Abstract
ZBED6 is a recently discovered transcription factor, unique to placental mammals, that has evolved from a domesticated DNA transposon. It acts as a repressor at the IGF2 locus. Here we show that ZBED6 acts as a transcriptional modulator in mouse myoblast cells, where more than 700 genes were differentially expressed after Zbed6-silencing. The most significantly enriched GO term was muscle protein and contractile fiber, which was consistent with increased myotube formation. Twenty small nucleolar RNAs all showed increased expression after Zbed6-silencing. The co-localization of histone marks and ZBED6 binding sites and the effect of Zbed6-silencing on distribution of histone marks was evaluated by ChIP-seq analysis. There was a strong association between ZBED6 binding sites and the H3K4me3, H3K4me2 and H3K27ac modifications, which are usually found at active promoters, but no association with the repressive mark H3K27me3. Zbed6-silencing led to increased enrichment of active marks at myogenic genes, in agreement with the RNA-seq findings. We propose that ZBED6 preferentially binds to active promoters and modulates transcriptional activity without recruiting repressive histone modifications.
Highlights
ZBED6 was recently discovered as a novel transcriptional repressor of IGF2 because a mutation disrupting one of its binding sites in porcine IGF2 intron 3 leads to greater postnatal IGF2 expression in skeletal and cardiac muscle and makes pigs grow more muscle and a bigger heart [1,2]
The present study demonstrates that ZBED6 is an important transcriptional modulator since Zbed6-silencing in mouse C2C12 cells combined with transcriptome analysis revealed consistent changes in expression levels for more than 700 genes
Many of these transcriptional changes are likely to be secondary effects but the finding of a significant enrichment of differentially expressed genes associated with one or more ZBED6 binding sites, for genes that are upregulated after ZBED6 silencing, strongly suggests that ZBED6 has many functional target sites in the genome besides the well-established and evolutionary conserved site in the third intron of IGF2
Summary
ZBED6 was recently discovered as a novel transcriptional repressor of IGF2 because a mutation disrupting one of its binding sites in porcine IGF2 intron 3 leads to greater postnatal IGF2 expression in skeletal and cardiac muscle and makes pigs grow more muscle and a bigger heart [1,2]. ZBED6 is unique to placental mammals and has evolved from a domesticated DNA transposon. The primary amino acid sequence of ZBED6, in particular the region comprising the DNA binding BED domains (residues 129–183 and 266–318), is highly conserved among all placental mammals for which sequence information is available (.26 species). ZBED6 contains one nucleolar localization signal (residues 61– 80), which targets ZBED6 protein into the nucleolus [1]. This lysine- and arginine-rich signal sequence is positively charged and extremely conserved among 26 placental mammals. This suggests that the nucleolar localization of ZBED6 is important for its function. A number of transcriptional regulators including MyoD and Myogenin repress rDNA transcription in the nucleolus during myogenesis of C2C12 cells [7]
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