Recent genetic and molecular studies indicated DUX4 as the likely candidate gene for FSHD (Dixit et al., 2007
; Lemmers et al., 2010
; Snider et al., 2010
). Although the abundance of DUX4-fl mRNA was extremely low in FSHD muscle, we previously showed that this represented relatively high expression of both DUX4-fl mRNA and protein in a small percentage of muscle nuclei at any time point, either because the gene was on transiently or the expressing nuclei were eliminated (Snider et al., 2010
). Yet, it remained unclear whether DUX4-fl expression had a biological consequence in FSHD. In our current study, we identify genes regulated by DUX4-fl and show that they are expressed at readily detectable levels in FSHD skeletal muscle, both cell lines and muscle biopsies, but not in control tissues, providing direct support for the model that misexpression of DUX4-fl is a causal factor for FSHD. The genes regulated by DUX4-fl suggest several specific mechanisms for FSHD pathophysiology.
Many of the genes highly upregulated by DUX4-fl normally function in the germline and/or early stem cells and are not present in healthy adult skeletal muscle. This supports a biological role for DUX4-fl in germ cell development and suggests potential disease mechanisms for FSHD. Activation of the gametogenic program might be incompatible with post-mitotic skeletal muscle, leading to apoptosis or cellular dysfunction. Also, the testis is an immune-privileged site and testis proteins misexpressed in cancers can induce an adaptive immune response (Simpson et al., 2005
). In fact, some of the genes regulated by DUX4-fl, such as the PRAME family (Chang et al., 2011
), are known cancer testis antigens, so it is reasonable to suggest that expression of these genes in skeletal muscle might also induce an adaptive immune response. An immune-mediated mechanism for FSHD is consistent with the focal inflammation and CD8+ T-cell infiltrates that characterize FSHD muscle biopsies (Frisullo et al., 2011
; Molnar et al., 1991
The induction of DEFB103 by DUX4 might influence both the adaptive and the innate immune response. DEFB103 can have a pro-inflammatory role in the adaptive immune response (Funderburg et al., 2007
) and can act as a chemo-attractant for monocytes, lymphocytes and dendritic cells (Lai and Gallo, 2009
). In this regard, it might enhance an adaptive immune response to germline antigens expressed in FSHD muscle. Though traditionally known for its role in antimicrobial defense (Sass et al., 2010
), DEFB103 has been shown to suppress the innate immune response to LPS and TLR4 stimulation in macrophages (Semple et al., 2011
; Semple et al., 2010
), and has also been shown to be an antagonistic ligand of the CXCR4 receptor (Feng et al., 2006
), which is important for muscle migration, regeneration, and differentiation (Griffin et al., 2010
; Melchionna et al., 2010
). In this study we show that DEFB103 inhibited the innate immune response to lentiviral infection in skeletal muscle cells, modestly induced myostatin in myoblasts, and impaired muscle cell differentiation. Therefore, DEFB103 might contribute to FSHD pathology by modulating the adaptive and innate immune response, as well as through inhibiting muscle differentiation. In this regard it is interesting to note that expression of murine Defb6 in the skeletal muscle of transgenic mice induced progressive muscle degeneration (Yamaguchi et al., 2007
), although the mechanism was not determined.
Reactivation of retroelements can result in genomic instability (Belancio et al., 2010
) and transcriptional deregulation (Schulz, 2006
). Therefore, DUX4 activation of MaLR transcripts might directly contribute to FSHD pathophysiology. It is interesting that DUX4 both activates retroelement transcription and suppresses the virally induced innate immune response. Although we have shown that DEFB103 can substitute for DUX4 to suppress the innate immune response, products of retroelements and endogenous retroviruses may do the same and, thus, the DUX4-mediated suppression of the innate immune response might be multi-factorial. Since DEFB103 is also expressed in the testis, it is interesting to consider whether the role of DUX4 in the germline might include a simultaneous activation of retroelement transcription and suppression of the innate immune response to those transcripts.
DUX4 regulated targets also include genes involved in RNA splicing, developmentally regulated components of the Pol II transcription complex, and ubiquitin-mediated protein degradation pathways, all of which may have pathophysiological consequences. A recent study indicated that induction of E3 ubiquitin ligases by DUX4 might cause muscle atrophy in FSHD (Vanderplanck et al., 2011
), consistent with our findings that multiple ubiquitin ligase family members are induced by DUX4. In addition, myostatin induces some of these ubiquitin ligases in skeletal muscle (Lokireddy et al., 2011
) and it is therefore possible that both DUX4 induction of ubiquitin ligases and the modest upregulation of myostatin by DEFB103 that we observed in this study can both contribute to muscle atrophy. DUX4 is also known to induce apoptosis in muscle cells and DUX-4 mediated myopathy in mice has been shown to be p53-dependent (Wallace et al., 2011
). As noted above, the activation of retrotransposons or reactivation of the gametogenic program, particularly inducers of cell cycle in post-mitotic muscle, might contribute to apoptosis. In addition, the altered expression of many factors involved in RNA transcription and splicing might affect cell differentiation and survival. In many human diseases a single mutation can effect multiple pathological pathways that collectively account for the complex disease phenotype. Our study of DUX4 regulated genes has identified several candidate pathways and future work will be necessary to determine their relative contributions to the disease phenotype.
In this regard, other genes have been identified as candidates for FSHD. For example, FRG1 expression has been reported to be elevated in FSHD muscle (Gabellini et al., 2002
) and FRG1 transgenic mice display a muscular dystrophy phenotype (Gabellini et al., 2006
). It is interesting that FRG1 is reported to alter RNA splicing in FSHD muscle (Gabellini et al., 2006
) and that our study shows that DUX4-fl also alters the expression of many genes that regulate splicing and RNA processing. It will be important to determine the relative contributions of DUX4 and FRG1 to FSHD pathophysiology; however, the human genetics shows a convincing linkage to polymorphisms necessary for the polyadenylation of the DUX4 mRNA (Lemmers et al., 2010
), indicating that DUX4 mRNA is a necessary component of the disease. Therefore, one therapeutic avenue to pursue for FSHD is to reduce the activity of DUX4, either by eliminating its expression in the muscle cells as we have done in vitro with an siRNA or by introducing a dominant negative, such as the DUX4-s splice form.
A previous study identified PITX1 as a DUX4 target gene expressed in FSHD skeletal muscle and in mouse cells transfected with DUX4 (Dixit et al., 2007
). Others have expressed DUX4 in mouse muscle cells and identified repression of the glutathione redox pathway (Bosnakovski et al., 2008
). Both of these findings are consistent with our expression array data. However, since many of the DUX4 binding sites reside in primate-specific MaLRs and some of the DUX4 targets are not conserved in mice, further studies are necessary to determine the conserved and primate-specific functions of DUX4, an important consideration for evaluating mouse models of FSHD.
In conclusion, our data support the model that inappropriate expression of DUX4 plays a causal role in FSHD skeletal muscle pathophysiology by activating germline gene expression, endogenous retrotransposons, and suppressors of differentiation in skeletal muscle. The set of genes robustly upregulated by DUX4 in FSHD skeletal muscle are candidate biomarkers because they are absent in control muscle and easily detected in FSHD1 and FSHD2 muscle. Furthermore, some target genes encode secreted proteins, which offer the potential for developing blood tests to diagnose FSHD or monitor response to interventions. Beyond their utilities as candidate biomarkers, the DUX4 targets identified in this study point to specific mechanisms of disease and may help guide the development of therapies for FSHD.