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.

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells.

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.

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.

Interaction of human mitochondrial transcription factor A in mitochondria: its involvement in the dynamics of mitochondrial DNA nucleoids.

Mitochondrial transcription factor A (TFAM) is a key regulator of mitochondrial DNA (mtDNA). TFAM interacts with itself and forms dimers; however, the precise interaction domain in vivo has not yet been determined. We herein showed that human TFAM formed oligomers in mitochondria by in situ chemical cross-linking. We used the separated fluorescent protein, monomeric Kusabira-Green, as a reporter to monitor their self-association in mitochondria. This reporter successfully detected the TFAM-TFAM interaction in cells as fluorescent signals on mitochondria. We also found that the N-terminal high-mobility group box domain was sufficient for this interaction. The expression of the dimer-defective mutant induced enlarged mtDNA nucleoids, suggesting the importance of dimerization in the distribution of mtDNA. The reporter system also supported the association and mixture between independent nucleoids through TFAM by a cell fusion assay using hemagglutinating virus of Japan. We here, for the first time, visualized the interaction of TFAM molecules in mitochondria and proposed its implications for the dynamics of mtDNA nucleoids.

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.

Dynamic regulation of mitochondrial genome maintenance in germ cells.

Mitochondria play a crucial role in the development and function of germ cells. Mitochondria contain a maternally inherited genome that should be transmitted to offspring without reactive oxygen species-induced damage during germ line development. Germ cells are also involved in the mitochondrial DNA (mtDNA) bottleneck; thus, the appropriate regulation of mtDNA in these cells is very important for this characteristic transmission. In this review, we focused on unique regulation of the mitochondrial genome in animal germ cells; paternal elimination and the mtDNA bottleneck in females. We also summarized the mitochondrial nucleoid factors involved in various mtDNA regulation pathways. Among them, mitochondrial transcription factor A (TFAM), which has pleiotropic and essential roles in mtDNA maintenance, appears to have putative roles in germ cell regulation.

Maintenance of mitochondrial genome distribution by mitochondrial AAA+ protein ClpX.

The segregation of mitochondrial DNA (mtDNA) is important for the maintenance and transmission of the genome between generations. Recently, we clarified that human mitochondrial transcription factor A (TFAM) is required for equal distribution and symmetric segregation of mtDNA in cultured cells; however, the molecular mechanism involved is largely unknown. ClpX is an ATPase associated with various cellular activities (AAA+) proteins that localize to the mitochondrial matrix and is suggested to associate with mtDNA. In this study, we found that RNAi-mediated knockdown of ClpX in HeLa cells resulted in enlarged mtDNA nucleoids, which is very similar to that observed in TFAM-knockdown cells in several properties. The expression of TFAM protein was not significantly reduced in ClpX-knockdown cells. However, the enlarged mtDNA nucleoids caused by ClpX-knockdown were suppressed by overexpression of recombinant TFAM and the phenotype was not observed in knockdown with ClpP, a protease subunit of ClpXP. Endogenous ClpX and TFAM exist in close vicinity, and ClpX enhanced DNA-binding activity of TFAM in vitro. These results suggest that human ClpX, a novel mtDNA regulator, maintains mtDNA nucleoid distribution through TFAM function as a chaperone rather than as a protease and its involvement in mtDNA segregation.

Human mitochondrial transcription factor A is required for the segregation of mitochondrial DNA in cultured cells.

The segregation and transmission of the mitochondrial genome in humans are complicated processes but are particularly important for understanding the inheritance and clinical abnormalities of mitochondrial disorders. However, the molecular mechanism of the segregation of mitochondrial DNA (mtDNA) is largely unclear. In this study, we demonstrated that human mitochondrial transcription factor A (TFAM) is required for the segregation of mtDNA in cultured cells. RNAi-mediated knockdown of TFAM in HeLa cells resulted in the enlarged mtDNA, as indicated by the assembly of fluorescent signals stained with PicoGreen. Fluorescent in situ hybridization confirmed the enlarged mtDNA and further showed the existence of increased numbers of mitochondria lacking mtDNA signals in TFAM knockdown cells. By complementation analysis, the C-terminal tail of TFAM, which enhances its affinity with DNA, was found to be required for the appropriate distribution of mtDNA. Furthermore, we found that TFAM knockdown induced asymmetric segregation of mtDNA between dividing daughter cells. These results suggest an essential role for human TFAM in symmetric segregation of mtDNA.

