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1.
Nat Commun ; 15(1): 5090, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918373

RESUMO

The development of haematopoiesis involves the coordinated action of numerous genes, some of which are implicated in haematological malignancies. However, the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here, we report a previously uncharacterised gene in haematopoiesis, identified by screening mutant embryonic stem cells. The gene, 'attenuated haematopoietic development (Ahed)', encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward, leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed-deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model, we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways, and published databases present cancer patients with somatic mutations in Ahed. Collectively, our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis, implicating its association with malignancies.


Assuntos
Hematopoese , Camundongos Knockout , Animais , Hematopoese/genética , Camundongos , Humanos , Feminino , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos Endogâmicos C57BL , Mutação , Anemia/genética , Masculino , Células-Tronco Embrionárias/metabolismo
2.
Hum Mol Genet ; 30(9): 758-770, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33607654

RESUMO

Posttranslational modification of a protein with glycosylphosphatidylinositol (GPI) is a conserved mechanism exists in all eukaryotes. Thus far, >150 human GPI-anchored proteins have been discovered and ~30 enzymes have been reported to be involved in the biosynthesis and maturation of mammalian GPI. Phosphatidylinositol glycan biosynthesis class A protein (PIGA) catalyzes the very first step of GPI anchor biosynthesis. Patients carrying a mutation of the PIGA gene usually suffer from inherited glycosylphosphatidylinositol deficiency (IGD) with intractable epilepsy and intellectual developmental disorder. We generated three mouse models with PIGA deficits specifically in telencephalon excitatory neurons (Ex-M-cko), inhibitory neurons (In-M-cko) or thalamic neurons (Th-H-cko), respectively. Both Ex-M-cko and In-M-cko mice showed impaired long-term fear memory and were more susceptible to kainic acid-induced seizures. In addition, In-M-cko demonstrated a severe limb-clasping phenotype. Hippocampal synapse changes were observed in Ex-M-cko mice. Our Piga conditional knockout mouse models provide powerful tools to understand the cell-type specific mechanisms underlying inherited GPI deficiency and to test different therapeutic modalities.


Assuntos
Glicosilfosfatidilinositóis , Ácido Caínico , Animais , Cognição , Glicosilfosfatidilinositóis/deficiência , Humanos , Ácido Caínico/metabolismo , Mamíferos , Camundongos , Camundongos Knockout , Mutação , Neurônios/metabolismo , Convulsões/genética , Convulsões/metabolismo
3.
PLoS One ; 14(12): e0225740, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31805151

RESUMO

When loss of heterozygosity (LOH) is correlated with loss or gain of a disease phenotype, it is often necessary to identify which gene or genes are involved. Here, we developed a region-specific LOH-inducing system based on mitotic crossover in human induced pluripotent stem cells (hiPSCs). We first tested our system on chromosome 19. To detect homozygous clones generated by LOH, a positive selection cassette was inserted at the AASV1 locus of chromosome 19. LOHs were generated by the combination of allele-specific double-stranded DNA breaks introduced by CRISPR/Cas9 and suppression of Bloom syndrome (BLM) gene expression by the Tet-Off system. The BLM protein inhibitor ML216 exhibited a similar crossover efficiency and distribution of crossover sites. We next applied this system to the short arm of chromosome 6, where human leukocyte antigen (HLA) loci are located. Genotyping and flow cytometric analysis demonstrated that LOHs associated with chromosomal crossover occurred at the expected positions. Although careful examination of HLA-homozygous hiPSCs generated from parental cells is needed for cancer predisposition and effectiveness of differentiation, they may help to mitigate the current shortcoming of hiPSC-based transplantation related to the immunological differences between the donor and host.


Assuntos
Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Sequência de Bases , Cromossomos Humanos Par 19/genética , Células Clonais , Troca Genética , Quebras de DNA de Cadeia Dupla , Regulação da Expressão Gênica , Genes Reporter , Antígenos de Histocompatibilidade Classe I/genética , Homozigoto , Humanos , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Telômero/genética
4.
Nat Commun ; 10(1): 5302, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811138

RESUMO

Although single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5'-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Proteínas Associadas a CRISPR/classificação , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Clivagem do DNA , DNA Helicases/metabolismo , Éxons , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes/métodos , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Distrofia Muscular de Duchenne/genética , Deleção de Sequência
5.
PLoS One ; 14(7): e0219592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31356639

