Tip60/KAT5 Histone Acetyltransferase Is Required for Maintenance and Neurogenesis of Embryonic Neural Stem Cells.

Epigenetic regulation via epigenetic factors in collaboration with tissue-specific transcription factors is curtail for establishing functional organ systems during development. Brain development is tightly regulated by epigenetic factors, which are coordinately activated or inactivated during processes, and their dysregulation is linked to brain abnormalities and intellectual disability. However, the precise mechanism of epigenetic regulation in brain development and neurogenesis remains largely unknown. Here, we show that Tip60/KAT5 deletion in neural stem/progenitor cells (NSCs) in mice results in multiple abnormalities of brain development. Tip60-deficient embryonic brain led to microcephaly, and proliferating cells in the developing brain were reduced by Tip60 deficiency. In addition, neural differentiation and neuronal migration were severely affected in Tip60-deficient brains. Following neurogenesis in developing brains, gliogenesis started from the earlier stage of development in Tip60-deficient brains, indicating that Tip60 is involved in switching from neurogenesis to gliogenesis during brain development. It was also confirmed in vitro that poor neurosphere formation, proliferation defects, neural differentiation defects, and accelerated astrocytic differentiation in mutant NSCs are derived from Tip60-deficient embryonic brains. This study uncovers the critical role of Tip60 in brain development and NSC maintenance and function in vivo and in vitro.

Activation of innate immune antiviral responses by Nod2.

Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs) and RIG-like helicase (RLH) receptors, are involved in innate immune antiviral responses. Here we show that nucleotide-binding oligomerization domain 2 (Nod2) can also function as a cytoplasmic viral PRR by triggering activation of interferon-regulatory factor 3 (IRF3) and production of interferon-beta (IFN-beta). After recognition of a viral ssRNA genome, Nod2 used the adaptor protein MAVS to activate IRF3. Nod2-deficient mice failed to produce interferon efficiently and showed enhanced susceptibility to virus-induced pathogenesis. Thus, the function of Nod2 as a viral PRR highlights the important function of Nod2 in host antiviral defense mechanisms.

MRG15 is required for pre-mRNA splicing and spermatogenesis.

Splicing can be epigenetically regulated and involved in cellular differentiation in somatic cells, but the interplay of epigenetic factors and the splicing machinery during spermatogenesis remains unclear. To study these interactions in vivo, we generated a germline deletion of MORF-related gene on chromosome 15 (MRG15), a multifunctional chromatin organizer that binds to methylated histone H3 lysine 36 (H3K36) in introns of transcriptionally active genes and has been implicated in regulation of histone acetylation, homology-directed DNA repair, and alternative splicing in somatic cells. Conditional KO (cKO) males lacking MRG15 in the germline are sterile secondary to spermatogenic arrest at the round spermatid stage. There were no significant alterations in meiotic division and histone acetylation. Specific mRNA sequences disappeared from 66 germ cell-expressed genes in the absence of MRG15, and specific intronic sequences were retained in mRNAs of 4 genes in the MRG15 cKO testes. In particular, introns were retained in mRNAs encoding the transition proteins that replace histones during sperm chromatin condensation. In round spermatids, MRG15 colocalizes with splicing factors PTBP1 and PTBP2 at H3K36me3 sites between the exons and single intron of transition nuclear protein 2 (Tnp2). Thus, our results reveal that MRG15 is essential for pre-mRNA splicing during spermatogenesis and that epigenetic regulation of pre-mRNA splicing by histone modification could be useful to understand not only spermatogenesis but also, epigenetic disorders underlying male infertile patients.

Developmentally Regulated RNA-binding Protein 1 (Drb1)/RNA-binding Motif Protein 45 (RBM45), a Nuclear-Cytoplasmic Trafficking Protein, Forms TAR DNA-binding Protein 43 (TDP-43)-mediated Cytoplasmic Aggregates.

Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.

Translational repression of the McKusick-Kaufman syndrome transcript by unique upstream open reading frames encoding mitochondrial proteins with alternative polyadenylation sites.

