Evolution of antagonism among DUX family members from an ancestral toxic single homeodomain protein

Double homeobox genes are unique to eutherian mammals, transiently expressed in early blastomere-stage embryos, and comprise 3 clades (DUXA, B, and C) evolved by homeodomain duplication and divergence from an ancestral single homeobox gene, sDUX, present in non-eutherian mammals and other vertebrates. We test platypus sDUX and find that it drives cellular phenotypes identical to DUX4, the human DUXC gene, including cytotoxicity and inhibition of myogenic differentiation. sDUX binds DNA as a head-to-head dimer, showing near overlap of protein core and DNA to the DUX4-DNA crystal structure. DUXA and sDUX bind both palindromic and nonpalindromic TAAT/TGAT double homeodomain motifs against DUX4 preference for non-palindromic. Although DUXA lacks transcriptional activation potential, we find significant similarities in transcriptional and chromatin profiles of sDUX with DUX4 and DUXA-VP64, including expression of ZGA genes and LTR elements. Furthermore, DUXA binds to a significant fraction of DUX4 target sites and is able to counteract transcriptional activation of DUX4 on its targets, potentiating a feedback inhibitory loop, including in cells from patients with facioscapulohumeral muscular dystrophy (FSHD). The DUX gene family therefore comprises cross-regulating members of opposing function, with implications for their roles in ZGA, FSHD, and cancer. Overall design: Doxycycline induced expression of DUX factor and uninduced control LHCN-M2 cells analyzed by RNA-seq and ATAC-seq with replicates.