Effects Of Tet Dioxygenases On DNA Methylation And Related Regulatory Mechanisms

As the primary form of DNA methylation modification,5mC plays an important role in regulation of chromatin structure and gene expression.Methylation of DNA is established and maintained by DNA methyltransferase(Dnmts),and the demethylation process has been recognized and studied in recent years.The ten-eleven translocation(Tet)protein family can catalyze the continuous oxidation of 5mC,gradually producing 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC)and 5-carboxylcytosine(5caC),which are eventually removed from the DNA and consequently restored to unmethylated cytosine.During the embryo development,Tet3 is highly expressed in oocytes and zygotes,and mediates the demethylation of the paternal DNA,while Tet1 and Tet2 begin to express at the late stages of preimplantation development.With the in-depth study on Tet proteins,it has been found that the regulation of DNA demethylation by Tet is a complex process..The differences in the action of three Tet proteins during embryonic development are not yet clear.Many issues such as the roles of different Tet protein on the paternal,maternal DNA in fertilized eggs and somatic DNA,and the functional mechanism of Tets remain to be further studied.To study the role of Stella in somatic cells,a cell line stably expressing Stella was established using HeLa cells,and Tet3 was transiently transfected into the cells.Immunofluorescence staining and Dot-blotting revealed that Stella inhibited the activity of Tet3 but not Tet1 and Tet2 in cells.To reduce the level of H3K9 dimethylation(H3K9 dimethylation,H3K9me2)in cells,cells were treated with histone methyltransferase inhibitor UNC0638.Immunofluorescence assays revealed that the decrease of H3K9me2 in somatic cells did not affect the Tet3 inhibition by exogenous Stella.In cloned embryos from UNC0638-treated cells,the reduction of H3K9me2 modification did not influence the distribution of Stella and the production of 5hmC during pronuclear development.Co-IP assays showed that Stella could not interact with H3K9me2 modified histones,and the levels of H3K9me2 in cells had no effect on the interaction intensity between Stella and Tet3.These results indicate that different from that in fertilized zygotes,H3K9me2 does not affect the interaction between Stella and Tet3 in somatic cell nuclear transfer embryos.Moreover,Stella has no interaction with Tetl or Tet2.To investigate the roles of Tet1 and Tet2 in zygotes,Tet1 and Tet2 mRNA were injected into the fertilized 1-cell embryos.Immunofluorescence staining showed that the 5hmC levels in both the paternal and maternal DNA were significantly increased by either Tet1 or Tet2 mRNA injection,but 5mC levels were not significantly altered.To further examine the effects of Tet1 and Tet2 on DNA methylation,single-cell methylation sequencing was performed on gametes and zygotes injected with GFP,Tet1,and Tet2 mRNAs,and the parental DNA were distinguished using parents' single nucleotide polymorphisms(SNPs).The results showed that Tet1 or Tet2 overexpression could result in further decrease in the methylation levels of maternal and paternal DNA.On the other hand,the methylation levels in some loci were elevated.In both the paternal and maternal genomes,Tet1 and Tet2 have their own specific recognition sites.Overexpression of Tetl inhibits the development of morula to blastocysts,while overexpression of Tet2 affects the division of embryos from two-cells to four-cells.Moreover,the quality of blastocysts were reduced in Tet1and Tet2 overexpressed embryos.Above results indicate that overexpressed Tet1 and Tet2 in 1-cell embryos can induce considerable changes in DNA nethylation profiles,and the altered DNA methylation in zygotes by Tet1 and Tet2 has an adverse effect on embryo development.To further investigate the regulating mechanism of Tet protein on 5hmC and 5mC accumulation,a Tet3 reversible expressing cell line was established in HEK293 cells using the "Tet-on advanced" system.Assays with immunofluorescence staining,Liquid chromatograph mass spectrometry(LC-MS)showed that Tet3 expression could lead to the overall increase of both 5hmC and 5mC.After the Tet3 expression was turned off,the decrease of 5hmC levels was accompanied by a simultaneous decrease of 5mC levels.Analysis of the H19 gene promoter region also showed that Tet3 caused elevation of both 5hmC and 5mC levels at specific sites.Detection of DNA methyltransferase activity in cells showed that overexpression of Tet3 could increase the activity of methyltransferases.Co-IP assays confirmed the interaction of Tet3 with DNMT1,DNMT3A and DNMT3B.These results suggest that Tet3 might enhance the activity of DNMTs,which in turn promotes the 5mC generation.In summary,the interaction between Stella and Tet3 in somatic cells or cloned embryos does not depend on H3K9me2 modification;Stella has no inhibitory effect on Tet1 and Tet2,and overexpression of Tet1 and Tet2 leads to abnormal DNA methylation in zygotes and hampers the embryo development;after fertilization,both DNA demethylation and remethylation occur in fertilized eggs;Tet3 can interact with DNMTs and may enhance the methyltransferase activity and.This study provides some new information for understanding the mechanism of DNA methylation reprogramming in fertilized and cloned embryos,and may also facilitate further elucidating the molecular mechanism of DNA modification mediated by Tet proteins.