BACKGROUND: Upstream open reading frames (uORFs) are commonly found in the 5'-untranslated region (UTR) of many genes and function in translational control. However, little is known about the existence of the proteins encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs of the McKusick-Kaufman syndrome (MKKS) gene that causes a genetic disorder. METHODS: Northern blotting, 3'-RACE, and bioinformatics were used for determining the length of transcripts and their 3' ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein products and their subcellular localization. RESULTS: The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the 5'-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial membrane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation sites at the 5'-UTR are atypical but surely exist in human transcripts. CONCLUSIONS: Multiple uORFs at the 5'-UTR of the MKKS long transcript function as translational repressor for MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane. GENERAL SIGNIFICANCE: Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs.

Rapid selection of XO embryonic stem cells using Y chromosome-linked GFP transgenic mice.

We developed a transgenic mouse line with Y chromosome-linked green fluorescent protein expressing transgenes (Y-GFP) by the conventional microinjection into the pronucleus of C57BL/6J fertilized oocytes. Embryonic stem (ES) cells derived from Y-GFP mice enabled not only sexing but also the identification of 39, XO karyotype by the lack of Y chromosome. Actually, when fluorescence activated cell sorting (FACS) was applied to Y-GFP ES cells, non-fluorescent ES cells were conveniently collected and showed the lack of Y chromosome by PCR genotyping and Southern blot analysis. FACS analysis revealed Y chromosome loss occurred at 2.9 % of 40, XY ES cells after five passages. These Y-GFP ES cells are potentially applicable to reduce the time, cost and effort needed to generate the gene-targeted mice by the production of male and female mice derived from the same ES cell clone.

Fatty Acids Play a Critical Role in Mitochondrial Oxidative Phosphorylation in Effector T Cells in Graft-versus-Host Disease.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.

MRG15 activates the cdc2 promoter via histone acetylation in human cells.

Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.

The cell senescence inducing gene product MORF4 is regulated by degradation via the ubiquitin/proteasome pathway.

After undergoing several rounds of divisions normal human fibroblasts enter a terminally non-dividing state referred to as cellular or replicative senescence. We cloned MORF4 (mortality factor on human chromosome 4), as a cellular senescence inducing gene that caused immortal cells assigned to complementation group B for indefinite division to stop dividing. To facilitate analyses of this gene, which is toxic to cells at low levels, we obtained stable clones of HeLa cells expressing a tetracycline-induced MORF4 construct that could be induced by doxycycline in a dose-dependent manner. MORF4 induction resulted in reduced colony formation after 14 days of culture, as previously observed. We determined that MORF4 protein was unstable and that addition of the proteasome inhibitor MG132 resulted in the accumulation of the protein. Following removal of MG132 the protein was rapidly degraded. Subcellular fractionation following MG132 treatment demonstrated that the protein accumulates primarily in the cytoplasm with some amounts present in the nucleus. It is therefore possible that MORF4 protein, which escapes degradation in the cytoplasm, is transported to the nucleus where it is functional. The results suggest that levels of MORF4 in cells must be tightly controlled and one mechanism involves stability of the protein.

Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo.

Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.

Mesenchymal Stromal Cells Inhibit Aerobic Glycolysis in Activated T Cells by Negatively Regulating Hexokinase II Activity Through PD-1/PD-L1 Interaction.

Mesenchymal stromal cells (MSCs) have been shown to inhibit aerobic glycolysis in activated T cells, leading to increased autophagy. Although tryptophan depletion induced by indoleamine 2,3-dioxygenase (IDO) generated by MSCs has been suggested as a potential mechanism, we found that this inhibition was completely abolished when T cells were physically separated from MSCs using the Transwell system. Instead, in the current study, we demonstrate that programmed cell death 1 receptor (PD-1) and its ligand PD-L1, the expression of which is induced on activated T cells and MSCs, respectively, in response to IFN-γ are involved in this inhibition. Blockade of PD-1/PD-L1 interaction by blocking antibodies significantly restored glucose uptake, glycolytic activity, and cluster formation of activated T cells, whereas a specific inhibitor of IDO, 1-methyl-DL-tryptophan, had no effect. Neither surface nor cytoplasmic glucose transporter-1 expression on T cells was changed by MSCs. In addition, glycolytic gene expression in activated T cells was not inhibited despite the presence of MSCs. However, we found that hexokinase II (HK2) protein expression was markedly decreased in activated T cells that had been cocultured with MSCs. PD-1 blocking antibody restored HK2 expression. Taken together, our findings indicate that the PD-1/PD-L1 axis is involved in the MSC-mediated suppression of T cell glycolysis by negatively regulating HK2 activity at the protein level, but not at the mRNA level.

