Repression of retrotransposal elements in mouse embryonic stem cells is primarily mediated by a DNA methylation-independent mechanism.

In defense of deleterious retrotransposition of intracisternal A particle (IAP) elements, IAP loci are heavily methylated and silenced in mouse somatic cells. To determine whether IAP is also repressed in pluripotent stem cells by DNA methylation, we examined IAP expression in demethylated mouse embryonic stem cells (mESCs) and epiblast-derived stem cells. Surprisingly, in demethylated ESC cultures carrying mutations of DNA methyltransferase I (Dnmt1), no IAP transcripts and proteins are detectable in undifferentiated Oct4(+) ESCs. In contrast, approximately 3.6% of IAP-positive cells are detected in Oct4(-) Dnmt1(-/-) cells, suggesting that the previously observed increase in IAP transcripts in the population of Dnmt1(-/-) ESCs could be accounted for by this subset of Oct4(-) Dnmt1(-/-) ESCs undergoing spontaneous differentiation. Consistent with this possibility, a dramatic increase of IAP mRNA (>100-fold) and protein expression was observed in Dnmt1(-/-) ESC cultures upon induction of differentiation through the withdrawal of leukemia-inhibitory factor for 6 or more days. Interestingly, both mRNAs and proteins of IAP can be readily detected in demethylated Oct4(+) epiblast-derived stem cells as well as differentiated mouse embryo fibroblasts, neurons, and glia upon conditional Dnmt1 gene deletion. These data suggest that mESCs are a unique stem cell type possessing a DNA methylation-independent IAP repression mechanism. This methylation-independent mechanism does not involve Dicer-mediated action of microRNAs or RNA interference because IAP expression remains repressed in Dnmt1(-/-); Dicer(-/-) double mutant ESCs. We suggest that mESCs possess a unique DNA methylation-independent mechanism to silence retrotransposons to safeguard genome stability while undergoing rapid cell proliferation for self-renewal.

DNA hypomethylation restricted to the murine forebrain induces cortical degeneration and impairs postnatal neuronal maturation.

DNA methylation is a major epigenetic factor regulating genome reprogramming, cell differentiation and developmental gene expression. To understand the role of DNA methylation in central nervous system (CNS) neurons, we generated conditional Dnmt1 mutant mice that possess approximately 90% hypomethylated cortical and hippocampal cells in the dorsal forebrain from E13.5 on. The mutant mice were viable with a normal lifespan, but displayed severe neuronal cell death between E14.5 and three weeks postnatally. Accompanied with the striking cortical and hippocampal degeneration, adult mutant mice exhibited neurobehavioral defects in learning and memory in adulthood. Unexpectedly, a fraction of Dnmt1(-/-) cortical neurons survived throughout postnatal development, so that the residual cortex in mutant mice contained 20-30% of hypomethylated neurons across the lifespan. Hypomethylated excitatory neurons exhibited multiple defects in postnatal maturation including abnormal dendritic arborization and impaired neuronal excitability. The mutant phenotypes are coupled with deregulation of those genes involved in neuronal layer-specification, cell death and the function of ion channels. Our results suggest that DNA methylation, through its role in modulating neuronal gene expression, plays multiple roles in regulating cell survival and neuronal maturation in the CNS.

CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain.

The postnatal forebrain subventricular zone (SVZ) harbors stem cells that give rise to olfactory bulb interneurons throughout life. The identity of stem cells in the adult SVZ has been extensively debated. Although, ependymal cells were once suggested to have stem cell characteristics, subsequent studies have challenged the initial report and postulated that subependymal GFAP(+) cells were the stem cells. Here, we report that, in the adult mouse forebrain, immunoreactivity for a neural stem cell marker, prominin-1/CD133, is exclusively localized to the ependyma, although not all ependymal cells are CD133(+). Using transplantation and genetic lineage tracing approaches, we demonstrate that CD133(+) ependymal cells continuously produce new neurons destined to olfactory bulb. Collectively, our data indicate that, compared with GFAP expressing adult neural stem cells, CD133(+) ependymal cells represent an additional-perhaps more quiescent-stem cell population in the mammalian forebrain.

Methyl-CpG binding proteins in the nervous system.

Classical methyl-CpG binding proteins contain the conserved DNA binding motif methyl-cytosine binding domain (MBD), which preferentially binds to methylated CpG dinucleotides. These proteins serve as transcriptional repressors, mediating gene silencing via DNA cytosine methylation. Mutations in methyl-CpG binding protein 2 (MeCP2) have been linked to the human mental retardation disorder Rett syndrome, suggesting an important role for methyl-CpG binding proteins in brain development and function. This mini-review summarizes the recent advances in studying the diverse functions of MeCP2 as a prototype for other methyl-CpG binding proteins in the development and function of the vertebrate nervous system.

Conditional Dnmt1 deletion in dorsal forebrain disrupts development of somatosensory barrel cortex and thalamocortical long-term potentiation.

The transcriptional mechanisms governing the development and plasticity of somatopic sensory maps in the cerebral cortex have not been extensively studied. In particular, no studies have addressed the role of epigenetic mechanisms in the development of sensory maps. DNA methylation is one the main epigenetic mechanisms available to mammalian cells to regulate gene transcription. As demethylation results in embryonic lethality, it has been very difficult to study the role of DNA methylation in brain development. We have used cre-lox technology to generate forebrain-specific deletion of DNA methyltransferase 1 (Dnmt1), the enzyme required for the maintenance of DNA methylation. We find that demethylation of neurons in the cerebral cortex results in the failure of development of somatosensory barrel cortex. We also find that in spite of functional thalamocortical neurotransmission, thalamocortical long-term potentiation cannot be induced in slices from Dnmt1 conditional mutants. These studies emphasize the importance of DNA methylation for the development of sensory maps and suggest epigenetic mechanisms may play a role in the development of synaptic plasticity.

DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling.

DNA methylation is a major epigenetic factor that has been postulated to regulate cell lineage differentiation. We report here that conditional gene deletion of the maintenance DNA methyltransferase I (Dnmt1) in neural progenitor cells (NPCs) results in DNA hypomethylation and precocious astroglial differentiation. The developmentally regulated demethylation of astrocyte marker genes as well as genes encoding the crucial components of the gliogenic JAK-STAT pathway is accelerated in Dnmt1-/- NPCs. Through a chromatin remodeling process, demethylation of genes in the JAK-STAT pathway leads to an enhanced activation of STATs, which in turn triggers astrocyte differentiation. Our study suggests that during the neurogenic period, DNA methylation inhibits not only astroglial marker genes but also genes that are essential for JAK-STAT signaling. Thus, demethylation of these two groups of genes and subsequent elevation of STAT activity are key mechanisms that control the timing and magnitude of astroglial differentiation.