Neuronal activity is altered in several neurological and psychiatric diseases. Upon depolarization not only neurotransmitters are released but also cytokines and other activators of signaling cascades. Unraveling their complex implication in transcriptional control in receiving cells will contribute to understand specific central nervous system (CNS) pathologies and will be of therapeutically interest. In this study we depolarized mature hippocampal neurons in vitro using KCl and revealed increased release not only of brain-derived neurotrophic factor (BDNF) but also of transforming growth factor beta (TGFB). Neuronal activity together with BDNF and TGFB controls transcription of DNA modifying enzymes specifically members of the DNA-damage-inducible (Gadd) family, Gadd45a, Gadd45b, and Gadd45g. MeDIP followed by massive parallel sequencing and transcriptome analyses revealed less DNA methylation upon KCl treatment. Psychiatric disorder-related genes, namely Tshz1, Foxn3, Jarid2, Per1, Map3k5, and Arc are transcriptionally activated and demethylated upon neuronal activation. To analyze whether misexpression of Gadd45 family members are associated with psychiatric diseases, we applied unpredictable chronic mild stress (UCMS) as established model for depression to mice. UCMS led to reduced expression of Gadd45 family members. Taken together, our data demonstrate that Gadd45 family members are new putative targets for UCMS treatments.
Growing evidence suggests that the lysine methyltransferase DOT1L/KMT4 has important roles in proliferation, survival, and differentiation of stem cells in development and in disease. We investigated the function of DOT1L in neural stem cells (NSCs) of the cerebral cortex. The pharmacological inhibition and shRNA-mediated knockdown of DOT1L impaired proliferation and survival of NSCs. DOT1L inhibition specifically induced genes that are activated during the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Chromatin-immunoprecipitation analyses revealed that two genes encoding for central molecules involved in the ER stress response, Atf4 and Ddit3 (Chop), are marked with H3K79 methylation. Interference with DOT1L activity resulted in transcriptional activation of both genes accompanied by decreased levels of H3K79 dimethylation. Although downstream effectors of the UPR, such as Ppp1r15a/Gadd34, Atf3, and Tnfrsf10b/ Dr5 were also transcriptionally activated, this most likely occurred in response to increased ATF4 expression rather than as a direct consequence of altered H3K79 methylation. While stem cells are particularly vulnerable to stress, the UPR and ER stress have not been extensively studied in these cells yet. Since activation of the ER stress program is also implicated in directing stem cells into differentiation or to maintain a proliferative status, the UPR must be tightly regulated. Our and published data suggest that histone modifications, including H3K4me3, H3K14ac, and H3K79me2, are implicated in the control of transcriptional activation of ER stress genes. In this context, the loss of H3K79me2 at the Atf4-and Ddit3-promoters appears to mark a point-of-no-return that activates the death program in NSCs. STEM CELLS 2016;34:233-245 SIGNIFICANCE STATEMENTPosttranslational histone modification control gene expression. They are means to pass on transcriptional information from one cell generation to another. We describe the impact of histone H3 dimethylation at lysine 79 (H3K79me2) in neural stem cells. Inhibiting the enzymatic activity mediating this modification leads to cell division defects and cell death by controlling expression of central transcription factors implicated in stress response. Histone methylations are a way to balance how stem cells can cope with the activation of the stress response, which can range from proliferation to differentiation and, in case of H3K79me2, to the control of cell death.
The disruptor of telomeric silencing 1-like (DOT1L) mediates methylation of histone H3 at position lysine 79 (H3K79). Conditional knockout of Dot1l in mouse cerebellar granule cells ( Dot1l -cKO Atoh1 ) led to a smaller external granular layer with fewer precursors of granule neurons. Dot1l -cKO Atoh1 mice had impaired proliferation and differentiation of granular progenitors, which resulted in a smaller cerebellum. Mutant mice showed mild ataxia in motor behavior tests. In contrast, Purkinje cell-specific conditional knockout mice showed no obvious phenotype. Genome-wide transcription analysis of Dot1l -cKO Atoh1 cerebella using microarrays revealed changes in genes that function in cell cycle, cell migration, axon guidance, and metabolism. To identify direct DOT1L target genes, we used genome-wide profiling of H3K79me2 and transcriptional analysis. Analysis of differentially methylated regions (DR) and differentially expressed genes (DE) revealed in total 12 putative DOT1L target genes in Dot1l -cKO Atoh1 affecting signaling ( Tnfaip8l3, B3galt5 ), transcription ( Otx1 ), cell migration and axon guidance ( Sema4a , Sema5a , Robo1 ), cholesterol and lipid metabolism ( Lss , Cyp51 ), cell cycle ( Cdkn1a ), calcium-dependent cell-adhesion or exocytosis ( Pcdh17 , Cadps2 ), and unknown function ( Fam174b ). Dysregulated expression of these target genes might be implicated in the ataxia phenotype observed in Dot1l -cKO Atoh1 . Electronic supplementary material The online version of this article (10.1007/s12035-018-1377-1) contains supplementary material, which is available to authorized users.
Cortical neurogenesis depends on the balance between self‐renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single‐cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self‐renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.
Zfp982 is a new mouse stem cell defining marker gene.• Zfp982 is co-expressed with Yap1 and stem cell marker genes in mESC.• ZFP982 binds to DNA and induces expression of master genes of stemness in mESC.• Expression of Zfp982 gene prevents neural differentiation and maintains stem cell characteristics.• ZFP982 and YAP1 interact in mESC and translocate to the cytoplasm upon neural differentiation.
Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.
Background The histone methyltransferase DOT1L catalyzes methylation of H3K79 and it is highly conserved in mammals. DOT1L plays a functional role in several biological processes including cell cycle regulation, DNA repair, RNA splicing and gene expression, suggesting a complex role in chromatin organization and regulation. Such a remarkable range of functions performed by DOT1L can be the result, at least partially, of its interaction with a plethora of proteins and presence in different complexes. Results Here, we characterized the cooperation of DOT1L with the nucleolar protein NPM1 and the impact of both proteins on peri-nucleolar heterochromatin activity. We show that i) DOT1L interacts preferentially with monomeric NPM1 in the nucleus; ii) DOT1L acts in concert with NPM1 to maintain each other’s protein homeostasis; iii) NPM1 depletion results in H3K79me2 upregulation at chromatin remodeling genes but does not affect their expression; iv) DOT1L and NPM1 preserved DNA satellite expression at peri-nucleolar heterochromatin via epigenetic mechanisms dependent on H3K27me3. Conclusions Our findings give insights into molecular mechanisms employed by DOT1L and NPM1 to regulate heterochromatin activities around the nucleoli and shed light on one aspect of the complex role of both proteins in chromatin dynamics.
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