Vigilin,a Multi-KH-Domain protein,cooperates with CTCF in regulation of IGF2/H19
【摘要】：正Previously,we found that CTCF play an important role in the coregulated imprin-ted genes,IGF2 and H19,and different methylation profiles for H19ICR were associated with aberrant imprinting of IGF2 and H19 in human hepatocellular carcinoma(HCC). We assume that CTCF would have an equally complex set of parameters in regulating genomic imprinting in human cancer.To this purpose,we sought to search for interaction proteins of CTCF.In a yeast two-hybrid screen,we identify a novel partner,VIGILIN, which has been characterized as a highly conserved multi-KH-domain protein that binds RNA and ssDNA.A specific interaction between CTCF and VIGILIN is confirmed in immunoprecipitation(IP) in human cells(HepG2 and CNEl).Overexpression of CTCF or VIGILIN downregulates IGF2 expression and enhances H19 expression in HepG2 and CNEl cells.In contrast,reduction of CTCF or VIGILIN with specific shRNA results in an increase of IGF2 and a decrease of H19.Simultaneously,overexpression of VIGILIN significantly increases CTCF expression both in mRNA and protein,and inverse,depletion of VIGILIN reduces CTCF expression. Nevertheless,expression alteration of CTCF shows no significant effect on VIGILIN.Moreover,CTCF and VIGILIN both regulate the imprinting of IGF2 in CNEl cells.These findings validate the functional relevance of CTCF and VIGILIN.Subsequent chromatin im-munoprecipitation(ChIP) analysis reveals that VIGILIN co-localizes with CTCF binding at IGF2/H19 locus.In addition, knockdown of VIGILIN markedly inhibits the cells viability in human HepG2 and CNEl cells by MTT assay.Together,our results reveal a new regulatory mechanism that VIGILIN contribute to regulating IGF2 and H19 at CTCF sites by interacting with CTCF. Our results also provide new insights into the role of CTCF and VIGILIN in the development of cancers,and implicate the potential application of VIGILIN in cancer ther-apy.