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  • Because Foxd regulates stem cell

    2018-11-12

    Because Foxd3 regulates stem cell properties in multiple lineages (Liu and Labosky, 2008; Hanna et al., 2002; Mundell et al., 2012; Mundell and Labosky, 2011; Teng et al., 2008; Tompers et al., 2005), Foxd3 target genes must regulate self-renewal, pluripotency, and/or survival of stem cells. Currently, only two direct targets of Foxd3 have been identified (Alb1 and the λ5-preB locus) (Liber et al., 2010; Xu et al., 2007). Therefore, we sought to identify additional targets of Foxd3. Using microarrays, qRT-PCR, and ChIP assays, we identified 6 novel targets of Foxd3: Sox4, Safb, Sox15, Fosb, Pmaip1 and Smarcd3. Additionally, we present data that Foxd3 functions upstream of genes required for skeletal muscle differentiation.
    Materials and methods
    Results
    Discussion We identified several genes, pathways, and biological functions that are misregulated in ESCs lacking Foxd3. Additionally, we identified 6 novel targets of Foxd3: Sox4, Safb, Sox15, Fosb, Pmaip1, and Smarcd3. We further characterized the Elesclomol of genes that function downstream of Sox15, and we showed that Foxd3 directly or indirectly regulates genes required for skeletal muscle development and regeneration, uncovering a novel role for Foxd3. The data presented in Fig. 3, together with previous work in the lab (Liu & Labosky, 2008), suggest that Foxd3 induced mutant ESCs precociously express genes required for mesoderm induction, but they are likely unable to differentiate into skeletal muscle. These data are consistent with the model shown in Fig. 3B in which Foxd3 represses Sox15 transcription resulting in increased Pax3 and decreased Myf5 expression in ESCs undergoing differentiation. An increase in Pax3 in skeletal muscle progenitors may result in increased self-renewal and decreased differentiation, limiting the number of mature skeletal muscle fibers (Epstein et al., 1995; Young & Wagers, 2010). Additionally, decreased Myf5 may result in decreased generation of skeletal muscle. The data presented here are consistent with a recent publication demonstrating the function of FOXD3 in hESCs; overexpression of FOXD3 in hESCs induces differentiation to paraxial mesoderm, including differentiation into skeletal myoblasts (Arduini & Brivanlou, 2012). Together, these data suggest a conserved function for Foxd3 in regulating skeletal muscle development in mammals. We hypothesize that the other targets of Foxd3 (Sox4, Safb, Fosb, Pmaip1 and Smarcd3) also regulate ES cell properties, and based on published accounts, several of these targets are of future interest. The transcription factor Sox4 is required for cardiac outflow tract development (Schilham et al., 1996; Ya et al., 1998; Maschhoff et al., 2003), a process regulated by the cardiac neural crest, another multipotent progenitor population in which Foxd3 function is critical (Mundell & Labosky, 2011; Teng et al., 2008; Nelms et al., 2011). The transcription factor FBJ osteosarcoma oncogene B (Fosb) promotes osteoblast differentiation while inhibiting adipogenesis (Sabatakos et al., 2000) suggesting that inhibition of Fosb by Foxd3 regulates differentiation of these lineages. Smarcd3 (also called Baf60c), a member of the Swi/Snf chromatin remodeling complex, associates with MyoD to promote transcription of genes required for myogenesis (Forcales et al., 2012; Ochi et al., 2008). While Sox15, Sox4, Fosb, and Smarcd3 have been implicated in regulating differentiation of disparate lineages, no one has carefully investigated the role of these proteins in maintaining ESC properties, and it is possible that Sox4, Fosb, and Sox15 are involved in maintaining pluripotency in ESCs. In addition to genes regulating pluripotency, two novel Foxd3 targets have the potential to regulate self-renewal of ESCs. Smarcd3 is a component of a Swi/Snf complex and is required to regulate self-renewal of neural stem cells (Lamba et al., 2008). While Smarcd3 mRNA can be detected in ESCs (Fig. 2B), to date, no one has analyzed the requirement for this protein in regulating self-renewal of ESCs. In addition, the function of the ubiquitously expressed nuclear scaffolding protein, Safb, has yet to be determined. It has been suggested that Safb may regulate the cell cycle, consistent with the possibility that Safb is required for ES cell proliferation and/or self-renewal (Huerta et al., 2007; Tapia et al., 2009; Debril et al., 2005). Together, this evidence from the literature is consistent with the hypothesis that these new targets of Foxd3 may regulate self-renewal in ESCs.