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Home arrow Health arrow DNA Modifications in the Brain. Neuroepigenetic Regulation of Gene Expression
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DNA METHYLTRANSFERASES

The first mammalian DNA methyltransferases cloned were the mouse maintenance enzyme Dnmt1 (Bestor et al., 1988) and its closest human homologue DNMT1 (Yen et al., 1992). Interestingly, DNMT1 mRNA was found to be most highly expressed in the brain. This high expression was rather surprising, considering the low proliferative potential of brain cells. A targeted mutation of Dnmtl does not affect morphology and growth rates of mouse embryonic stem cells (ESCs) in tissue culture, but in mouse embryos it results in severe developmental abnormalities and lethality (Li et al., 1992). The next mammalian Dnmt gene cloned was logically termed Dnmt2 (Okano, Xie, & Li, 1998a). It contained all conserved methyltransferase motifs and, thus, could likely encode a functional cytosine methyltransferase. Dnmt2 has weak DNA methylation activity and seemed to be a dual-function protein capable of methylating both DNA and a cytosine residue in the anticodon loop of tRNAAsp (Jeltsch, Nellen, & Lyko, 2006). The next mouse genes cloned were Dnmt3a and Dnmt3b, encoding two highly similar proteins of 908 and 859 amino acids, respectively (Okano et al., 1998b). Cloned Dnmt3 proteins are the long-sought de novo DNA methyltransferases. Dnmt3a and Dnmt3b genes are highly expressed in ESCs and at a much lower levels in adult somatic tissues. Both were found to be required for genome-wide de novo methylation and essential for mammalian development (Okano, Bell, Haber, & Li, 1999).

The Dnmtl gene is highly expressed throughout the entire neuraxis at embryonic day 13 (E13) (Goto et al., 1994). Its expression in the brain decreases at E18 and postnatal day 1 (P1), although relatively high levels are retained in the forebrain. Expression is further attenuated at P7 throughout the entire brain, except for the granular layers of the cerebellum and the neuronal layer of the olfactory bulb. Only weak expression is discernible throughout the entire gray matter of the brain at P21 and later. Thus, the Dnmtl gene is expressed in postmitotic neurons, which after migration from the neurogenic zones have already reached their final destination in the brain. Its expression is maintained in most differentiated neurons in the adult and even aged brain, including the cerebellar granular layer and hippocampal neuronal layers. Considering that differentiated neurons are thought to no longer synthesize DNA, the persistent expression of Dnmtl indicates the occurrence of DNA methylation in neuronal cells in the absence of cell proliferation. After a conditional postmitotic neuron-specific knockout (KO) Dnmtl mutation in mice Dnmtl-deficient neurons were present in adult mutant brain at a constant percentage at all ages (Fan et al., 2001). When Dnmtl gene deletion occurred in ~30% neurons at midgestation, newborn mutants were born alive and normal, but the percentage of mutant cells in brain tissue was decreased significantly by P14 and not detectable at all by 3 weeks of age. Thus, Dnmt1 is required for the survival of mitotic neuronal precursor cells and their daughter cells, but not for the survival of postmitotic neurons.

Dnmt3a and Dnmt3b mRNAs are readily detectable in the newborn mouse cortex (Feng, Chang, Li, & Fan, 2005). In the cortex of adult mice Dnmt3a is expressed at a high level, whereas expression of Dnmt3b is not detectable. Thus, Dnmt3a and Dnmt3b play distinct roles in brain development. In the adult brain Dnmt3a is present in postmitotic neurons and oligodendrocytes and nearly absent in astroglial cells. A double knockout (DKO) of Dnmtl and Dnmt3a at P14 with the efficiency ~50% did not affect mouse life span and behavior, but significantly attenuated long-term potentiation in hippocampus and impaired learning and memory ability (Feng et al., 2010). Thus, Dnmt1 and Dnmt3a are required for normal synaptic plasticity. A gene expression analysis showed 84 genes to be upregulated and seven genes downregulated in DKO mice. An upregulated gene Statl undergoes demethylation at the -895 to -1010 bp position of its promoter after deletion of Dnmtl and Dnmt3a.The cortex and hippocampus of DKO mice exhibit an ~20% reduction of total 5mC, suggesting that demethylation in DKO neurons could be widespread. Thus, Dnmt1 and Dnmt3a are essential for maintaining proper DNA methylation patterns at certain genomic loci in postmitotic CNS neurons.

Analysis of the Dnmt3a KO mice showed that Dnmt3a is required for neurogenesis (Wu et al., 2010). Two groups of Dnmt3a binding sites were found in the postnatal neuronal stem cell genome. The first group consists of genes in which CpG islands along their promotes are enriched with H3K4me3 (eg, Dlx2, Gbx2). On these genes Dnmt3a is generally excluded from H3K4me3-high, CpG-rich proximal promoter sequences, but it is enriched in flanking regions. The other group includes genes with CpG-poor promoters and low levels of H3K4me3 (eg, Gfap). The Dnmt3a binding sites on these genes frequently overlap with proximal promoter sequences. Thus, Dnmt3a may occupy and methylate defined genomic regions associated with both transcriptionally active and inactive genes. Gene ontology analysis showed that genes functionally related to neurogenesis are significantly enriched within H3K4me3-high group, whereas genes involved in development of nonneuronal lineage are enriched in H3K4me3-low group. Gene expression profiling of Dnmt3a KO mice identified both upregulated and down- regulated genes. The Dnmt3a binding sites within regulatory regions were detected in 942 of these genes; notably, genes with known roles in postnatal neurogenesis (eg, Dlx2, Sp8, and Neurog2) were downregulated, whereas those involved in astroglial and oligo- dendroglial differentiation (eg, Sparcll, Nkx2-2) were upregulated. Thus, Dnmt3a binding can either promote transcription (neurogenic genes) or repress it (glial differentiation genes). Dnmt3a deficiency was found to increase the H3K27me3 levels associated with many downregulated targets. Antagonism between Dnmt3a and H3K27me3 may facilitate the expression of many H3K4me3-high targets by reducing H3K27me3 levels. Apparently, Dnmt3a not only mediates gene repression by methylating proximal promoters, but also promotes transcription of neurogenic genes by antagonizing Polycomb repression through nonproximal promoter methylation.

 
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