DUX4 expression in embryonic stem cells

Our group has identified the Double Homeobox 4 (DUX4) gene within the D4Z4 repeated DNA elements in the 4q35 region linked to the FSHD muscular dystrophy (1). The DUX4 gene is derepressed in FSHD muscle cells and expresses a transcription factor that initiates a large gene dysregulation cascade. Dr. S. Tapscott's group has published that DUX4 mRNAs were expressed in embryonic stem cells (hESC) and in the germ line (testis) of healthy individuals, as well as in induced pluripotent stem cells (hiPS) derived from healthy fibroblasts (2).
We used cultures of hESC lines derived from healthy embryos or from embryos carrying the FSHD genetic defect (in collaboration with Prof. Karen Sermon, Department of Embryology and Genetics, VUB, Brussels). We have optimized the preparation of hESC nuclear extracts, protein separation by 4-12% gradient PAGE-SDS, western blotting and specific immunodetection (mouse monoclonal: 9A12 MAb) of the low abundance DUX4 protein. We could thus reproducibly immunodetect several protein bands after 3 weeks of differentiation.
Moreover, an immunofluorescence signal was detected with the same antibody at the periphery of ES cell colonies in which differentiation initiates. We could specifically amplify DUX4 mRNAs by 3'RACE in differentiating control and FSHD ES cell lines but only in rare samples of proliferating ES cells.
These observations suggest that DUX4 is only expressed at a very low level or not at all during ES cell proliferation and that its expression is induced during early differentiation. This study was funded by the FSHD Global Research Fundation (Australia).

References :
1)Dixit et al. DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1. 2007, Proc Natl Acad Sci USA. 104(46):18157-62.
2)Snider et al. Facioscapulohumeral dystrophy: incomplete suppression of a retrotransposed gene. 2010, PLoS Genet. 6(10):e1001181