Dissecting methylation profiles of imprinted and/or non-imprinted genes in cloned animal genomes

• Main Authors: Chih-Jie Shen, 沈志傑
• Other Authors: Chuan-Mu Chen
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/00784109109728862653

博士 === 國立中興大學 === 生命科學系所 === 100 === DNA methylation is a major epigenetic modification found in mammalian genomes and it regulates crucial aspects of gene functions. Mammal cloning by somatic cell nuclear transfer (SCNT) often results in gestational or neonatal failure with only a few percentage of manipulated embryos producing live births. Many of the cloned embryos that survive to term succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. The uniparentally expressed imprinted genes control the development of embryos. This study has shown the aberrant putative differentiated methylation regions (DMRs) of imprinted genes existed in the genome of cloned animal, even their appearance looks like normal. In this study, we firstly defined the several putative DMRs of imprinted genes in cloned porcine and bovine genomes. The putative DMRs of imprinted genes are vulnerable during somatic cell nuclear transfer (SCNT) process. The skipping of the reprogramming process happened in cloning animals by nuclear transfer of somatic cells may cause the inappropriate methylation patterns. The differentiated somatic cells, unlike germ cells, do not back to the necessary reprogramming process to erase the epigenetic markers correctly and timely. These abnormal modifications could affect the expression of genes and cause the unexpected pathologies. The OPN and H19 were identified by the multimeric methyl-binding domain (MBD) protein pull down assay. We also demonstrated three CpG sites (CpG1, CpG13, and CpG20) which regulate the OPN transcription by their DNA methylation status. In cloned pigs, these OPN and H19 genes showed aberrant methylation patterns in their ear tissues. Furthermore, differentially methylated hybridization chip (DMH chip) was also performed to screen the aberrant methylated DNA elements in cloned animals. Either hypermethylation or hypomethylation frequently appeared in the tissues of cloned animals. Five genes, EXD3, PDZD4, MTMR7, IGF2R, and PEG3 were selected for further study. Especially, the PEG3 with aberrant methylation status was selected from cloned pig CP5 testis with azoospermia phenomenon. The immunohistochemistry (IHC) staining of PEG3 showed the extremely different distribution in testis between wild-type (WT) and CP5 pigs. The PEG3 interacts with NF-κB, β-catenin, and E-cadherin. The data showed that CP5 testis lacked of the E-cadherin expression inside testicular cord. The aberrant location of PEG3 expression may affect the maturation of spermatogonia through the down-regulated β-catenin and E-cadherin. Connectively, we identified four DMRs of imprinted genes in the wild-type pig genomes, including two maternal imprinted loci (INS and IGF2) and two paternal imprinted loci (H19 and IGF2R). Aberrant DNA methylation phenomenon, either hypermethylation or hypomethylation, commonly appeared in H19 (45% vs. 30%), IGF2 (40% vs. 0%), INS (50% vs. 5%), and IGF2R (15% vs. 45%) imprinted loci, in cloned pigs and also found in cloned bovine genomes: H19 (0% vs. 29%), IGF2 (21% vs. 29%), XIST (7.9% vs. 7.9%). Our data also revealed that aberrant methylation frequently occurred in imprinted genes, but not satellite loci, Satellite I, Satellite II, VNTR, and Art2, in cloned bovine. It indicated that the methylation between imprinted genes and satellite loci is regulated by different mechanisms. Furthermore, quantitative RT-PCR was applied to assess the expression of these imprinted genes in the cloned bovine NT-6. We found that IGF2 and H19 extremely overexpressed in the liver, vein, ear, skin, and uterus of the NT-6 bovine. In conclustion, even the successful produced of cloned swine and bovine fortunately avoid the preneonatal or postnatal death, the perturbation and intervention of mehtylation in imprinted genes still exists. It perhaps may be the reasons of adult pathologies and short life occurred in cloned animals. Here, we provided the results that the cloned animals still have the several risks of health and unexpected defects. Finding and correcting the aberrant pattern of imprinted genes after SCNT would offer insights into the improvement in future cloning animal technique.