In a study published in Nature Structural & Molecular Biology, the team led by Prof. DU Yarui and Prof. XU Guoliang from the Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences revealed the spatiotemporal regulation mechanism of Tet dioxygenases by low-complexity domain (LCD) located in the catalytic active center.
DNA methylation, predominantlyrepresented by 5-methylcytosine (5mC), serves as a remarkable epigenetic marker playing a critical role in the regulation of gene expression, genomic imprinting, X chromosome inactivation, and transposon silencing in mammals. The establishment of DNA methylation patterns is jointly regulated by DNA methyltransferases (Dnmts) and Tet dioxygenases. Aberrant oxidation of 5-methylcytosine in the mammalian genome links to developmental failure and tumorigenesis.
The previous work (Gu et al., The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477, 2011) showed that Tet3 appeared to be largely inactive despite its high abundance in GV oocytes, indicating the presence of a mechanism to keep the Tet3 activity in check. One recent study (Chen et al., Maternal inheritance of glucose intolerance via oocyte TET3 insufficiency. Nature 605, 2022) highlighted the necessity also for adequate Tet3 activity to achieve parental gene demethylation and avoid transmission of adverse maternal exposure to the offspring. Thus, there must be a mechanism to ensure a tight control of Tet3 to determine where and when to exert its oxidation activity. The regulation of Tet enzymatic activity could allow for target-specific 5mC oxidation, but the mechanism underlying such a control system and its detailed workings remain unknown.
In this study, the researchers found that the recombinant Tet3 lacking LCD was hyperactive in converting 5mC into oxidized species in vitro. Subsequently, the researchers obtained the mouse model with conditional knockout of LCD in the female germline using the unique AG-haESCs technology and CRISPR/Cas9 gene editing technology. Using UHPLC-MS/MS analysis and immunofluorescence staining, the researchers demonstrated that the absence of LCD resulted in a significant increase in 5mC oxidation products in the oocyte genome.
Although the number of mature oocytes produced by conditional knockout (CKO) females remains unchanged, their female fertility was significantly reduced. The researchers further analyzed the phenotypes of oocytes expressing Tet3 ΔLCD protein and found that the maturation process of LCD KO oocytes appeared to be compromised as reflected by a delayed germinal vesicle (GV) breakdown (GVBD) and their severely impaired developmental competence.
To further explore how LCD deletion caused impaired oocyte maturation, the researchers combined the results of RNA-seq, WGBS, ACE-seq and Cut&Tag and found that without the inhibition by LCD, 5mC oxidation by Tet3 occurs extensively across the oocyte genome. As oocytes are at the meiosis arrest stage, the oxi-mC derivatives such as 5fC and5caC accumulated on the originally hypermethylated ERVK elements, accompanied by a decrease in H3K9me3 levels, which destroyed the dual inhibition of 5mC and H3K9me3 on retrotransposable elements such as ERVK, affected the expression of oogenesis related genes adjacent to ERVK, supposedly contributing to defects in oogenesis and preimplantation development.
The study reveals that LCD plays a suppressive role critical for the regulation of Tet3 activity, shielding the oocyte methylome from oxidative erosion. Aberrant DNA oxidation conferred by hyperactive TET enzymes may also provide a new paradigm for human infertility, reproductive aging or other pathologies, especially leukemia with altered DNA methylation.