ZBED6, a Novel Transcription Factor Derived from a Domesticated DNA Transposon Regulates IGF2 Expression and Muscle Growth

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Abstract
A single nucleotide substitution in intron 3 of IGF2 in pigs abrogates a binding site for a repressor and leads to a 3-fold up-regulation of IGF2 in skeletal muscle. The mutation has major effects on muscle growth, size of the heart, and fat deposition. Here, we have identified the repressor and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (ChIP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications, including development and transcriptional regulation. The phenotypic effects in mutant pigs and ZBED6-silenced C2C12 myoblasts, the extreme sequence conservation, its nucleolar localization, the broad tissue distribution, and the many target genes with essential biological functions suggest that ZBED6 is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth. The molecular identification of genes and mutations affecting complex traits and disorders has proven to be very challenging in humans as well as in model organisms. These so-called quantitative traits arise from interactions between two or more genes and their environment, and can be mapped to their underlying genes via closely linked stretches of DNA called quantitative trait loci (QTL). Previously, we identified a single nucleotide substitution in a noncoding region of the insulin-like growth factor 2 gene (IGF2) in pigs that is underlying a major QTL affecting muscle growth, heart size, and fat deposition. The mutation disrupts interaction with an unknown nuclear protein acting as a repressor of IGF2 transcription. In the present study, we have isolated a zinc finger protein of unknown function and show that it regulates the expression of IGF2. The protein, which we named ZBED6, is encoded by a domesticated DNA transposon that was inserted into the genome prior to the radiation of placental mammals. ZBED6 is exclusive to placental mammals and highly conserved among species. Our functional characterization of ZBED6 shows that it has a broad tissue distribution and may affect the expression of thousands of other genes, besides IGF2, that control fundamental biological processes. We postulate that ZBED6 is an important transcription factor affecting development, cell proliferation, and growth in placental mammals.