Mouse model to understand the role of Dux4 in FSHD

FSHD is an autosomal dominant disease that affects 1:20000 individuals. Mapping studies have associated the disease with a reduced number (1-10) of the D4Z4 macrosatellite repeats from the usual ~100. These repeats lie adjacent to the telomeres and are usually present in a highly silenced epigenetic state. It is not clear which genes are affected or how DNA methylation patterns affect the disease. Within the D4Z4 repeat is an ORF encoding a putative transcription factor named Dux4, containing two homeodomains. Although the function of Dux4 is unknown, the Dux4 homeodomains are similar to those of Pax7, a protein known to be involved in muscle development, proliferation and differentiation. We have previously reported that Dux4 is toxic when misexpressed at high levels in many cell types, and blocks differentiation of myoblasts when expressed at low levels, and competes with Pax7 for regulation of myogenic target genes. To model Dux4 function in vivo, we made a Dux4-inducible mES cell line by inserting a doxycycline-inducible Dux4 allele (iDux4+3’UTR) onto the X chromosome at a euchromatic region (HPRT). High-level induction of the Dux4 was toxic to mES cells but low-level Dux4 resulted in altered differentiation. When iDux4+3’UTR mice where generated and bred, this allele demonstrated leaky phenotypes in females, and male-specific lethality. Rare live-born males were small and underdeveloped with abnormal skin and defective sperm development and showed changes to muscle fibers, but no overt muscle degeneration. However, mice died within 1 month, well before the stage degeneration usually begins in FSHD. Dux4 protein could be induced and observed in cultured primary cells, and we are evaluatingpups and embryos for Dux4 expression in vivo. Dux4 carrier females were smaller and displayed the skin phenotype in transverse stripes. We hypothesize that the 3’ UTR contains an enhancer which drives leaky expression in some embryonic cell types and that X chromosome inactivation combined with selective survival of XDUX4-inactive cells protects the females from the lethality. To test for selectively biased X inactivation, we crossed Dux4 carrier females with XGFP males. Upon FACS analysis of the GFP+ cells in XGFP/ X+ vs. XGFP/ XDux4 female progeny, we found that the latter had an elevated frequency of GFP positive cells in most tissues, including the satellite cell compartment of skeletal muscle, confirming our hypothesis of selective XDUX4 inactivation. This mouse model suggests that Dux4 is a dominant lethal gene even when expressed at very low levels and can cause a variety of developmental defects in EBs and in embryo development