MRG15, a component of HAT and HDAC complexes, is essential for proliferation and differentiation of neural precursor cells.

Neurogenesis during development depends on the coordinated regulation of self-renewal and differentiation of neural precursor cells (NPCs). Chromatin regulation is a key step in self-renewal activity and fate decision of NPCs. However, the molecular mechanism or mechanisms of this regulation is not fully understood. Here, we demonstrate for the first time that MRG15, a chromatin regulator, is important for proliferation and neural fate decision of NPCs. Neuroepithelia from Mrg15-deficient embryonic brain are much thinner than those from control, and apoptotic cells increase in this region. We isolated NPCs from Mrg15-deficient and wild-type embryonic whole brains and produced neurospheres to measure the self-renewal and differentiation abilities of these cells in vitro. Neurospheres culture from Mrg15-deficient embryo grew less efficiently than those from wild type. Measurement of proliferation by means of BrdU (bromodeoxyuridine) incorporation revealed that Mrg15-deficient NPCs have reduced proliferation ability and apoptotic cells do not increase during in vitro culture. The reduced proliferation of Mrg15-deficient NPCs most likely accounts for the thinner neuroepithelia in Mrg15-deficient embryonic brain. Moreover, we also demonstrate Mrg15-deficient NPCs are defective in differentiation into neurons in vitro. Our results demonstrate that MRG15 has more than one function in neurogenesis and defines a novel role for this chromatin regulator that integrates proliferation and cell-fate determination in neurogenesis during development.

Comprehensive Metabolomic Analysis of IDH1R132H Clinical Glioma Samples Reveals Suppression of ß-oxidation Due to Carnitine Deficiency.

Gliomas with Isocitrate dehydrogenase 1 (IDH1) mutation have alterations in several enzyme activities, resulting in various metabolic changes. The aim of this study was to determine a mechanism for the better prognosis of gliomas with IDH mutation by performing metabolomic analysis. To understand the metabolic state of human gliomas, we analyzed clinical samples obtained from surgical resection of glioma patients (grades II-IV) with or without the IDH1 mutation, and compared the results with U87 glioblastoma cells overexpressing IDH1 or IDH1R132H. In clinical samples of gliomas with IDH1 mutation, levels of D-2-hydroxyglutarate (D-2HG) were increased significantly compared with gliomas without IDH mutation. Gliomas with IDH mutation also showed decreased intermediates in the tricarboxylic acid cycle and pathways involved in the production of energy, amino acids, and nucleic acids. The marked difference in the metabolic profile in IDH mutant clinical glioma samples compared with that of mutant IDH expressing cells includes a decrease in ß-oxidation due to acyl-carnitine and carnitine deficiencies. These metabolic changes may explain the lower cell division rate observed in IDH mutant gliomas and may provide a better prognosis in IDH mutant gliomas.

MrgX is not essential for cell growth and development in the mouse.

MRGX is one of the members of MORF4/MRG family of transcriptional regulators, which are involved in cell growth regulation and cellular senescence. We have shown that MRGX and MRG15 associate with Rb in nucleoprotein complexes and regulate B-myb promoter activity. To elucidate the functions of MRGX and to explore its potential role in modulating cell growth in vivo, we have generated MrgX-deficient mice. Characterization of the expression pattern of mouse MrgX demonstrated it was ubiquitously expressed in all tissues of adult mice and also during embryogenesis and overlapped with its homolog Mrg15. MRGX and MRG15 proteins localize predominantly to the chromatin fraction in the nucleus, although a small amount of both proteins localized to the nuclear matrix. Whereas disruption of Mrg15 results in embryonic lethality, absence of MrgX did not impair mouse development and MrgX null mice are healthy and fertile. MrgX-deficient and wild-type mouse embryonic fibroblasts (MEFs) also had similar growth rates and showed no differences in cell cycle-related gene expression in response to serum stimulation. Mrg15 expression in MrgX-deficient tissues and MEFs was not upregulated compared with wild-type tissues and MEFs. MRG15 is highly conserved with orthologs present from humans to yeast and is essential for survival of mice. In contrast, MRGX, which evolved later, is expressed only in vertebrates, suggesting that the lack of phenotype of MrgX-deficient mice is secondary to a compensatory effect by the evolutionarily conserved MRG15 protein but not vice versa.

