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Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons

Dnmt1 and Dnmt3a are important DNA methyltransferases that are expressed in postmitotic neurons, but their function in the CNS is unclear. We generated conditional mutant mice that lack Dnmt1, Dnmt3a or both exclusively in forebrain excitatory neurons and found that only double knockout (DKO) mice showed abnormal long-term plasticity in the hippocampal CA1 region together with deficits in learning and memory. Although we found no neuronal loss, hippocampal neurons in DKO mice were smaller than in the wild type; furthermore, DKO neurons showed deregulated expression of genes, including the class I MHC genes and Stat1, that are known to contribute to synaptic plasticity. In addition, we observed a significant decrease in DNA methylation in DKO neurons. We conclude that Dnmt1 and Dnmt3a are required for synaptic plasticity, learning and memory through their overlapping roles in maintaining DNA methylation and modulating neuronal gene expression in adult CNS neurons.

DNA Methylation and Its Basic Function

In the mammalian genome, DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine. DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factor(s) to DNA. During development, the pattern of DNA methylation in the genome changes as a result of a dynamic process involving both de novo DNA methylation and demethylation. As a consequence, differentiated cells develop a stable and unique DNA methylation pattern that regulates tissue-specific gene transcription. In this chapter, we will review the process of DNA methylation and demethylation in the nervous system. We will describe the DNA (de)methylation machinery and its association with other epigenetic mechanisms such as histone modifications and noncoding RNAs. Intriguingly, postmitotic neurons still express DNA methyltransferases and components involved in DNA demethylation. Moreover, neuronal activity can modulate their pattern of DNA methylation in response to physiological and environmental stimuli. The precise regulation of DNA methylation is essential for normal cognitive function. Indeed, when DNA methylation is altered as a result of developmental mutations or environmental risk factors, such as drug exposure and neural injury, mental impairment is a common side effect. The investigation into DNA methylation continues to show a rich and complex picture about epigenetic gene regulation in the central nervous system and provides possible therapeutic targets for the treatment of neuropsychiatric disorders.

Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain

DNA methylation has critical roles in the nervous system and has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes. Here we show that the single base–resolution DNA methylome from adult mouse dentate neurons consists of both CpG (∼75%) and CpH (∼25%) methylation (H = A/C/T). Neuronal CpH methylation is conserved in human brains, enriched in regions of low CpG density, depleted at protein-DNA interaction sites and anticorrelated with gene expression. Functionally, both methylated CpGs (mCpGs) and mCpHs can repress transcription in vitro and are recognized by methyl-CpG binding protein 2 (MeCP2) in neurons in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNA methyltransferase 3A (DNMT3A) for active maintenance in postmitotic neurons. These characteristics of CpH methylation suggest that a substantially expanded proportion of the neuronal genome is under cytosine methylation regulation and provide a new foundation for understanding the role of this key epigenetic modification in the nervous system.

Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in various embryonic and adult tissues. Mice mutant for either Tet1 or Tet2 are viable, raising the question of whether these enzymes have overlapping roles in development. Here we have generated Tet1 and Tet2 double-knockout (DKO) embryonic stem cells (ESCs) and mice. DKO ESCs remained pluripotent but were depleted of 5hmC and caused developmental defects in chimeric embryos. While a fraction of double-mutant embryos exhibited midgestation abnormalities with perinatal lethality, viable and overtly normal Tet1/Tet2-deficient mice were also obtained. DKO mice had reduced 5hmC and increased 5mC levels and abnormal methylation at various imprinted loci. Nevertheless, animals of both sexes were fertile, with females having smaller ovaries and reduced fertility. Our data show that loss of both enzymes is compatible with development but promotes hypermethylation and compromises imprinting. The data also suggest a significant contribution of Tet3 to hydroxylation of 5mC during development.

Loss of Tet enzymes compromises proper differentiation of embryonic stem cells.

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and are dynamically expressed during development. Whereas loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development is not established. We have generated Tet1/2/3 triple-knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential. Combined deficiency of all three Tets depleted 5hmC and impaired ESC differentiation, as seen in poorly differentiated TKO embryoid bodies (EBs) and teratomas. Consistent with impaired differentiation, TKO ESCs contributed poorly to chimeric embryos, a defect rescued by Tet1 reexpression, and could not support embryonic development. Global gene-expression and methylome analyses of TKO EBs revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet- and 5hmC-mediated DNA demethylation in proper regulation of gene expression during ESC differentiation and development.

A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

The recent discovery of 5-hydroxymethyl-cytosine (5 hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5 mC) and 5 hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC-ESI-MS/MS-MRM) to simultaneously measure levels of 5 mC and 5 hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5 hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5 hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5 mC and 5 hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.

