Maternal inheritance of glucose intolerance via oocyte TET3 insufficiency.

Diabetes mellitus is prevalent among women of reproductive age, and many women are left undiagnosed or untreated1. Gestational diabetes has profound and enduring effects on the long-term health of the offspring2,3. However, the link between pregestational diabetes and disease risk into adulthood in the next generation has not been sufficiently investigated. Here we show that pregestational hyperglycaemia renders the offspring more vulnerable to glucose intolerance. The expression of TET3 dioxygenase, responsible for 5-methylcytosine oxidation and DNA demethylation in the zygote4, is reduced in oocytes from a mouse model of hyperglycaemia (HG mice) and humans with diabetes. Insufficient demethylation by oocyte TET3 contributes to hypermethylation at the paternal alleles of several insulin secretion genes, including the glucokinase gene (Gck), that persists from zygote to adult, promoting impaired glucose homeostasis largely owing to the defect in glucose-stimulated insulin secretion. Consistent with these findings, mouse progenies derived from the oocytes of maternal heterozygous and homozygous Tet3 deletion display glucose intolerance and epigenetic abnormalities similar to those from the oocytes of HG mice. Moreover, the expression of exogenous Tet3 mRNA in oocytes from HG mice ameliorates the maternal effect in offspring. Thus, our observations suggest an environment-sensitive window in oocyte development that confers predisposition to glucose intolerance in the next generation through TET3 insufficiency rather than through a direct perturbation of the oocyte epigenome. This finding suggests a potential benefit of pre-conception interventions in mothers to protect the health of offspring.

Loss of TET reprograms Wnt signaling through impaired demethylation to promote lung cancer development.

Oncogenic imbalance of DNA methylation is well recognized in cancer development. The ten-eleven translocation (TET) family of dioxygenases, which facilitates DNA demethylation, is frequently dysregulated in cancers. How such dysregulation contributes to tumorigenesis remains poorly understood, especially in solid tumors which present infrequent mutational incidence of TET genes. Here, we identify loss-of-function mutations of TET in 7.4% of human lung adenocarcinoma (LUAD), which frequently co-occur with oncogenic KRAS mutations, and this co-occurrence is predictive of poor survival in LUAD patients. Using an autochthonous mouse model of KrasG12D -driven LUAD, we show that individual or combinational loss of Tet genes markedly promotes tumor development. In this Kras-mutant and Tet-deficient model, the premalignant lung epithelium undergoes neoplastic reprogramming of DNA methylation and transcription, with a particular impact on Wnt signaling. Among the Wnt-associated components that undergo reprogramming, multiple canonical Wnt antagonizing genes present impaired expression arising from elevated DNA methylation, triggering aberrant activation of Wnt signaling. These impairments can be largely reversed upon the restoration of TET activity. Correspondingly, genetic depletion of ß-catenin, the transcriptional effector of Wnt signaling, substantially reverts the malignant progression of Tet-deficient LUAD. These findings reveal TET enzymes as critical epigenetic barriers against lung tumorigenesis and highlight the therapeutic vulnerability of TET-mutant lung cancer through targeting Wnt signaling.

Uracil-DNA Glycosylase UNG Promotes Tet-mediated DNA Demethylation.

In mammals, active DNA demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active demethylation in embryonic stem cells and induced pluripotent stem cells, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA demethylation. To search for other factors that might contribute to demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA demethylation.

Cbx4 regulates the proliferation of thymic epithelial cells and thymus function.

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

Auto-suppression of Tet dioxygenases protects the mouse oocyte genome from oxidative demethylation.

DNA cytosine methylation plays a vital role in repressing retrotransposons, and such derepression is linked with developmental failure, tumorigenesis and aging. DNA methylation patterns are formed by precisely regulated actions of DNA methylation writers (DNA methyltransferases) and erasers (TET, ten-eleven translocation dioxygenases). However, the mechanisms underlying target-specific oxidation of 5mC by TET dioxygenases remain largely unexplored. Here we show that a large low-complexity domain (LCD), located in the catalytic part of Tet enzymes, negatively regulates the dioxygenase activity. Recombinant Tet3 lacking LCD is shown to be hyperactive in converting 5mC into oxidized species in vitro. Endogenous expression of the hyperactive Tet3 mutant in mouse oocytes results in genome-wide 5mC oxidation. Notably, the occurrence of aberrant 5mC oxidation correlates with a consequent loss of the repressive histone mark H3K9me3 at ERVK retrotransposons. The erosion of both 5mC and H3K9me3 causes ERVK derepression along with upregulation of their neighboring genes, potentially leading to the impairment of oocyte development. These findings suggest that Tet dioxygenases use an intrinsic auto-regulatory mechanism to tightly regulate their enzymatic activity, thus achieving spatiotemporal specificity of methylome reprogramming, and highlight the importance of methylome integrity for development.

Tet1 regulates adult hippocampal neurogenesis and cognition.

DNA hydroxylation catalyzed by Tet dioxygenases occurs abundantly in embryonic stem cells and neurons in mammals. However, its biological function in vivo is largely unknown. Here, we demonstrate that Tet1 plays an important role in regulating neural progenitor cell proliferation in adult mouse brain. Mice lacking Tet1 exhibit impaired hippocampal neurogenesis accompanied by poor learning and memory. In adult neural progenitor cells deficient in Tet1, a cohort of genes involved in progenitor proliferation were hypermethylated and downregulated. Our results indicate that Tet1 is positively involved in the epigenetic regulation of neural progenitor cell proliferation in the adult brain.

Deficiency in a glutamine-specific methyltransferase for release factor causes mouse embryonic lethality.

Biological methylation is a fundamental enzymatic reaction for a variety of substrates in multiple cellular processes. Mammalian N6amt1 was thought to be a homologue of bacterial N(6)-adenine DNA methyltransferases, but its substrate specificity and physiological importance remain elusive. Here, we demonstrate that N6amt1 functions as a protein methyltransferase for the translation termination factor eRF1 in mammalian cells both in vitro and in vivo. Mass spectrometry analysis indicated that about 70% of the endogenous eRF1 is methylated at the glutamine residue of the conserved GGQ motif. To address the physiological significance of eRF1 methylation, we disrupted the N6amt1 gene in the mouse. Loss of N6amt1 led to early embryonic lethality. The postimplantation development of mutant embryos was impaired, resulting in degeneration around embryonic day 6.5. This is in contrast to what occurs in Escherichia coli and Saccharomyces cerevisiae, which can survive without the N6amt1 homologues. Thus, N6amt1 is the first glutamine-specific protein methyltransferase characterized in vivo in mammals and methylation of eRF1 by N6amt1 might be essential for the viability of early embryos.