Maintaining Proper Levels of DNA Methylation Marks Through the TET Family is Critical for Normal Embryo Development in Pigs
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DNA methylation is one of the principal epigenetic modifications that plays an essential role in transcriptional regulation. After fertilization, mammalian embryos undergo dynamic changes in genome-wide DNA methylation patterns and the changes are essential for normal embryo development. Ten-eleven translocation (TET) methylcytosine dioxygenases are implicated in DNA demethylation by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The three members of TET protein family, TET1, TET2, and TET3, are highly expressed in preimplantation embryos in a stage-specific manner. Previous studies demonstrated that TET proteins are involved in diverse biological processes such as gene regulation, pluripotency maintenance, and cell differentiation by mediating 5mC oxidation. My dissertation research was conducted to elucidate the mechanistic roles of TET proteins in epigenetic reprogramming of mammalian embryos using porcine embryos as a model. The first set of studies focused on the relationship between TET proteins and pluripotency. To understand the role of TET proteins in establishing pluripotency in preimplantation embryos, CRISPR/Cas9 technology and TET-specific inhibitors were applied. TET1 depletion unexpectedly resulted in an increased expression of NANOG and ESRRB genes in blastocysts, although the DNA methylation levels of NANOG promoter were not changed. Interestingly, transcript abundance of TET3 was increased in blastocysts carrying inactivated TET1, which might have had an effect on the increase of NANOG and ESRRB. When the activity of TET enzymes was inhibited to eliminate the compensatory increase of TET3 under the absence of functional TET1, the expression levels of NANOG and ESRRB were decreased and methylation level of NANOG promoter was increased. In addition, ICM specification was impaired by the inhibition of TET enzymes. These results suggest that the TET family is a critical component of the pluripotency network of porcine embryos by regulating expression of genes involved in pluripotency and early lineage specification. In the next set of studies, the presence of TET3 isoforms in porcine oocytes and cumulus cells was investigated to dissect the gene structure of TET3 that could assist in understanding mechanistic actions of TET3 in the DNA demethylation process. Among the three TET3 isoforms identified in cumulus cells, only the pTET3L isoform, which contains CXXC domain that carry DNA binding property, was verified in mature porcine oocytes. Expression level of the pTET3L isoform was much higher in mature oocytes compared to that in somatic cells and tissues. In addition, the transcript level of this isoform was significantly increased during oocyte maturation. These results suggest that pTET3L isoform is predominantly present in mature porcine oocytes and that CXXC domain may play an important role in DNA demethylation in zygotes. In a follow-up study, the role of the TET3 CXXC domain in controlling post-fertilization demethylation in porcine embryos was investigated by injecting TET3 GFP-CXXC into mature porcine oocytes. The injected CXXC was exclusively localized in the pronuclei, indicating that the CXXC domain may localize TET3 to the nucleus. The CXXC overexpression reduced the 5mC level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, the transcript abundance of NANOG and ESRRB was increased in blastocysts derived from GFP-CXXC overexpressing zygotes. These results provide an evidence that the CXXC domain of TET3 is critical for post-fertilization demethylation of porcine embryos and proper expression of pluripotency related genes in blastocysts. In the last set of studies, the impact of MBD proteins on porcine embryo development was examined under the hypothesis that competitive binding of MBD and TET proteins to 5mC contributes to the proper maintenance of DNA methylation levels in embryos. Cloning of porcine MBD1, MBD3, and MBD4 from mature oocytes indicates that the genes are highly conserved among different species, implying the involvement of porcine MBD proteins in the maintenance of DNA methylation. MBD1 overexpression in oocytes impaired preimplantation development of porcine embryos, suggesting that the MBD1 overexpression may have negatively affected porcine embryo development because proper DNA methylation levels were not preserved under the high level of MBD1. Collectively, the studies in my dissertation demonstrate that TET family proteins are important epigenetic players involved in the regulation of pluripotency and reprogramming of DNA methylation, and are thus crucial for normal embryo development. The findings in the dissertation will improve our understanding of epigenetic events occurring in mammalian embryos, and have the potential to overcome epigenetic defects that are observed in pluripotent stem cells and in-vitro derived embryos.
General Audience Abstract
Epigenetic modifications are heritable changes affecting the level of gene expression without changing the sequence of the genome. DNA methylation, one of the biggest epigenetic marks in mammalian genome, is often correlated to gene repression. In mammals, DNA methylation patterns are dramatically changed during preimplantation development to acquire embryonic developmental potential. Understanding of the epigenetic changes occurring in preimplantation embryos is necessary for producing healthy domestic animals in agriculture and for developing strategies for the treatment of epigenetic defects in human. Ten-eleven translocation (TET) family enzymes, TET1, TET2, and TET3, are known to function as a DNA methylation modifier by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). My dissertation research was performed to elucidate the role of TET family during preimplantation development using porcine embryos as a model. Pluripotency refers to the ability of cells to differentiate into all cell types of a mature organism. Pluripotent cells emerge in embryos as embryonic cells acquire lineage-specific characteristics. The first set of studies focused on the role of TET enzymes in regulating the pluripotency of porcine embryos. The impacts of inhibited activities of TET enzymes on the expression of pluripotency related genes were examined. We found that the inhibition of all TET enzymes leads to a decreased expression of pluripotency related genes, an altered DNA methylation level on a gene segment controlling pluripotency, and the impaired formation of pluripotent cell lineage in porcine embryos. This study demonstrates that the TET family is critical for the acquisition of pluripotency in porcine embryos. In the following sets of studies, the function of TET3 protein in the demethylation process occurring in preimplantation embryos was investigated. Fertilized mammalian embryos undergo genome-wide demethylation process to reset germ cell specific epigenetic marks into the embryonic epigenome. Previous studies indicate that TET3 is responsible for the demethylation process in mammalian embryos, although detailed mechanistic action of TET3 is still elusive. Here, we identified a predominant expression of a specific TET3 gene in porcine oocytes. The TET3 gene contained a CXXC domain, a potential DNA binding module, suggesting that TET3 may mediate DNA demethylation through its DNA binding property. To examine the function of the CXXC domain in TET3-mediated DNA demethylation, isolated CXXC domain was injected into porcine oocytes. The injection of CXXC domain facilitated DNA demethylation in embryos, demonstrating that the DNA binding property of TET3 is important for its functionality. In the last study, we investigated the importance of genes known to interact with TET enzymes in porcine embryos. Methyl-CpG-binding domain proteins (MBDs) have the ability to bind methylated region on the genome and play a critical role in mediating DNA methylation and gene repression. Our hypothesis was that a competitive binding of MBD and TET proteins to methylated regions was critical for proper DNA methylation levels in embryos. We identified that porcine MBD sequences were very similar to other species in terms of gene structure, indicating that the genes could also possess gene repressing activity by competing with TET enzymes during porcine embryo development. Injection of MBD1 mRNA to oocytes impaired normal embryo development, suggesting that the injected MBD1 may have negatively affected early embryo development in pigs by disrupting the proper maintenance of DNA methylation levels. My dissertation researches demonstrate that maintaining proper DNA methylation levels through the TET family is critical for normal embryo development in pigs. This research assists in improving our understating of epigenetic dynamics occurring in mammalian embryos and offers a potential solution to the epigenetic defects frequently observed in mammalian embryos produced through artificial reproductive technologies and pluripotent stem cells reprogrammed from somatic cells.
- Doctoral Dissertations