Role of Tet1 and 5-hydroxymethylcytosine in cocaine action

Ten-eleven translocation (TET) enzymes mediate the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is enriched in brain, and its ultimate DNA demethylation. However, the influence of TET and 5hmC on gene transcription in brain remains elusive. We found that ten-eleven translocation protein 1 (TET1) was downregulated in mouse nucleus accumbens (NAc), a key brain reward structure, by repeated cocaine administration, which enhanced behavioral responses to cocaine. We then identified 5hmC induction in putative enhancers and coding regions of genes that have pivotal roles in drug addiction. Such induction of 5hmC, which occurred similarly following TET1 knockdown alone, correlated with increased expression of these genes as well as with their alternative splicing in response to cocaine administration. In addition, 5hmC alterations at certain loci persisted for at least 1 month after cocaine exposure. Together, these reveal a previously unknown epigenetic mechanism of cocaine action and provide new insight into how 5hmC regulates transcription in brain in vivo.

Skeletal muscle action of estrogen receptor α is critical for the maintenance of mitochondrial function and metabolic homeostasis in females

ERα action in skeletal muscle is involved in the preservation of mitochondrial health and insulin sensitivity in female mice and can serve as a defense against metabolic disease in women. Postmenopausal muscle and mitochondrial mayhem Menopause ushers in a host of changes that range from unpleasant to undesirable. One undesirable shift is a loss of protection against insulin resistance, which brings with it a constellation of consequences in the form of chronic disease associated with metabolic dysfunction. Now, Ribas et al. investigate the mechanism underlying the postmenopausal chinks in a woman’s energy homeostasis armor. The estrogen receptor (ER) is known to participate in the preservation of mitochondrial health and insulin sensitivity in mice, but the precise tissue-specific mechanisms remain unclear. Because skeletal muscle is a main tissue responsible for insulin-stimulated glucose disposal, the authors first showed that ERα expression in muscle correlated with metabolic health in human females. They then created a muscle-specific ERα knockout (MERKO) mouse and found that glucose homeostasis was disrupted, fat accumulation increased, and mitochondrial function impaired. These findings imply that ERα action in skeletal muscle helps maintain mitochondrial function and metabolic homeostasis in females. Impaired estrogen receptor α (ERα) action promotes obesity and metabolic dysfunction in humans and mice; however, the mechanisms underlying these phenotypes remain unknown. Considering that skeletal muscle is a primary tissue responsible for glucose disposal and oxidative metabolism, we established that reduced ERα expression in muscle is associated with glucose intolerance and adiposity in women and female mice. To test this relationship, we generated muscle-specific ERα knockout (MERKO) mice. Impaired glucose homeostasis and increased adiposity were paralleled by diminished muscle oxidative metabolism and bioactive lipid accumulation in MERKO mice. Aberrant mitochondrial morphology, overproduction of reactive oxygen species, and impairment in basal and stress-induced mitochondrial fission dynamics, driven by imbalanced protein kinase A–regulator of calcineurin 1–calcineurin signaling through dynamin-related protein 1, tracked with reduced oxidative metabolism in MERKO muscle. Although muscle mitochondrial DNA (mtDNA) abundance was similar between the genotypes, ERα deficiency diminished mtDNA turnover by a balanced reduction in mtDNA replication and degradation. Our findings indicate the retention of dysfunctional mitochondria in MERKO muscle and implicate ERα in the preservation of mitochondrial health and insulin sensitivity as a defense against metabolic disease in women.

Regulation of nucleotide metabolism by mutant p53 contributes to its gain-of-function activities

SUMMARY Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP dependent protein (GTPase) activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. Additionally, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase (dCK) for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53 harboring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53’s control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

Dnmt3a Regulates Both Proliferation and Differentiation of Mouse Neural Stem Cells

DNA methylation is known to regulate cell differentiation and neuronal function in vivo. Here we examined whether deficiency of a de novo DNA methyltransferase, Dnmt3a, affects in vitro differentiation of mouse embryonic stem cells (mESCs) to neuronal and glial cell lineages. Early‐passage neural stem cells (NSCs) derived from Dnmt3a‐deficient ESCs exhibited a moderate phenotype in precocious glial differentiation compared with wild‐type counterparts. However, successive passaging to passage 6 (P6), when wild‐type NSCs become gliogenic, revealed a robust phenotype of precocious astrocyte and oligodendrocyte differentiation in Dnmt3a−/− NSCs, consistent with our previous findings in the more severely hypomethylated Dnmt1−/− NSCs. Mass spectrometric analysis revealed that total levels of methylcytosine in Dnmt3a−/− NSCs at P6 were globally hypomethylated. Moreover, the Dnmt3a−/− NSC proliferation rate was significantly increased compared with control from P6 onward. Thus, our work revealed a novel role for Dnmt3a in regulating both the timing of neural cell differentiation and the cell proliferation in the paradigm of mESC‐derived‐NSCs.