The microtubule destabilizing protein stathmin controls the transition from dividing neuronal precursors to postmitotic neurons during adult hippocampal neurogenesis

The hippocampus is one of the two areas in the mammalian brain where adult neurogenesis occurs. Adult neurogenesis is well known to be involved in hippocampal physiological functions as well as pathophysiological conditions. Microtubules (MTs), providing intracellular transport, stability, and transmitting force, are indispensable for neurogenesis by facilitating cell division, migration, growth, and differentiation. Although there are several examples of MT‐stabilizing proteins regulating different aspects of adult neurogenesis, relatively little is known about the function of MT‐destabilizing proteins. Stathmin is such a MT‐destabilizing protein largely restricted to the CNS, and in contrast to its developmental family members, stathmin is also expressed at significant levels in the adult brain, notably in areas involved in adult neurogenesis. Here, we show an important role for stathmin during adult neurogenesis in the subgranular zone of the mouse hippocampus. After carefully mapping stathmin expression in the adult dentate gyrus (DG), we investigated its role in hippocampal neurogenesis making use of stathmin knockout mice. Although hippocampus development appears normal in these animals, different aspects of adult neurogenesis are affected. First, the number of proliferating Ki‐67+ cells is decreased in stathmin knockout mice, as well as the expression of the immature markers Nestin and PSA‐NCAM. However, newborn cells that do survive express more frequently the adult marker NeuN and have a more mature morphology. Furthermore, our data suggest that migration in the DG might be affected. We propose a model in which stathmin controls the transition from neuronal precursors to early postmitotic neurons. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1226–1242, 2014     

Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase

Cytosine methylation of genomic DNA controls gene expression and maintains genome stability. How a specific DNA sequence is targeted for methylation by a methyltransferase is largely unknown. Here, we show that histone H3 tails lacking lysine 4 (K4) methylation function as an allosteric activator for methyltransferase Dnmt3a by binding to its plant homeodomain (PHD). In vitro, histone H3 peptides stimulated the methylation activity of Dnmt3a up to 8-fold, in a manner reversely correlated with the level of K4 methylation. The biological significance of allosteric regulation was manifested by molecular modeling and identification of key residues in both the PHD and the catalytic domain of Dnmt3a whose mutations impaired the stimulation of methylation activity by H3 peptides but not the binding of H3 peptides. Significantly, these mutant Dnmt3a proteins were almost inactive in DNA methylation when expressed in mouse embryonic stem cells while their recruitment to genomic targets was unaltered. We therefore propose a two-step mechanism for de novo DNA methylation - first recruitment of the methyltransferase probably assisted by a chromatin- or DNA-binding factor, and then allosteric activation depending on the interaction between Dnmt3a and the histone tails - the latter might serve as a checkpoint for the methylation activity.     

Copernican stem cells: regulatory constellations in adult hippocampal neurogenesis

In the adult, neurogenesis occurs where constellations of signaling molecules are correctly orchestrated and where competent cells are present to interpret these signals. As the instruments used to observe adult neurogenesis become more sophisticated, the concept of a discrete competent "stem cell" has become less concrete. Neural progenitor cells once thought committed to a single lineage can be influenced to become multipotent and somatic tissues appear to yield cells capable of tremendous peripheral and central lineage potential. The variety of cell types that appear competent to generate neurons suggests that the "Hilios" of adult neurogenesis may not necessarily be a single cellular entity but rather the sum of signals that dictate, "Make a new neuron here." These signals may not be limited to the recruitment of preexisting neural stem cells but may also, in some subtle way, reprogram local precursors to create "stem-like cells," where needed.     

