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

Double homeobox genes are unique to eutherian mammals, transiently expressed in early blastomere-stage embryos, and comprise 3 clades (DUXA, B, C) evolved by homeobox duplication and divergence from an ancestral single homeobox gene, sDUX, present in other vertebrates. We test platypus sDUX and find that it drives cytotoxicity and inhibition of myogenesis identical to DUX4, the human DUXC gene. sDUX binds DNA as a head-to-head dimer, with protein core and DNA nearly overlapping the DUX4-DNA crystal structure. Although DUXA lacks transcriptional activity, DUXA-VP64 transcriptome and chromatin accessibility profiles significantly overlap those of DUX4 and sDUX, including expression of ZGA genes and LTR elements. Furthermore, DUXA binds DUX4 sites at most DUX4 target genes and counteracts transcriptional activity of DUX4, including in FSHD patient cells, potentiating a feedback inhibitory loop. The DUX gene family therefore comprises cross-regulating members of opposing function, with implications for their roles in ZGA, FSHD, and cancer. Overall design: ChIP-seq was performed in duplicates starting from HEK293 cells that were transfected with a plasmid encoding for a V5-epitope tagged DUX4.