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.

A synthetic male-specific sterilization system using the mammalian pro-apoptotic factor in a malaria vector mosquito.

Conditional cell death systems are useful for various aspects of basic science with a wide range of applications, including genetic pest control. We recently demonstrated that expression of the mammalian pro-apoptotic factor, B-cell leukaemia/lymphoma 2-associated X protein (Bax), can induce apoptosis in specific tissues by using tissue specific promoters in silkworm and mosquito. Here, we newly identified a functional promoter in the Asian malaria vector, Anopheles stephensi, which enables gene expression specifically in the testis. We produced a transgenic mosquito line that expresses mouse Bax under the control of this testis-specific promoter. Transgenic mosquito males exhibited aberrant testes without functional sperm and complete sterility, whereas transgenic females maintained normal fecundity. Despite their abnormal testes, the transgenic males maintained normal function of male accessory glands and typical mating behaviour. As a result of mating with these males, females showed refractoriness to further mating. These results suggest that transgenic males induce female sterility via mating. The mosquito is one of the most important disease vectors, and the control of their population benefits global public health. Thus, this Bax-mediated synthetic male-specific sterilization system could be applied to population control of mosquitoes.

Characterization of Bombyx mori mitochondrial transcription factor A, a conserved regulator of mitochondrial DNA.

In the present study, we initially cloned and characterized a mitochondrial transcription factor A (Tfam) homologue in the silkworm, Bombyx mori. Bombyx mori TFAM (BmTFAM) localized to mitochondria in cultured silkworm and human cells, and co-localized with mtDNA nucleoids in human HeLa cells. In an immunoprecipitation analysis, BmTFAM was found to associate with human mtDNA in mitochondria, indicating its feature as a non-specific DNA-binding protein. In spite of the low identity between BmTFAM and human TFAM (26.5%), the expression of BmTFAM rescued mtDNA copy number reductions and enlarged mtDNA nucleoids in HeLa cells, which were induced by human Tfam knockdown. Thus, BmTFAM compensates for the function of human TFAM in HeLa cells, demonstrating that the mitochondrial function of TFAM is highly conserved between silkworms and humans. BmTfam mRNA was strongly expressed in early embryos. Through double-stranded RNA (dsRNA)-based RNA interference (RNAi) in silkworm embryos, we found that the knockdown of BmTFAM reduced the amount of mtDNA and induced growth retardation at the larval stage. Collectively, these results demonstrate that BmTFAM is a highly conserved mtDNA regulator and may be a good candidate for investigating and modulating mtDNA metabolism in this model organism.

Complex formation of the neuron-specific ELAV-like Hu RNA-binding proteins.

Hu proteins are RNA-binding proteins that are the vertebrate homologs of Drosophila ELAV, and are implicated in stabilization or enhanced translation of specific mRNAs with AU-rich elements (AREs) in the 3'-untranslated region. Here, using the yeast two-hybrid system, we show that neuron-specific Hu proteins can interact with themselves. Immuno precipitation assays demonstrated that the interaction between Hu proteins occurs in mammalian cells and is strongly enhanced in the presence of cellular RNA. Furthermore, using in situ chemical crosslinking assays, we found that HuD, one of the neuron-specific Hu proteins, multimerizes in cells. The crosslinked HuD multimers retain specific RNA-binding ability and can interact with additional Hu proteins. Consistent with this biochemical property, HuD showed granular distribution in two neurogenic cell lines. These results suggest that the RNA-bound form of HuD multimerizes cooperatively to form a specific granular structure that may serve as a site of post-transcriptional regulation of ARE-containing mRNAs.

Discrimination of target by siRNA: designing of AML1-MTG8 fusion mRNA-specific siRNA sequences.