PAGE separation of hemi-methylated or unmethylated oligonucleotide substrates to distinguish between maintenance and de novo DNA methyltranferase activity

DNA methyltransferase (DNMT) enzymes catalyze the addition of a methyl group to cytosine residues in DNA. Appropriate cytosine methylation of CpG dinucleotides is required for normal mammalian development and homeostasis, and quantitative methods are necessary to assess DNMT activity in various cell extracts. The method described in this report utilizes incorporation of S-[methyl-(3)H]-adenosyl-L-methionine into hemi-methylated or unmethylated oligonucleotides to distinguish between maintenance and de novo DNMT activity, respectively. However, unlike previously described methods, this protocol uses native polyacrylamide gel electrophoresis to detect the incorporation of radioactivity into substrate oligonucleotides. This approach distinguishes between incorporation of radioactivity into target substrate oligonucleotides and incorporation into non-specific cellular DNA that often contaminates nuclear extracts, and permits the reproducible quantitation and comparison of de novo and maintenance DNMT activities in various cell lines. Electrophoretic separation of the methylated substrates is a cost-effective, specific, and reproducible approach to quantitate DNMT activities in nuclear extracts.     

Impact of exercise and a complex environment on hippocampal dendritic morphology,Bdnf gene expression,and DNA methylation in male rat pups neonatally exposed to alcohol

Alcohol exposure in utero can result in Fetal Alcohol Spectrums Disorders (FASD). Measures of hippocampal neuroplasticity, including long‐term potentiation, synaptic and dendritic organization, and adult neurogenesis, are consistently disrupted in rodent models of FASD. The current study investigated whether third trimester‐equivalent binge‐like alcohol exposure (AE) [postnatal days (PD) 4–9] affects dendritic morphology of immature dentate gyrus granule cells, and brain‐derived neurotrophic factor (Bdnf ) gene expression and DNA methylation in hippocampal tissue in adult male rats. To understand immediate impact of alcohol, DNA methylation was measured in the PD10 hippocampus. In addition, two behavioral interventions, wheel running (WR) and environmental complexity (EC), were utilized as rehabilitative therapies for alcohol‐induced deficits. AE significantly decreased dendritic complexity of the immature neurons, demonstrating the long‐lasting impact of neonatal alcohol exposure on dendritic morphology of immature neurons in the hippocampus. Both housing conditions robustly enhanced dendritic complexity in the AE animals. While Bdnf exon I DNA methylation was lower in the AE and sham‐intubated animals compared with suckle controls on PD10, alterations to Bdnf DNA methylation and gene expression levels were not present at PD72. In control animals, exercise, but not exercise followed by housing in EC, resulted in higher levels of hippocampal Bdnf gene expression and lower DNA methylation. These studies demonstrate the long‐lasting negative impact of developmental alcohol exposure on hippocampal dendritic morphology and support the implementation of exercise and complex environments as therapeutic interventions for individuals with FASD. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 708–725, 2017     

Sexually divergent DNA methylation patterns with hippocampal aging

DNA methylation is a central regulator of genome function, and altered methylation patterns are indicative of biological aging and mortality. Age‐related cellular, biochemical, and molecular changes in the hippocampus lead to cognitive impairments and greater vulnerability to neurodegenerative disease that varies between the sexes. The role of hippocampal epigenomic changes with aging in these processes is unknown as no genome‐wide analyses of age‐related methylation changes have considered the factor of sex in a controlled animal model. High‐depth, genome‐wide bisulfite sequencing of young (3 month) and old (24 month) male and female mouse hippocampus revealed that while total genomic methylation amounts did not change with aging, specific sites in CG and non‐CG (CH) contexts demonstrated age‐related increases or decreases in methylation that were predominantly sexually divergent. Differential methylation with age for both CG and CH sites was enriched in intergenic and intronic regions and under‐represented in promoters, CG islands, and specific enhancer regions in both sexes, suggesting that certain genomic elements are especially labile with aging, even if the exact genomic loci altered are predominantly sex‐specific. Lifelong sex differences in autosomal methylation at CG and CH sites were also observed. The lack of genome‐wide hypomethylation, sexually divergent aging response, and autosomal sex differences at CG sites was confirmed in human data. These data reveal sex as a previously unappreciated central factor of hippocampal epigenomic changes with aging. In total, these data demonstrate an intricate regulation of DNA methylation with aging by sex, cytosine context, genomic location, and methylation level.     