RESUMO

Chromosome abnormalities induces profound alterations in gene expression, leading to various disease phenotypes. Recent studies on yeast and mammalian cells have demonstrated that aneuploidy exerts detrimental effects on organismal growth and development, regardless of the karyotype, suggesting that aneuploidy-associated stress plays an important role in disease pathogenesis. However, whether and how this effect alters cellular homeostasis and long-term features of human disease are not fully understood. Here, we aimed to investigate cellular stress responses in human trisomy syndromes, using fibroblasts and induced pluripotent stem cells (iPSCs). Dermal fibroblasts derived from patients with trisomy 21, 18 and 13 showed a severe impairment of cell proliferation and enhanced premature senescence. These phenomena were accompanied by perturbation of protein homeostasis, leading to the accumulation of protein aggregates. We found that treatment with sodium 4-phenylbutyrate (4-PBA), a chemical chaperone, decreased the protein aggregates in trisomy fibroblasts. Notably, 4-PBA treatment successfully prevented the progression of premature senescence in secondary fibroblasts derived from trisomy 21 iPSCs. Our study reveals aneuploidy-associated stress as a potential therapeutic target for human trisomies, including Down syndrome.


Assuntos
Senescência Celular , Fibroblastos/patologia , Agregados Proteicos , Trissomia/patologia , Aneuploidia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glucose/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactatos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Fenilbutiratos/farmacologia , Agregados Proteicos/efeitos dos fármacos , RNA/metabolismo , Trissomia/genética
6.
J Allergy Clin Immunol ; 143(1): 213-228.e10, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29596938

RESUMO

BACKGROUND: A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α-dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α-independent cell death remains unclear. OBJECTIVE: We investigated contribution of TNF-α-dependent and TNF-α-independent signals to the development of dermatitis in epidermis-specific Cflar-deficient (CflarE-KO) mice. METHODS: We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE-KO;Tnfrsf1a+/- and CflarE-KO;Tnfrsf1a-/- mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α-independent cell death of CflarE-KO;Tnfrsf1a-/- mice. RESULTS: CflarE-KO;Tnfrsf1a-/- mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE-KO;Tnfrsf1a+/- mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE-KO;Tnfrsf1a-/- mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of CflarE-KO;Tnfrsf1a-/- mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE-KO;Tnfrsf1a-/- mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. CONCLUSION: TNF receptor superfamily 1 (TNFR1)-dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti-TNF-α antibody alone is not sufficient.


Assuntos
Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Dermatite/imunologia , Epiderme/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Apoptose/genética , Apoptose/imunologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Dermatite/genética , Dermatite/patologia , Epiderme/patologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia
7.
Cell Rep ; 23(11): 3223-3235, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29898394

RESUMO

Hematopoietic stem cells (HSCs) comprise a heterogeneous population exhibiting self-renewal and differentiation capabilities; however, the mechanisms involved in maintaining this heterogeneity remain unclear. Here, we show that SATB1 is involved in regulating HSC heterogeneity. Results in conditional Satb1-knockout mice revealed that SATB1 was important for the self-renewal and lymphopoiesis of adult HSCs. Additionally, HSCs from Satb1/Tomato-knockin reporter mice were classified based on SATB1/Tomato intensity, with transplantation experiments revealing stronger differentiation toward the lymphocytic lineage along with high SATB1 levels, whereas SATB1- HSCs followed the myeloid lineage in agreement with genome-wide transcription and cell culture studies. Importantly, SATB1- and SATB1+ HSC populations were interconvertible upon transplantation, with SATB1+ HSCs showing higher reconstituting and lymphopoietic potentials in primary recipients relative to SATB1- HSCs, whereas both HSCs exhibited equally efficient reconstituted lympho-hematopoiesis in secondary recipients. These results suggest that SATB1 levels regulate the maintenance of HSC multipotency, with variations contributing to HSC heterogeneity.


Assuntos
Células-Tronco Hematopoéticas/citologia , Proteínas de Ligação à Região de Interação com a Matriz/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Antígeno B7-2/metabolismo , Diferenciação Celular , Linhagem da Célula , Autorrenovação Celular , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Linfopoese , Proteínas de Ligação à Região de Interação com a Matriz/deficiência , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/citologia , Linfócitos T/metabolismo
8.
BMC Genomics ; 19(1): 318, 2018 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720086

RESUMO

BACKGROUND: CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive. RESULTS: Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats. CONCLUSIONS: The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.


