Id3 Is a Direct Transcriptional Target of Pax7 in Quiescent Satellite Cells

Abstract

INTRODUCTIONPax3 and Pax7 are two closely related transcription factors that are expressed in the dermomyotome, and they have been shown to be essential for generation of all fetal trunk musculature . Pax3/7-expressing dermomyotomal cells have recently been shown to give rise to skeletal muscle progenitors, termed satellite cells, in the adult (Gros et al., 2005;Kassar-Duchossoy et al., 2005;Relaix et al., 2005;Schienda et al., 2006). Satellite cells are a small population of myogenic progenitors that reside between the sarcolemma and basal lamina of the muscle fiber, and they play a crucial role in postnatal muscle growth and regeneration (reviewed in Zammit et al., 2006). Following skeletal muscle injury or exercise-induced activation, the normally quiescent Pax7-expressing satellite cells proliferate extensively and up-regulate expression of MyoD (Smith et al., 2001;Yan et al., 2003;Montarras et al., 2005) and Myf-5 (Conboy and Rando, 2002;Yan et al., 2003), before differentiation into skeletal muscle.Pax3 and Pax7 are members of the Pax transcription factor family and contain both paired (PD) and homeodomain (HD) DNA binding motifs. Pax3 and Pax7 can bind to DNA sequences containing either a consensus paired domain binding site (GTCAC A/G C/G A/T T/C) or a homeodomain binding site (ATTA) (Chalepakis et al., 1994;Chalepakis and Gruss, 1995). Pax3 and Pax7 activate MyoD expression (Maroto et al., 1997;Tajbakhsh et al., 1997;Relaix et al., 2003Relaix et al., , 2004, and recently Pax3 has been shown to directly bind sequences that regulate the expression of either MyoD in C2C12 cells (Hu et al., 2008) or Myf-5 during development of hypaxial muscle (Bajard et al., 2006). Although it is clear that Pax3/7 can directly induce the expression of Myf5 (Bajard et al., 2006), MyoD (Hu et al., 2008), and fibroblast growth factor receptor 4 (Lagha et al., 2008), other relevant targets for Pax3/7 in satellite cells have yet to be identified. To identify potential targets for Pax3/7 in satellite cells, we examined the transcriptional profile of genes induced by these transcription factors in the C2C12 muscle cell line. Of the genes identified, we found that a subset were also expressed in quiescent satellite cells and therefore they could potentially be direct targets of Pax7 in these cells. In this report, we focus on two such putative Pax3/7 transcriptional targets, inhibitor of DNA binding (Id) 2 and Id3, which we found to be expressed in quiescent satellite cells. We report that Pax3/7 can drive expression of both Id2 and Id3 in C2C12 cells under low serum conditions, that the Id3 promoter contains a conserved Pax3/7 binding site, and that Pax3/7 can activate expression of a reporter construct driven by the Id3 promoter. In addition, we demonstrate that Pax7 is normally bound to the Id3 promoter in quiescent satellite cells and that short hairpin RNA (shRNA)-mediated knockdown of Pax7 expression in cultured satellite cells Abbreviations used: C1R, complement component 1 R subunit; ChIP, chromatin immunoprecipitation; CS...

Pax3 induces differentiation of juvenile skeletal muscle stem cells without transcriptional upregulation of canonical myogenic regulatory factors

“…This was consistent with previous reports (Olguin and Olwin, 2004;Zammit et al, 2006;Olguin et al, 2007). The majority of SMPs express Pax7 protein in vivo and in vitro just after isolation (Cerletti et al, 2008), but Pax7 mRNA expression is reduced substantially soon after placing SMPs in culture (Kumar et al, 2009) (Fig. 4F).…”

miR-206 and -486 Induce Myoblast Differentiation by Downregulating Pax7

“…Our next question was whether Pax7 prevented its own repression via inhibition of miR-206 and -486. Pax7 directly upregulates a number of repressors of myogenesis, including Id2 and Id3 (22) (Fig. 9).…”

BMP-mediated induction of GATA4/5/6 blocks somitic responsiveness to SHH

“…−/− MEFs Sufu −/− MEFs were infected with retroviruses encoding pOZ-FlagHA-puro (Kumar et al, 2009), pOZ-GATA4-FlagHA-puro or pOZ-GATA5-FlagHApuro. Stable polyclones were selected using puromycin.…”

“…−/− MEFs that were infected with either pOZ-FlagHA-puro (the parental retrovirus) (Kumar et al, 2009), pOZ-GATA4-FlagHA-puro or pOZ-GATA5-FlagHA-puro were employed for ChIP as described previously (Carroll et al, 2005). Rabbit anti-FOG1 (as above), rabbit anti-HA (Santa Cruz Biotechnology, as above) or control rabbit IgG (Jackson Lab; 1:1000) was employed for ChIP.…”