Systemically delivered Erythropoietin transiently enhances adult hippocampal neurogenesis

Erythropoietin is a primary regulator of erythropoiesis in the hematopoietic system. More recently erythropoietin has been shown to play a role in neurogenesis and provide neurotrophic support to injured CNS tissue. Here the effects of large systemic doses of erythropoietin on basal levels of adult hippocampal neurogenesis in mice were examined. A 7-day period of recombinant human erythropoietin (rhEPO) administration increased the number of bromodeoxyuridine [BrdU(+)] cells in the sub-granular zone (SGZ) by 30%. Analysis of cell phenotype revealed an increase in mitotically active doublecortin(+) neuronal progenitor cells and glial fibrillary acidic protein(+) SGZ radial astrocytes/stem cells but not mature S100beta(+) astrocytes. These effects appeared to be mediated, in part, by mitogen-activated protein kinase signaling and potentially regulated by suppressor of cytokine signaling-3. Hippocampal levels of phosphorylated extracellular signal-related kinase 42/44 and suppressor of cytokine signaling-3 were increased 2-6 h after a single systemic rhEPO injection. However, rhEPO had no observed effect on the long-term survival of new born cells in the SGZ, with similar numbers of BrdU(+) cells and BrdU(+)/NeuN(+) co-labeled cells after 4 weeks. Therefore, systemically delivered rhEPO transiently increased adult hippocampal neurogenesis without any apparent long-term effects.     

Novel anticancer drug curaxin CBL0137 impairs DNA methylation by eukaryotic DNA methyltransferase Dnmt3a

Novel DNA intercalating anticancer drug curaxin CBL0137 significantly inhibited in vitro DNA methylation by eukaryotic DNA methyltransferase Dnmt3a catalytic domain (Dnmt3a-CD) at low micromolar concentrations (IC₅₀ 3–9 µM). CBL0137 reduced the binding affinity of Dnmt3a-CD to its DNA target, causing up to four-fold increase in the K_d of the enzyme/DNA complex. Binding of CBL0137 to Dnmt3a-CD was not observed. The observed decrease in methylation activity of Dnmt3a-CD in the presence of CBL0137 can be explained by curaxin’s ability to intercalate into DNA.     

Oligomerization of DNMT3A controls the mechanism of de novo DNA methylation

DNMT3A is one of two human de novo DNA methyltransferases essential for regulating gene expression through cellular development and differentiation. Here we describe the consequences of single amino acid mutations, including those implicated in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes, at the DNMT3A·DNMT3A homotetramer and DNMT3A·DNMT3L heterotetramer interfaces. A model for the DNMT3A homotetramer was developed via computational interface scanning and tested using light scattering and electrophoretic mobility shift assays. Distinct oligomeric states were functionally characterized using fluorescence anisotropy and steady-state kinetics. Replacement of residues that result in DNMT3A dimers, including those identified in AML patients, show minor changes in methylation activity but lose the capacity for processive catalysis on multisite DNA substrates, unlike the highly processive wild-type enzyme. Our results are consistent with the bimodal distribution of DNA methylation in vivo and the loss of clustered methylation in AML patients. Tetramerization with the known interacting partner DNMT3L rescues processive catalysis, demonstrating that protein binding at the DNMT3A tetramer interface can modulate methylation patterning. Our results provide a structural mechanism for the regulation of DNMT3A activity and epigenetic imprinting.     

Biochemical fractionation reveals association of DNA methyltransferase (Dnmt) 3b with Dnmt1 and that of Dnmt 3a with a histone H3 methyltransferase and Hdac1

De novo DNA methyltransferases, Dnmt3a and 3b, were purified by fractionation of S-100 extract from mouse lymphosarcoma cells through several chromatographic matrices followed by glycerol density gradient centrifugation. Dnmt3a was separated from Dnmt3b and Dnmt1 in the first column, Q-Sepharose whereas Dnmt3b co-purified with Dnmt1 after further fractionation through Mono-S and Mono-Q columns and glycerol density gradient centrifugation. Following purification, the majority of de novo DNA methyltransfearse activity was associated with Dnmt3b/Dnmt1 fractions. By contrast, the fractions containing Dnmt3a alone exhibited markedly reduced activity, which correlated with diminished expression of this isoform in these cells. Histone deacetylase 1(Hdac1) cofractionated with Dnmt3a throughout purification whereas Hdac1 was separated from Dnmt3b/Dnmt1 following chromatography on Mono-Q column. Dnmt3a purified through glycerol gradient centrifugation was also associated with a histone H3 methyltransferase (HMTase) activity whereas purified Dnmt3b/Dnmt1 was devoid of any HMTase activity. The activity of this HMTase was abolished when lysine 9 of N-terminal histone H3 peptide was replaced by leucine whereas mutation of lysine 4 to leucine inhibited this activity only partially. This is the first report on the identification of a few key co-repressors associated with endogenous Dnmt3a and of a complex containing Dnmt3b and a minor form of Dnmt1 following extensive biochemical fractionation.     

