Differential RNA-seq analyses in MB135iDUX4_KLF18 KO_i3xFLAG-KLF18_AAVSI short and long cell lines

DUX4 initiates the first wave of zygotic genome activation (ZGA) in the human embryo and its misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). However, other factors that help regulate ZGA-like transcription in human development and disease are still not fully understood. Here we identify Krüppel-like factor 18 (KLF18) as a component of a DUX4 feed-forward network that is necessary for expression of KLF17 and a subset of the DUX4-regulated totipotency-associated genes. We mapped the genome-wide binding profile of KLF18 downstream of DUX4 and showed that its activity is influenced by DUX4 and chromatin accessibility. We find that in contrast to the rodent ortholog, human KLF18 has a predicted beta-solenoid structure composed of a variable number of tandem repeats (VNTRs) with multiple different structural variants in the human population. Expression of different KLF18 variants in combination with DUX4 showed similar transcriptional activity, whereas assessment of KLF18 structural variants in individuals with FSHD1 showed inconsistent association with disease severity that requires further study as a modifier locus. Together these findings establish the polymorphic transcription factor KLF18 as a critical component of a DUX4-initiated feed-forward network and a candidate locus for human diversity.