RESUMO
The placenta is a unique organ for ensuring normal embryonic growth in the uterine. Here, we found that maternal RNA transcription in Dlk1-Dio3 imprinted domain is essential for placentation. PolyA signals were inserted into Gtl2 to establish a mouse model to prevent the expression of maternal RNAs in the domain. The maternal allele knock-in (MKI) and homozygous (HOMO) placentas showed an expanded junctional zone, reduced labyrinth and poor vasculature impacting both fetal and maternal blood spaces. The MKI and HOMO models displayed dysregulated gene expression in the Dlk1-Dio3 domain. In situ hybridization detected Dlk1, Gtl2, Rtl1, miR-127 and Rian dysregulated in the labyrinth vasculature. MKI and HOMO induced Dlk1 to lose imprinting, and DNA methylation changes of IG-DMR and Gtl2-DMR, leading to abnormal gene expression, while the above changes didn't occur in paternal allele knock-in placentas. These findings demonstrate that maternal RNAs in the Dlk1-Dio3 domain are involved in placental vasculature, regulating gene expression, imprinting status and DNA methylation.
Assuntos
Proteínas de Ligação ao Cálcio , Impressão Genômica , RNA Longo não Codificante , Animais , Feminino , Camundongos , Gravidez , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Placenta/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Meg8-DMR is the first maternal methylated DMR to be discovered in the imprinted Dlk1-Dio3 domain. The deletion of Meg8-DMR enhances the migration and invasion of MLTC-1 depending on the CTCF binding sites. However, the biological function of Meg8-DMR during mouse development remains unknown. In this study, a CRISPR/Cas9 system was used to generate 434 bp genomic deletions of Meg8-DMR in mice. High-throughput and bioinformatics profiling revealed that Meg8-DMR is involved in the regulation of microRNA: when the deletion was inherited from the mother (Mat-KO), the expression of microRNA was unchanged. However, when the deletion occurred from the father (Pat-KO) and homozygous (Homo-KO), the expression was upregulated. Then, differentially expressed microRNAs (DEGs) were identified between WT with Pat-KO, Mat-KO, and Homo-KO, respectively. Subsequently, these DEGs were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) term enrichment analysis to explore the functional roles of these genes. In total, 502, 128, and 165 DEGs were determined. GO analysis showed that these DEGs were mainly enriched in axonogenesis in Pat-KO and Home-KO, while forebrain development was enriched in Mat-KO. Finally, the methylation levels of IG-DMR, Gtl2-DMR, and Meg8-DMR, and the imprinting status of Dlk1, Gtl2, and Rian were not affected. These findings suggest that Meg8-DMR, as a secondary regulatory region, could regulate the expression of microRNAs while not affecting the normal embryonic development of mice.
Assuntos
MicroRNAs , Animais , Feminino , Camundongos , Gravidez , Proteínas de Ligação ao Cálcio/genética , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Impressão Genômica , Peptídeos e Proteínas de Sinalização Intercelular/genética , MicroRNAs/genética , Sequências Reguladoras de Ácido NucleicoRESUMO
The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains three well-characterized paternally methylated differentially methylated regions (DMRs): IG-DMR, Gtl2-DMR, and Dlk1-DMR. These DMRs control the expression of many genes involved in embryonic development, inherited diseases, and human cancer in this domain. The first maternal methylation DMR discovered in this domain was the Meg8-DMR, the targets and biological function of which are still unknown. Here, using an enhancer-blocking assay, we first dissected the functional parts of the Meg8-DMR and showed that its insulator activity is dependent on the CCCTC-binding factor (CTCF) in MLTC-1. Results from RNA-seq showed that the deletion of the Meg8-DMR and its compartment CTCF binding sites, but not GGCG repeats, lead to the downregulation of numerous genes on chromosome 12, in particular the drastically reduced expression of Dlk1 and Rtl1 in the Dlk1-Dio3 domain, while differentially expressed genes are enriched in the MAPK pathway. In vitro assays revealed that the deletion of the Meg8-DMR and CTCF binding sites enhances cell migration and invasion by decreasing Dlk1 and activating the Notch1-Rhoc-MAPK/ERK pathway. These findings enhance research into gene regulation in the Dlk1-Dio3 domain by indicating that the Meg8-DMR functions as a long-range regulatory element which is dependent on CTCF binding sites and affects multiple genes in this domain.