Assuntos
Engenharia Genética/métodos , Alelos , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Injeções , Camundongos , Camundongos Knockout , Zigoto/metabolismo
9.
Nucleic Acids Res ; 46(10): e63, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29554276

RESUMO

Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.


Assuntos
Homozigoto , Células-Tronco Embrionárias Murinas/fisiologia , Mutagênese/genética , Animais , Células Cultivadas , Elementos de DNA Transponíveis , Diploide , Citometria de Fluxo , Vetores Genéticos , Gentamicinas/farmacologia , Haploidia , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Poli A , Puromicina/farmacologia , Reprodutibilidade dos Testes
10.
Neuropharmacology ; 131: 291-303, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29274751

RESUMO

Na+/Ca2+ exchangers (NCXs) are expressed primarily in the plasma membrane of most cell types, where they mediate electrogenic exchange of one Ca2+ for three Na+ ions, depending on Ca2+ and Na+ electrochemical gradients across the membrane. Three mammalian NCX isoforms (NCX1, NCX2, and NCX3) are each encoded by a distinct gene. Here, we report that NCX2 and NCX3 protein and mRNA levels are relatively reduced in hippocampal CA1 of APP23 and APP-KI mice. Likewise, NCX2+/- or NCX3+/- mice exhibited impaired hippocampal LTP and memory-related behaviors. Moreover, relative to controls, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in NCX2+/- mouse hippocampus but increased in hippocampus of NCX3+/- mice. NCX2 or NCX3 heterozygotes displayed impaired maintenance of hippocampal LTP, a phenotype that in NCX2+/- mice was correlated with elevated calcineurin activity and rescued by treatment with the calcineurin (CaN) inhibitor FK506. Likewise, FK506 treatment significantly restored impaired hippocampal LTP in APP-KI mice. Moreover, Ca2+ clearance after depolarization following high frequency stimulation was slightly delayed in hippocampal CA1 regions of NCX2+/- mice. Electron microscopy revealed relatively decreased synaptic density in CA1 of NCX2+/- mice, while the number of spines with perforated synapses in CA1 significantly increased in NCX3+/- mice. We conclude that memory impairment seen in NCX2+/- and NCX3+/- mice reflect dysregulated hippocampal CaMKII activity, which alters dendritic spine morphology, findings with implications for memory deficits seen in Alzheimer's disease model mice.


Assuntos
Doença de Alzheimer/metabolismo , Região CA1 Hipocampal/metabolismo , Disfunção Cognitiva/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/patologia , Calcineurina/metabolismo , Inibidores de Calcineurina/farmacologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/patologia , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Memória/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Trocador de Sódio e Cálcio/genética , Sinapses/metabolismo , Sinapses/patologia , Tacrolimo/farmacologia
11.
J Mol Cell Cardiol ; 114: 93-104, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29129702

RESUMO

Protein quality control in cardiomyocytes is crucial to maintain cellular homeostasis. The accumulation of damaged organelles, such as mitochondria and misfolded proteins in the heart is associated with heart failure. During the process to identify novel mitochondria-specific autophagy (mitophagy) receptors, we found FK506-binding protein 8 (FKBP8), also known as FKBP38, shares similar structural characteristics with a yeast mitophagy receptor, autophagy-related 32 protein. However, knockdown of FKBP8 had no effect on mitophagy in HEK293 cells or H9c2 myocytes. Since the role of FKBP8 in the heart has not been fully elucidated, the aim of this study is to determine the functional role of FKBP8 in the heart. Cardiac-specific FKBP8-deficient (Fkbp8-/-) mice were generated. Fkbp8-/- mice showed no cardiac phenotypes under baseline conditions. The Fkbp8-/- and control wild type littermates (Fkbp8+/+) mice were subjected to pressure overload by means of transverse aortic constriction (TAC). Fkbp8-/- mice showed left ventricular dysfunction and chamber dilatation with lung congestion 1week after TAC. The number of apoptotic cardiomyocytes was dramatically elevated in TAC-operated Fkbp8-/- hearts, accompanied with an increase in protein levels of cleaved caspase-12 and endoplasmic reticulum (ER) stress markers. Caspase-12 inhibition resulted in the attenuation of hydrogen peroxide-induced apoptotic cell death in FKBP8 knockdown H9c2 myocytes. Immunocytological and immunoprecipitation analyses indicate that FKBP8 is localized to the ER and mitochondria in the isolated cardiomyocytes, interacting with heat shock protein 90. Furthermore, there was accumulation of misfolded protein aggregates in FKBP8 knockdown H9c2 myocytes and electron dense deposits in perinuclear region in TAC-operated Fkbp8-/- hearts. The data suggest that FKBP8 plays a protective role against hemodynamic stress in the heart mediated via inhibition of the accumulation of misfolded proteins and ER-associated apoptosis.


