DNA methyltransferases 3A and 3B target specific sequences during mouse gastrulation
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ABSTRACT:
PROVIDER: PRJNA821521 | ENA |
REPOSITORIES: ENA
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2012-02-27 | E-GEOD-35200 | biostudies-arrayexpress
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Project description:Polyamines are essential for various cell functions and are produced in most cells, as well as being taken up from extracellular sources. Polyamines, especially spermine, in blood cells increase when polyamines are supplied from the digestive tract. Altered polyamine metabolism has an impact on substrate concentrations and enzyme activities involved in gene methylation, and may affect gene expression. Therefore, we investigated the effect of decreased polyamine synthesis and extracellular spermine supply on substrate concentrations and enzyme activities involved in gene methylation and on changes of methylation in promoter regions using methylation arrays. In Jurkat cells and human mammary epithelial cells, changes in polyamine concentrations differed after polyamine synthesis inhibition. However, S-adenosylmethionine decarboxylase activity and the decarboxylated S-adenosylmethionine to S-adenosyl-L-methionine ratio were decreased by spermine addition in both cells. After inhibiting polyamine synthesis in Jurkat cells by D,L-alpha-difluoromethylornithine, the protein levels of DNA methyltransferase (DNMT) 1, 3A and 3B were not changed, but the activity of the three enzymes markedly decreased. The protein levels of these enzymes were not changed by addition of spermine; however, DNMT 3B was markedly activated and DNMT 3A was also activated. DNMT 1 was not activated. Effects on methylation of promoter regions included significant changes for tumor suppressor genes in response to altered polyamine metabolism. However, no significant changes in methylation status were found on genes of which methylation status and their related protein levels were affected by spermine. When considering these results, there are many genes for which polyamines have an impact on expression via methylation of promoter regions.
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Project description:Targeted disruption of DNMT1, 3A and 3B in human embryonic stem cells
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Project description:Base editor is also a powerful tool to introduce early stop codons to simultaneously silence multiple genes to study family gene function in embryo development. Here, we identified the highest efficiency base editing system (hA3A-eBE-Y130F) with few sgRNA independent off-target sites among six well-established base editors in mouse embryo. Further, we developed a Multi-Stop system to simultaneously knock-out all Ten-eleven translocation (TET) family of dioxygenase genes (Tet1, Tet2 and Tet3) in zygotic genome leads embryo die before 10.5 days of gestation. Moreover, we showed that inactivation of three DNA methyltransferases genes (Dnmt1, Dnmt3a and Dnmt3b) in zygote leads to gastrulation failure. We also obtained multiple TET and DNMT family genes (6KO: Dnmt1/3a/3b and Tet1/2/3; 5KO: Dnmt3a/3b and Tet1/2/3; 4KO: Dnmt1 and Tet1/2/3) deactivated mouse embryos in one step, which indicated that Dnmt1 or Dnmt3a/b is indispensable for mouse gastrulation.
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Project description:CpG methylation by the de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes yet cannot methylate the DNA wrapped around the nucleosome core2, and favor the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-EM structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3, and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur. CpG methylation by the de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes yet cannot methylate the DNA wrapped around the nucleosome core2, and favor the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-EM structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3, and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.
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