| The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains three paternally expressed protein-coding genes, multiple maternally expressed long noncoding RNA genes and numerous mi RNAs and sno RNAs. There are three known paternally methylated DMRs(IG-DMR, Gtl2-DMR and Dlk1-DMR). mi R-341 and mi R-1188 are two mi RNAs that located 226 bp apart in intron 2 of Meg8. Previous studies found that insertional mutagenesis by AAV vector or Sleeping Beauty transposon around mi R-341 in this domain leads to overexpression of adjacent genes and facilitates hepatocarcinogenesis. We assumed that there might be unknown regulatory sequences in close proximity to mi R-341. There are two small Cp G islands(named CGI-1 and CGI-2) in the vicinity of the mi R-341/mi R-1188 locus. While CGI-1 overlaps mi R-341, CGI-2 is located ~800 bp downstream of mi R-1188. In this study, we focus on mi R-341/mi R-1188 and CGI-1/CGI-2, explore the epigenetic status including DNA methylation and post-translational histone modifications, and show that CGI-2 is a CTCF-binding differentially methylated region that displays enhancer-blocking activity.Firstly, we analyzed the expression patterns and imprinting status of mi R-341 and mi R-1188. Results from in situ hybridization analysis showed that the primary transcripts of mi R-341 and mi R-1188 share a similar expression pattern, with expression signals predominantly detected in the forebrain, hindbrain, tongue, thymus, lung, liver and vertebral cartilage at E15.5. Quantitative PCR analysis of mi RNA expression levels showed that the mature forms of mi R-341 and mi R-1188 share similar expression patterns with their primary transcripts, with high expression in tongue and liver, medium expression in brain and lung, and low expression in heart and kidney. Imprinting analysis showed that mi R-341 and mi R-1188 are expressed only from the maternal allele.Secondly, bisulfite sequencing analysis was carried out to determine the DNA methylation patterns and dynamics of CGI-1 and CGI-2 during mouse embryonic development. Results showed that CGI-2 is a novel maternally methylated DMR in fetal tissues at E15.5 and E18.5. However, CGI-2 is highly methylated in both sperm and oocytes, and then demethylated during pre-implantation development. CGI-2 is re-methylated after implantation, but it is interesting that the paternal allele of CGI-2 is firstly re-methylated, and then demethylated again from E5.5 to E7.5. CGI-2 acquires differential methylation prior to E7.5. CGI-1 is highly methylated in mouse gametes, pre- and post-implantation embryos except blastocysts, and is not a DMR.Thirdly, Ch IP assays were performed to detect the enrichments of histone modifications and the binding of CTCF. Extensive histone H3K9me3 enrichments were observed at CGI-2 and its adjacent regions. In liver, both of histone H3K4me3 and H3K9me3 were enriched at CGI-2, but in placenta, no H3K4me3 enrichment was found at CGI-2. There are four highly conserved predicted CTCF binding sites around CGI-2. CTCFBS#3 is located in the differentially methylated CGI-2. The results of Ch IP assays showed that CTCF only binds to CGI-2 in vivo, but not to the other three sites.Lastly, we constructed a series of vectors and analyzed the function of CGI-2 in an enhancer-blocking assay. The result showed that CGI-2 displays enhancer-blocking activity dependent upon the CTCF binding site.Taken together, CGI-2 is the first maternally methylated DMR found in the mouse Dlk1-Dio3 imprinted domain. The methylation landscape of CGI-2 changes dynamically during pre- and post-implantation development. CGI-2 acquires differential methylation prior to E7.5. Both of histone H3K4me3 and H3K9me3 are enriched at CGI-2. CTCF binds to CGI-2 in vivo. CGI-2 displays enhancer-blocking activity dependent upon the CTCF binding site. These results will contribute to the further investigation of imprinting regulation in the Dlk1-Dio3 domain. |
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