MRG15 regulates embryonic development and cell proliferation.

MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15-/- embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15-/- mouse embryonic fibroblasts. The hearts of the Mrg15-/- embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15-/- embryos appeared pale, and microarray analysis revealed that alpha-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the alpha-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation.

Polyethylene glycol-based homologated ligands for nicotinic acetylcholine receptors.

A homologous series of polyethylene glycol (PEG) monomethyl ethers were conjugated with three ligand series for nicotinic acetylcholine receptors. Conjugates of acetylaminocholine, the cyclic analog 1-acetyl-4,4-dimethylpiperazinium, and pyridyl ether A-84543 were prepared. Each series was found to retain significant affinity at nicotinic receptors in rat cerebral cortex with tethers of up to six PEG units. Such compounds are hydrophilic ligands which may serve as models for fluorescent/affinity probes and multivalent ligands for nAChR.

Multipurpose MRG domain involved in cell senescence and proliferation exhibits structural homology to a DNA-interacting domain.

The ubiquitous MRG/MORF family of proteins is involved in cell senescence, or the terminal loss of proliferative potential, a model for aging and tumor suppression at the cellular level. These proteins are defined by the approximately 20 kDa MRG domain that binds a plethora of transcriptional regulators and chromatin-remodeling factors, including the histone deacetylase transcriptional corepressor mSin3A and the novel nuclear protein PAM14, and they are also known components of the Tip60/NuA4 complex via interactions with the MRG binding protein (MRGBP). We present here the crystal structure of a prototypic MRG domain from human MRG15 whose core consists of two orthogonal helix hairpins. Despite the lack of sequence similarity, the core structure has surprisingly striking homology to a DNA-interacting domain of the tyrosine site-specific recombinases XerD, lambda integrase, and Cre. Site-directed mutagenesis studies based on the X-ray structure and bioinformatics identified key residues involved in the binding of PAM14 and MRGBP.

Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit DNA-repair defects post exposure to gamma ionizing radiation.

MORF4-related gene on chromosome 15 (MRG15) is a core component of the NuA4/Tip60 histone acetyltransferase complex that modifies chromatin structure. We here demonstrate that Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit an impaired DNA-damage response post gamma irradiation, when compared to wild-type cells. Defects in DNA-repair and cell growth, and delayed recruitment of repair proteins to sites of damage were observed. Formation of phosphorylated H2AX and 53BP1 foci was delayed in Mrg15 mutant versus wild-type cells following irradiation. These data implicate a novel role for MRG15 in DNA-damage repair in mammalian cells.

PAM14, a novel MRG- and Rb-associated protein, is not required for development and T-cell function in mice.

PAM14 has been found to associate in complexes with the MORF4/MRG family of proteins as well as Rb, the tumor suppressor protein. This suggested that it might be involved in cell growth, immortalization, and/or senescence. To elucidate the in vivo function of PAM14, we characterized the expression pattern of mouse Pam14 and generated PAM14-deficient (Pam14(-/-)) mice. Pam14 was widely expressed in all mouse tissues and as early as 7 days during embryonic development. Despite this ubiquitous expression in wild-type mice, Pam14(-/-) mice were healthy and fertile. Response to mitogenic stimulation and production of interleukin-2 were the same in stimulated splenic T cells from Pam14(-/-) mice as in control littermates. Cell growth rates of mouse embryonic fibroblasts (MEFs) from all three genotypes were the same, and immortalized cells were obtained from all cell cultures during continuous culture. There was also no difference in expression of growth-related genes in response to serum stimulation in the null versus control MEFs. These data demonstrate that PAM14 is not essential for normal mouse development and cell cycle control. PAM14 likely acts as an adaptor protein in nucleoprotein complexes and is probably compensated for by another functionally redundant protein(s).