Abstract
The expression of Igf2 in mammals shows a complex regulation involving multiple promoters and epigenetic mechanisms. We previously identified a novel regulatory mechanism based on the interaction between the transcriptional factor ZBED6 and Igf2 intron. Disruption of the ZBED6-Igf2 interaction leads to a dramatic up-regulation of IGF2 expression postnatally. In the current study we characterize an additional layer of regulation involving miR483 encoded by another Igf2 intron. We found a highly significant up-regulation of miR483 expression when the ZBED6-Igf2 axis is disrupted in transgenic mice. Furthermore, CRISPR/Cas9 mediated knock-out of miR483 in C2C12 myoblast cells, both wild-type and cells with disrupted ZBED6-Igf2 axis (Igf2dGGCT), resulted in down-regulation of Igf2 expression and a reduced proliferation rate. This was further validated using miR483 mimics and inhibitors. RNA-seq analysis revealed a significant enrichment of genes involved in the PI3K-Akt signaling pathway among genes down-regulated in miR483−/− cells, including Igf2 down-regulation. The opposite pattern was observed in Igf2dGGCT cells, where Igf2 is up-regulated. Our data suggest a positive feedback between miR483 and Igf2 promoter activity, strongly affecting how ZBED6 controls Igf2 expression in various cell types.
Highlights
The expression of Igf[2] in mammals shows a complex regulation involving multiple promoters and epigenetic mechanisms
Because Igf[2] appears to be the most important downstream target for ZBED69, we performed a screen using our Insulin-like growth factor 2 (Igf2)-KI mice in which the ZBED6 binding site in Igf[2] is disrupted. This experimental design allows us to dissect if an altered expression of a miRNA in Zbed6-KO mice is mediated through the interaction with its binding site in Igf[2] (Figs. 1 and S1)
Neither miR483-3p nor miR483-5p were expressed at detectable levels in liver (Supplementary Table 2). qRT-PCR analysis confirmed the striking difference between muscle and liver tissue as regards the induction of Igf[2] mRNA expression when the ZBED6-Igf[2] axis is disrupted (Fig. 1e, right)
Summary
The expression of Igf[2] in mammals shows a complex regulation involving multiple promoters and epigenetic mechanisms. CRISPR/Cas[9] mediated knock-out of miR483 in C2C12 myoblast cells, both wild-type and cells with disrupted ZBED6-Igf[2] axis (Igf2dGGCT), resulted in down-regulation of Igf[2] expression and a reduced proliferation rate. This was further validated using miR483 mimics and inhibitors. Transcriptional regulation of Igf[2] is complex and occurs at different levels starting from its genomic locus, which includes the H19 gene, an imprinted control region (ICR), and a set of enhancers. It has been reported that miR483-5p binds directly to the 5’UTR region of Igf[2] and thereby enhances Igf2 transcription[17]
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