De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes

In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting.     

DNA甲基化在精子发生中的作用

精子发生(spermatogenesis)是一个高度特化的细胞复杂分化过程,其中DNA二核苷酸CpG甲基化变化与基因转录激活、染色质改构以及遗传印记相关,并且该甲基化与基因表达之间的关系是非直接的,其可通过染色质结构的改变或DNA与蛋白质的相互作用来介导。本文着重介绍精子发生过程中DNA甲基化及其跨代遗传风险、DNA甲基转移酶的调控机制以及DNA甲基化与男性不育之间的关系等,为不育症的防治、精子表观遗传质量评价以及降低辅助生殖技术后代表观遗传疾病风险等提供基础资料。     

Age‐related decline in BubR1 impairs adult hippocampal neurogenesis

Aging causes significant declines in adult hippocampal neurogenesis and leads to cognitive disability. Emerging evidence demonstrates that decline in the mitotic checkpoint kinase BubR1 level occurs with natural aging and induces progeroid features in both mice and children with mosaic variegated aneuploidy syndrome. Whether BubR1 contributes to age‐related deficits in hippocampal neurogenesis is yet to be determined. Here we report that BubR1 expression is significantly reduced with natural aging in the mouse brain. Using established progeroid mice expressing low amounts of BubR1, we demonstrate these mice exhibit deficits in neural progenitor proliferation and maturation, leading to reduction in new neuron production. Collectively, our identification of BubR1 as a new and critical factor controlling sequential steps across neurogenesis raises the possibility that BubR1 may be a key mediator regulating aging‐related hippocampal pathology. Targeting BubR1 may represent a novel therapeutic strategy for age‐related cognitive deficits.     

Mammalian DNA methyltransferases show different subnuclear distributions

In mammalian cells, DNA methylation patterns are precisely maintained after DNA replication with defined changes occurring during development. The major DNA methyltransferase (Dnmt1) is associated with nuclear replication sites during S-phase, which is consistent with a role in maintenance methylation. The subcellular distribution of the recently discovered de novo DNA methyltransferases, Dnmt3a and Dnmt3b, was investigated by immunofluorescence and by epitope tagging. We now show that both Dnmt3a and Dnmt3b are distributed throughout the nucleoplasm but are not associated with nuclear DNA replication sites during S-phase. These results suggest that de novo methylation by Dnmt3a and Dnmt3b occurs independently of the replication process and might involve an alternative mechanism for accessing the target DNA. The different subcellular distribution of mammalian DNA methyltransferases might thus contribute to the regulation of DNA methylation.     

DNA methylation‐mediated epigenetic control

During differentiation and development cells undergo dramatic morphological and functional changes without any change in the DNA sequence. The underlying changes of gene expression patterns are established and maintained by epigenetic processes. Early mechanistic insights came from the observation that gene activity and repression states correlate with the DNA methylation level of their promoter region. DNA methylation is a postreplicative modification that occurs exclusively at the C5 position of cytosine residues (5mC) and predominantly in the context of CpG dinucleotides in vertebrate cells. Here, three major DNA methyltransferases (Dnmt1, 3a, and 3b) establish specific DNA methylation patterns during differentiation and maintain them over many cell division cycles. CpG methylation is recognized by at least three protein families that in turn recruit histone modifying and chromatin remodeling enzymes and thus translate DNA methylation into repressive chromatin structures. By now a multitude of histone modifications have been linked in various ways with DNA methylation. We will discuss some of the basic connections and the emerging complexity of these regulatory networks. J. Cell. Biochem. 108: 43–51, 2009. © 2009 Wiley‐Liss, Inc.