Year: 2008
Type: conference-paper
Abstract: By targeting each FSHD candidate gene to a doxycycline-inducible locus in murine C2 myoblasts, we have shown that DUX4 is uniquely toxic, that this toxicity is caused by sensitizing cells to oxidative stress, and that both Pax3 and Pax7 act as dominant inhibitors of the DUX4 phenotype. At low, non-toxic, levels of expression, DUX4 interferes with the expression of myogenic regulators and inhibits differentiation, a feature shared by DUX4c. Using a set of deletion and frameshift mutants, we show that both repression of MyoD and inhibition of differentiation require translation of the homeodomains but not the c-terminus, while toxicity requires translation of both the homeodomains and the c-terminus. Based on these results, we introduced DUX4, in the context of the terminal D4Z4 repeat, into mouse ES cells downstream of a doxycycline-inducible promoter on the X chromosome, proximal to the ubiquitously-expressed HPRT locus. We had previously tried, unsuccessfully, to generate transgenic mice carrying DUX4 and D4Z4 in various constructs by pronuclear injection (random integration) without success. Using the X-linked inducible locus, we have now derived strains carrying D4Z4/DUX4, however surprisingly, the D4Z4/DUX4 X chromosome (XD) is malespecific lethal, even without dox. Female carriers are fertile but suffer a growth disadvantage and a severe skin phenotype. Although they do not display an obvious muscular dystrophy, these are the first mice to carry phenotypically active D4Z4 sequences. We hypothesize that D4Z4 sequences interact with our basal promoter, resulting in DUX4 expression, and consequent growth defects. We interpret previous failures to generate mice carrying active D4Z4 repeats to be due to strong selection against D4Z4 integrations into open chromatin where DUX4 may be expressed. Females may be partially protected from the dominant-lethal gene by X-inactivation, an unintended consequence of our X-linked approach. We discuss these results in the context of developing an animal model for FSHD.
Authors: Bosnakovski, Darko, Kyba, Michael
Institutions: Fred Hutch Cancer Center, University of Minnesota
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W204463207