AML1-MTG8 is a chimeric transcription factor produced by t(8;21) chromosome translocation and causes AML. AML1-MTG8 acts as a dominant negative effector on normal AML1 protein, a key transcriptional regulator of hematopoietic differentiation, but its precise mechanism is not known. To analyze the function of AML1-MTG8 in leukemic cells and to explore the possibility of AML1-MTG8-targeted therapy, we designed nine small interfering RNAs (siRNAs) targeting a 25-nucleotide region spanning the fusion point of AML1 and MTG8. Two different siRNAs (AM2 and AM4) significantly reduced AML1-MTG8 expression from a transfected reporter plasmid at both the mRNA and protein levels. Both siRNAs did not reduce AML1b expression, but AM2 siRNA showed slightly reducing activity against MTG8b mRNA that is 86% homologous to the corresponded region of AML1-MTG8 mRNA. Moreover, using a cationic lipid reagent, the siRNAs were efficiently introduced into leukemia cell lines with t(8;21), SKNO-1 (30-40%) and Kasumi-1 (60-70%) cells, and reduced specifically the endogenous AML1-MTG8 expression. The siRNAs reduced neither the wild type AML1 in Kasumi-1 cells nor wild type MTG8b in human erythroblastic leukemia (HEL) cells. These results indicated that the two siRNAs are highly specific for the fusion mRNA. The knockdown of AML1-MTG8 in Kasumi-1 cells resulted in the activation of p14(ARF) promoter activity and increased the expression of integrin alphaIIb, whose expression is related to megakaryocytic differentiation. However, the knockdown of AML1-MTG8 in Kasumi-1 cells did not inhibit the cell growth, suggesting that the siRNA-mediated knockdown of AML1-MTG8 is useful for the functional analysis of the gene, but it alone might not be sufficient for gene therapy of the leukemia.

Paraneoplastic cerebellar degeneration associated with an onconeural antibody against creatine kinase, brain-type.

Onconeural immunity, a cancer-stimulated immune reaction that cross-reacts with neural tissues, is considered to be the principal pathological mechanism for paraneoplastic neurological syndromes (PNS). A common PNS is paraneoplastic cerebellar degeneration (PCD). We had encountered a PCD patient with urothelial carcinomas (UC) of the urinary bladder who was negative for the well-characterized PNS-related onconeural antibodies. In the present study, we aimed to identify a new PCD-related onconeural antibody, capable of recognizing both cerebellar neurons and cancer tissues from the patient, and applied a proteomic approach using mass spectrometry. We identified anti-creatine kinase, brain-type (CKB) antibody as a new autoantibody in the serum and cerebrospinal fluid from the patient. Immunohistochemistry indicated that anti-CKB antibody reacted with both cerebellar neurons and UC of the urinary bladder tissues. However, anti-CKB antibody was not detected in sera from over 30 donors, including bladder cancer patients without PCD, indicating that anti-CKB antibody is required for onset of PCD. We also detected anti-CKB antibody in sera from three other PCD patients. Our study demonstrated that anti-CKB antibody may be added to the list of PCD-related autoantibodies and may be useful for diagnosis of PCD.

Biochemical properties of Caenorhabditis elegans HMG-5, a regulator of mitochondrial DNA.

Caenorhabditis elegans HMG-5, which is encoded by F45E4.9, contains two high mobility group (HMG) box domains and shows sequence similarity with mammalian mitochondrial transcription factor A (TFAM). In this study, using soaking RNA interference, we found that knockdown of HMG-5 reduced the amount of mtDNA in P0 hermaphrodites, suggesting it as functional orthologue of mammalian TFAM. We also examined the biochemical property of HMG-5 in mammalian cells and in vitro. We found that HMG-5 localized to the mitochondria in human cultured cells and was included in the NP-40-insoluble fraction in which mtDNA and TFAM were enriched. By immunoprecipitation analysis, HMG-5 was found to associate with human mitochondrial DNA (mtDNA) in the cells. In vitro binding experiment also showed that HMG-5 binds to C. elegans mtDNA and plasmid DNA, indicating its feature as a non-specific DNA-binding protein. Furthermore, it was found that HMG-5 can interact with itself. These results demonstrate that HMG5 shares similar biochemical properties with mammalian TFAM as a nucleoid factor. HMG-5 could be a good candidate for investigating mtDNA metabolism in multicellular organisms.

Microtubule association of a neuronal RNA-binding protein HuD through its binding to the light chain of MAP1B.