Assuntos
Apoptose , Cardiotônicos/metabolismo , Retículo Endoplasmático/metabolismo , Coração/fisiopatologia , Hemodinâmica , Estresse Fisiológico , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Aorta/patologia , Apoptose/efeitos dos fármacos , Caspase 12/metabolismo , Constrição Patológica , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Coração/efeitos dos fármacos , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mitofagia/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos , Pressão , Ligação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a Tacrolimo/deficiência , Remodelação Ventricular/efeitos dos fármacos
12.
Mol Neurobiol ; 55(5): 3889-3900, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28547530

RESUMO

Among five members of the K+-dependent Na+/Ca2+ exchanger (NCKX) family (NCKX1-5), only NCKX2 is highly expressed in mouse brain. NCKX2 in plasma membranes mediates cytosolic calcium excretion through electrogenic exchange of 4 Na+ for 1 Ca2+ and 1 K+. Here, we observed significantly decreased levels of NCKX2 protein and mRNA in the CA1 region of APP23 mice, a model of Alzheimer's disease. We also found that, like APP23 mice, heterozygous NCKX2-mutant mice exhibit mildly impaired hippocampal LTP and memory acquisition, the latter based on novel object recognition and passive avoidance tasks. When we addressed underlying mechanisms, we found that both CaMKII autophosphorylation and CaMKIV phosphorylation significantly decreased in CA1 regions of NCKX2+/- relative to control mice. Likewise, phosphorylation of GluA1 (Ser-831) and CREB (Ser-133), respective downstream targets of CaMKII and CaMKIV, also significantly decreased in the CA1 region. BDNF protein and mRNA levels significantly decreased in CA1 of NCKX2+/- relative to control mice. Finally, CaN activity increased in CA1 of NCKX2+/- mice. Our findings suggest that like APP23 mice, NCKX2+/- mice may exhibit impaired learning and hippocampal LTP due to decreased CaM kinase II and CaM kinase IV activities.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Transtornos Cognitivos/enzimologia , Trocador de Sódio e Cálcio/genética , Animais , Astrócitos/metabolismo , Comportamento Animal , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Região CA1 Hipocampal/metabolismo , Calcineurina/metabolismo , Transtornos Cognitivos/patologia , Transtornos Cognitivos/fisiopatologia , Heterozigoto , Humanos , Potenciação de Longa Duração , Masculino , Memória , Camundongos Transgênicos , Modelos Biológicos , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sinapses/metabolismo
13.
Sci Rep ; 7(1): 764, 2017 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-28396582

RESUMO

Eukaryotic genomes are organised into complex higher-order structures within the nucleus, and the three-dimensional arrangement of chromosomes is functionally important for global gene regulation. The existence of supernumerary chromosome 21 in Down syndrome may perturb the nuclear architecture at different levels, which is normally optimised to maintain the physiological balance of gene expression. However, it has not been clearly elucidated whether and how aberrant configuration of chromosomes affects gene activities. To investigate the effects of trisomy 21 on nuclear organisation and gene expression, we performed three-dimensional fluorescent imaging analysis of chromosome-edited human induced pluripotent stem cells (iPSCs), which enabled identification of the parental origin of the three copies of chromosome 21. We found that two copies of maternal chromosomes resulting from meiotic nondisjunction had a higher tendency to form an adjacent pair and were located relatively distant from the nuclear membrane, suggesting the conserved interaction between these homologous chromosomes. Transcriptional profiling of parental-origin-specific corrected disomy 21 iPSC lines indicated upregulated expression of the maternal alleles for a group of genes, which was accompanied by a fluctuating expression pattern. These results suggest the unique effects of a pair of maternal chromosomes in trisomy 21, which may contribute to the pathological phenotype.


