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.

Dnmt1 and Dnmt3a, two major DNA methyltransferases, are expressed in postmitotic neurons, but their function in the central nervous system (CNS) is unclear. We generated conditional mutant mice that lack either Dnmt1, or Dnmt3a, or both exclusively in forebrain excitatory neurons and found only double knockout (DKO) mice exhibited abnormal hippocampal CA1 long-term plasticity and deficits of learning and memory. While no neuronal loss was found, the size of hippocampal neurons in DKO was smaller; furthermore, DKO neurons showed a deregulation of gene expression including class I MHC and Stat1 that are known to play a role in 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.The long-lasting changes in synaptic plasticity underlying learning and memory require changes in neuronal gene expression 1 . Epigenetic mechanisms such as histone modification 2 and DNA methylation are thought to contribute to this adaptive neuronal gene expression 3 . It has been shown that different histone modifications are associated with various neuronal gene expression states 2, 4 ; moreover, increasing histone acetylation by histone deacetylase inhibitors promotes recovery of learning and memory in a mouse model of neurodegeneration 5,6 . However, the function of DNA methylation in the nervous system is

DNA methylation plays 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 low CpG-density regions, depleted at protein-DNA interaction sites, and anti-correlated with gene expression. Functionally, both mCpGs and mCpHs can repress transcription in vitro and are recognized by MeCP2 in neurons in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNMT3A for active maintenance in post-mitotic neurons. These characteristics of CpH methylation suggest a significantly expanded proportion of the neuronal genome under cytosine methylation regulation and provide a new foundation for understanding the role of this key epigenetic modification in the nervous system.

SUMMARY To investigate the cell-intrinsic aging mechanisms that erode the function of somatic stem cells during aging, we have conducted a comprehensive integrated genomic analysis of young and aged cells. We profiled the transcriptome, DNA methylome, and histone modifications of young and old murine hematopoietic stem cells (HSCs). Transcriptome analysis indicated reduced TGFβ signaling and perturbation of genes involved in HSC proliferation and differentiation. Aged HSCs exhibited broader H3K4me3 peaks across HSC identity and self-renewal genes, and showed increased DNA methylation at transcription factor binding sites associated with differentiation-promoting genes combined with a reduction at genes associated with HSC maintenance. Together these changes reinforce HSC self-renewal and diminish differentiation, paralleling phenotypic HSC aging behavior. Ribosomal biogenesis emerged as a particular target of aging, with increased transcription of ribosomal protein and RNA genes, and hypomethylation of rRNA genes. This dataset will serve as a reference for future epigenomic analysis of stem cell aging.

Summary 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 whether these enzymes have overlapping roles in development. Here, we have generated Tet1 and Tet2 double knockout (DKO) 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 mid-gestation 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. It also suggests a significant contribution of Tet3 to hydroxylation of 5mC during development.