RNA-binding proteins (RBPs) play a vital role in the post-transcriptional regulation of gene expression during neuronal differentiation and synaptic plasticity. One such RBP family, the neuronal Hu protein family, serves as an early marker of neuronal differentiation and targets several mRNAs containing adenine/uridine-rich elements. Recently, we reported that one of the neuronal Hu proteins, HuD stimulates cap-dependent translation through interactions with eIF4A and poly (A) tail. Nevertheless, little is known with respect to how neuronal Hu proteins contribute to the local translation of target mRNAs in neuronal differentiation. Here, we found that neuronal Hu proteins, but not the ubiquitously expressed HuR protein, directly interact with the light chain of microtubule-associated proteins MAP1B (LC1). We also show that HuD simultaneously binds both RNA and LC1 in vitro and that it tightly associates with microtubules in cells in an LC1-dependent manner, raising the possibility that HuD recruits target mRNAs to microtubules. These results uncover the neuronal binding partners for neuron-specific Hu proteins and suggest the involvement of Hu proteins in microtubule-mediated regulation of mRNA expression within neuronal processes.

Association of a novel mitochondrial protein M19 with mitochondrial nucleoids.

We have identified a novel mitochondrial protein, termed M19, by proteomic analysis of mitochondrial membrane proteins from HeLa cells. M19 is highly conserved among vertebrates, and possesses no homologous domains with other known proteins. By northern and western blotting, mouse M19 was shown to be expressed in various tissues, and to be especially abundant in the brain. Human M19 (hM19) is present in mitochondria, and protease-protection experiment showed it to be sublocalized in the matrix space. Carboxy-terminally tagged hM19 appeared as spotted signals within mitochondria and co-localized with signals arising from mitochondrial DNA (mtDNA), suggesting the inclusion of M19 in the mtDNA-protein complex (mitochondrial nucleoids). Fractionation of mitochondrial nucleoids from HeLa cells revealed that hM19 has a similar distribution pattern like that of known nucleoid components, such as mtSSB and PHBs, and surely exists in the nucleoid fraction. Furthermore, expression of M19 is closely related to the amount of mtDNA, because it was down-regulated in mtDNA-depleted rho(0) HeLa cells. These results indicate that M19 associates with the nucleoid and likely regulates the organization and metabolism of mtDNA.

Human prohibitin 1 maintains the organization and stability of the mitochondrial nucleoids.

Mitochondrial prohibitin (PHB) proteins have diverse functions, such as the regulation of apoptosis and the maintenance of mitochondrial morphology. In this study, we clarified a novel mitochondrial function of PHB1 that regulates the organization and maintenance of mitochondrial DNA (mtDNA). In PHB1-knockdown cells, we found that mtDNA is not stained by fluorescent dyes, such as ethidium bromide and PicoGreen, although the mitochondrial membrane potential still maintains. We also demonstrated that mtDNA, which is predominantly found in the NP-40-insoluble fraction when isolated from normal mitochondria, is partially released into the soluble fraction when isolated from PHB1-knockdown cells, indicating that the organization of the mitochondrial nucleoids has been altered. Furthermore, we found that PHB1 regulates copy number of mtDNA by stabilizing TFAM protein, a known protein component of the mitochondrial nucleoids. However, TFAM does not affect the organization of mtDNA as observed in PHB1-knockdown cells. Taken together, these results demonstrate that PHB1 maintains the organization and copy number of the mtDNA through both TFAM-independent and -dependent pathways.

Mitochondrial functions and estrogen receptor-dependent nuclear translocation of pleiotropic human prohibitin 2.

Proteins with multiple cellular functions provide biological diversity to eukaryotic cells. In the current studies, we identified the mitochondrial functions of human prohibitin 2 (PHB2), which was initially identified as a repressor of estrogen-dependent transcriptional activity. The mitochondrial complex of PHB2 consists of PHB1, voltage-dependent anion channel 2, adenine nucleotide translocator 2, and the anti-apoptotic Hax-1, which is a novel binding partner for PHB2. RNA interference-mediated knockdown of PHB2 in HeLa cells resulted in caspase-dependent apoptosis through down-regulation of Hax-1 and fragmentation of mitochondria. We also found that, although PHB2 is predominantly expressed in the mitochondria of HeLa cells, it translocates to nucleus in the presence of estrogen receptor alpha and estradiol. Here, we first demonstrated the roles of mammalian PHB2 in mitochondria and the molecular mechanism of its nuclear targeting and showed that PHB2 is a possible molecule directly coupling nuclear-mitochondrial interaction.