Assuntos
Cromossomos Humanos Par 21 , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Herança Materna , Meiose , Não Disjunção Genética , Transcrição Gênica , Linhagem Celular , Núcleo Celular/genética , Regulação da Expressão Gênica , Marcação de Genes , Loci Gênicos , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/metabolismo , Fenótipo , Trissomia
14.
Sci Rep ; 7: 43613, 2017 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-28252665

RESUMO

DNA transposons and retroviruses are versatile tools in functional genomics and gene therapy. To facilitate their application, we conducted a genome-wide insertion site profiling of the piggyBac (PB), Tol2 and Sleeping Beauty (SB) transposons and the murine leukemia virus (MLV) in mouse embryonic stem cells (ESCs). PB and MLV preferred highly expressed genes, whereas Tol2 and SB preferred weakly expressed genes. However, correlations with DNase I hypersensitive sites were different for all vectors, indicating that chromatin accessibility is not the sole determinant. Therefore, we analysed various chromatin states. PB and MLV highly correlated with Cohesin, Mediator and ESC-specific transcription factors. Notably, CTCF sites were correlated with PB but not with MLV, suggesting MLV prefers smaller promoter-enhancer loops, whereas PB insertion encompasses larger chromatin loops termed topologically associating domains. Tol2 also correlated with Cohesin and CTCF. However, correlations with ESC-specific transcription factors were weaker, suggesting that Tol2 prefers transcriptionally weak chromatin loops. Consistently, Tol2 insertions were associated with bivalent histone modifications characteristic of silent and inducible loci. SB showed minimum preference to all chromatin states, suggesting the least adverse effect on adjacent genes. These results will be useful for vector selection for various applications.


Assuntos
Cromatina/genética , Elementos de DNA Transponíveis , Vetores Genéticos/genética , Vírus da Leucemia Murina/fisiologia , Mutagênese Insercional , Integração Viral , Animais , Montagem e Desmontagem da Cromatina , Expressão Gênica , Regulação da Expressão Gênica , Ordem dos Genes , Genoma , Genômica/métodos , Histonas/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição
15.
PLoS One ; 11(6): e0156904, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27258152

RESUMO

Epithelial to mesenchymal transition (EMT) is a biological process of metastatic cancer. However, an effective anticancer therapy that directly targets the EMT program has not yet been discovered. Recent studies have indicated that mesenchymal to epithelial transition (MET), the reverse phenomenon of EMT, is observed in fibroblasts during the generation of induced pluripotent stem cells. In the present study, we investigated the effects of reprogramming factors (RFs) on squamous cell carcinoma (SCC) cells. RFs-introduced cancer cells (RICs) demonstrated the enhanced epithelial characteristics in morphology with altered expression of mRNA and microRNAs. The motility and invasive activities of RICs in vitro were significantly reduced. Furthermore, xenografts of RICs exhibited no lymph node metastasis, whereas metastasis was detected in parental SCC-inoculated mice. Thus, we concluded that RICs regained epithelial properties through MET and showed reduced cancer malignancy in vitro and in vivo. Therefore, the understanding of the MET process in cancer cells by introduction of RFs may lead to the designing of a novel anticancer strategy.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Transição Epitelial-Mesenquimal/genética , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Cicatrização/genética , Cicatrização/fisiologia
16.
Cell Rep ; 15(6): 1228-41, 2016 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-27134169

RESUMO

Chromosomal aneuploidy and specific gene mutations are recognized early hallmarks of many oncogenic processes. However, the net effect of these abnormalities has generally not been explored. We focused on transient myeloproliferative disorder (TMD) in Down syndrome, which is characteristically associated with somatic mutations in GATA1. To better understand functional interplay between trisomy 21 and GATA1 mutations in hematopoiesis, we constructed cellular disease models using human induced pluripotent stem cells (iPSCs) and genome-editing technologies. Comparative analysis of these engineered iPSCs demonstrated that trisomy 21 perturbed hematopoietic development through the enhanced production of early hematopoietic progenitors and the upregulation of mutated GATA1, resulting in the accelerated production of aberrantly differentiated cells. These effects were mediated by dosage alterations of RUNX1, ETS2, and ERG, which are located in a critical 4-Mb region of chromosome 21. Our study provides insight into the genetic synergy that contributes to multi-step leukemogenesis.


