to chromatin containing this region of the Id3 gene in C2C12 cells programmed to express either Pax3 or Pax7 and that endogenous Pax7 is similarly bound to this region of the Id3 gene in quiescent satellite cells. Together, our findings suggest that Id2 and Id3 are transcriptional targets of Pax7 in satellite cells.
Previous work has indicated that >90% of CSM4B cells isolated from skeletal muscle express Pax7 and that such cells exhibit the functional characteristics of quiescent skeletal muscle stem cells or satellite cells (Cerletti et al., 2008
). When freshly isolated, this cell population displays robust expression of Pax7, Myf5, and both Id2 and Id3, yet it does not express appreciable levels of Id1. Culturing these cells in vitro leads to both their entry into the cell cycle (Cerletti et al., 2008
), induction of MyoD expression, and subsequent loss of Pax7 expression (A). The expression of Id2 and Id3 eventually declines in these cultures as well, in parallel with loss of Pax7 expression and induction of skeletal muscle differentiation. Previous work has indicated that expression of Id1 is high in proliferating C2C12 cells and significantly decreases when these cells are induced to differentiate in low serum-containing medium (Benezra et al., 1990
). The induction of Id genes by Pax3/Pax7 in C2C12 cells cultured in low serum, or expression of Id2 and Id3 in quiescent satellite cells, suggests that the expression of Id factors can be divorced from cellular proliferation. Interestingly, however, maximal induction of the Id3-luciferase reporter by cotransfected Pax3/7 required the presence of high serum.
Chromatin IP analysis demonstrated that Pax7 binds to a conserved binding site adjacent to the promoter of the Id3 gene in freshly isolated, quiescent satellite cells, and mutagenesis of the Id3 promoter has indicated that this Pax7 binding site is necessary for Pax7-mediated induction of an Id3-promoter-luciferase construct in transfected C2C12 cells. Based upon these findings, we propose that Pax7 may act to maintain the expression of Id3 (and potentially Id2) specifically in quiescent satellite cells (outlined in ). Once satellite cells are induced to enter the cell cycle and express MyoD, expression of Id2/3 may be maintained either by signaling pathways downstream of serum factors and/or by Pax7 that is also expressed in proliferating satellite cells. Indeed knockdown of Pax7 attenuated the expression of both Id2 and Id3 in cultures of proliferating satellite cells. Consistent with the notion that Pax7 may be necessary to maintain the expression of Id2/3 in growth arrested cells, expression of both Pax7 and Id2/3 is lost in differentiated myotubes.
Figure 5. Pax7 activates the expression of Id2 and Id3 in quiescent satellite cells. Based on our findings we speculate that Pax7 induces the expression of Id2/3 in quiescent satellite cells, where these dominant-negative HLH factors may act to block premature (more ...)
The Id family of HLH proteins have been demonstrated to block the function of bHLH proteins such as MyoD and Myf5, by titrating their E protein binding partners (Benezra et al., 1990
); and to also block negative autoregulation of the Notch effector Hes1 (Bai et al., 2007
) and thereby maintain high level expression of Hes1 and potentially other members of the hairy/enhancer of split family. In this light, it is interesting that quiescent satellite cells express relatively robust levels of both Id2 and Id3 (this study) and several members of the hairy/enhancer of split family, including Heyl (Fukada et al., 2007
), Hes1 and Hey1 (Wagers, unpublished observations). It is possible that high-level expression of Id2 and Id3 in quiescent satellite cells acts to either block the activity of Myf5 that is readily expressed (at least at the RNA level) in at least 90% of quiescent satellite cells (Kuang et al., 2007
) or to maintain the expression of the Notch-induced transcriptional repressors Hes1, Hey1, and/or Heyl, which in turn may block expression of MyoD and Myf5. Thus, by inducing the expression of Id2 and Id3, Pax7 may act to block the premature differentiation of quiescent satellite cells. In addition, Pax7 may also block skeletal muscle differentiation by targeting myogenic bHLH proteins for degradation (Olguin et al., 2007
). In contrast, in activated satellite cells, Pax7 may act to directly induce the expression of Myf5 (Bajard et al., 2006
) and MyoD (Hu et al., 2008
), which in turn will compete with Id proteins for E protein dimerization. In this scenario, Pax7 serves two roles in satellite cells: blocking their differentiation via Id2/3 induction during quiescence and promoting their differentiation via Myf5/MyoD induction during activation (). Interestingly, Id family members have been implicated in both promoting self-renewal and blocking differentiation of hematopoietic (Jankovic et al., 2007
) and neural (Robert Benezra, personal communication) stem cells, and our work suggests that they may play a similar role in satellite cells as well. Although mice deleted for Id3 are viable and show no discernible muscle phenotype (Pan et al., 1999
), mice lacking both Id2 and Id3 are embryonic lethal (Robert Benezra, personal communication). Because quiescent satellite cells express both Id2 and Id3 (), it is possible that the lack of a skeletal muscle phenotype in mice lacking only Id3 (Pan et al., 1999
) is due to functional redundancy with Id2. Therefore, to determine whether Id2 and Id3 are indeed necessary to block differentiation of quiescent satellite cells, it will be necessary to conditionally delete both these genes in this cell type.
In addition to Id2 and Id3, we noted that several other genes induced by Pax7 in C2C12 cells were also specifically expressed in quiescent satellite cells. These genes include ITM2A, the chemokine receptor CXCR4, BMP4, the sugar binding protein chondrolectin, C1R, PTGIS, and MGP. Interestingly, the expression of many of these Pax7 inducible genes was greatest in quiescent satellite cells and expression of these genes was extinguished during either proliferation or differentiation of these cells. CXCR4 has already been demonstrated to play a crucial role in migration of skeletal muscle cells during embryogenesis (Vasyutina et al., 2005
). It will be interesting to determine the role of both Id2/Id3 and these other putative Pax7 transcriptional targets in quiescent satellite cells.