Assuntos
Cromossomos Humanos Par 21/genética , Síndrome de Down/genética , Epistasia Genética , Fator de Transcrição GATA1/genética , Hematopoese/genética , Modelos Biológicos , Mutação/genética , Pareamento de Bases/genética , Sequência de Bases , Diferenciação Celular/genética , Linhagem da Célula/genética , Eritropoese/genética , Técnicas de Inativação de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/patologia , Edição de RNA/genética , Deleção de Sequência , Fatores de Transcrição/metabolismo , Regulação para Cima/genética
17.
Genes Cells ; 21(5): 392-5, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27028186

RESUMO

From November 17 to 20 in 2015, the Conference on Transposition and Genome Engineering 2015 (TGE 2015) was held at Nara Kasugano International Forum-IRAKA-in Nara, Japan, located at the center of Nara Park. All of the presentations were carried out at Nohgaku hall in Nara Kasugano International Forum-IRAKA. Participation totaled 148 persons (30 international, 118 domestic), who were able to engage in lively scientific discussions over the 4-day period. The guest speaker list consisted of many top-notch international researchers, an achievement for which the conference received praise from the attendees. There were 36 oral presentations including the keynote lecture (22 presentations from guest speakers, complemented with 14 selected from abstract submissions). Additionally, there were 46 poster presentations. The conference uniquely combined research mainly from two different genomics approaches: (i) transposon technology allowing random genomic integration followed by gene discovery-related phenotypes and (ii) genome editing technology with designer nuclease allowing precise modification of a gene-of-interest.


Assuntos
Elementos de DNA Transponíveis , Engenharia Genética/métodos , Genômica/métodos , Japão
18.
Nucleic Acids Res ; 44(8): e78, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-26833260

RESUMO

An important challenge in cancer genomics is precise detection of structural variations (SVs) by high-throughput short-read sequencing, which is hampered by the high false discovery rates of existing analysis tools. Here, we propose an accurate SV detection method named COSMOS, which compares the statistics of the mapped read pairs in tumor samples with isogenic normal control samples in a distinct asymmetric manner. COSMOS also prioritizes the candidate SVs using strand-specific read-depth information. Performance tests on modeled tumor genomes revealed that COSMOS outperformed existing methods in terms of F-measure. We also applied COSMOS to an experimental mouse cell-based model, in which SVs were induced by genome engineering and gamma-ray irradiation, followed by polymerase chain reaction-based confirmation. The precision of COSMOS was 84.5%, while the next best existing method was 70.4%. Moreover, the sensitivity of COSMOS was the highest, indicating that COSMOS has great potential for cancer genome analysis.


Assuntos
Biologia Computacional/métodos , Células-Tronco Embrionárias/citologia , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Animais , Variação Genética/genética , Genoma Humano/genética , Humanos , Camundongos
19.
J Dermatol Sci ; 81(3): 173-81, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26655443

RESUMO

BACKGROUND: Keratin 5 (K5) is a cytoskeletal tissue-specific protein expressed in the epithelial cells of skin and esophagus and ectopic K5 expression in lymphocytes has never been reported. OBJECTIVE: Here we demonstrate an ectopic epidermal self-protein expression in B-1 B cell by fate mapping of K5-expressing cells. METHODS: K5-Cre×CAG-CAT-loxP-EGFP double Tg (K5×GFP) mice that express enhanced GFP under the control of the K5 promoter were employed. RESULTS: Unexpectedly, B220(+)GFP(+) cells were found in LN, spleen, peripheral blood and peritoneal cavity. These cells were IgM(+)IgD(low)CD23(-)CD43(+)CD19(+)CD93(-), indicating that they were B-1 B cells. The number of B220(+)GFP(+) cells was significantly larger in spleen than in the other tissues tested. Although GFP(+) B-1 cells did not express K5 in the periphery, Lin(-)CD93(+)B220(low-neg)CD19(+) B-1 B cell progenitors expressed GFP and B220(+)CD93(+) progenitor cells expressed K5 and MHC-class II in BM, indicating that GFP(+) B-1 cells transiently expressed K5 and the progenitor cells were potential APC. GFP(+) B-1 cells in the periphery continued expressing MHC class II and had exogenous antigen-presenting capacity comparable to non-follicular B cells. GFP(+) B-1 cells spontaneously secreted more IgM than GFP(-) B-1 cells in vitro. CONCLUSION: These results indicate that B-1 B cells transiently and partially express K5 in BM and are potent for both natural antibody production and antigen presentation.


Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Queratina-15/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Animais , Formação de Anticorpos , Apresentação de Antígeno , Biomarcadores/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Linhagem da Célula , Células Cultivadas , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoglobulina M/biossíntese , Queratina-15/genética , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/imunologia , Regiões Promotoras Genéticas , Fatores de Tempo
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