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1.
Nat Commun ; 11(1): 3339, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620764

RESUMO

Chromosomal NUP98-PHF23 translocation is associated with an aggressive form of acute myeloid leukemia (AML) and poor survival rate. Here, we report the molecular mechanisms by which NUP98-PHF23 recognizes the histone mark H3K4me3 and is inhibited by small molecule compounds, including disulfiram that directly targets the PHD finger of PHF23 (PHF23PHD). Our data support a critical role for the PHD fingers of NUP98-PHF23, and related NUP98-KDM5A and NUP98-BPTF fusions in driving leukemogenesis, and demonstrate that blocking this interaction in NUP98-PHF23 expressing AML cells leads to cell death through necrotic and late apoptosis pathways. An overlap of NUP98-KDM5A oncoprotein binding sites and H3K4me3-positive loci at the Hoxa/b gene clusters and Meis1 in ChIP-seq, together with NMR analysis of the H3K4me3-binding sites of the PHD fingers from PHF23, KDM5A and BPTF, suggests a common PHD finger-dependent mechanism that promotes leukemogenesis by this type of NUP98 fusions. Our findings highlight the direct correlation between the abilities of NUP98-PHD finger fusion chimeras to associate with H3K4me3-enriched chromatin and leukemic transformation.


Assuntos
Cromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Doença Aguda , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Cromatina/genética , Dissulfiram/farmacologia , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Dedos de Zinco PHD/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Translocação Genética/efeitos dos fármacos , Translocação Genética/genética
2.
Oncogene ; 39(25): 4884-4895, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32451433

RESUMO

Chromatin remodeling factors contribute to establish aberrant gene expression programs in cancer cells and therefore represent valuable targets for therapeutic intervention. BPTF (Bromodomain PhD Transcription Factor), a core subunit of the nucleosome remodeling factor (NURF), modulates c-MYC oncogenic activity in pancreatic cancer. Here, we analyze the role of BPTF in c-MYC-driven B-cell lymphomagenesis using the Eµ-Myc transgenic mouse model of aggressive B-cell lymphoma. We find that BPTF is required for normal B-cell differentiation without evidence of haploinsufficiency. In contrast, deletion of one Bptf allele is sufficient to delay lymphomagenesis in Eµ-Myc mice. Tumors arising in a Bptf heterozygous background display decreased c-MYC levels and pathway activity, together with increased activation of the NF-κB pathway, a molecular signature characteristic of human diffuse large B-cell lymphoma (DLBCL). In human B-cell lymphoma samples, we find a strong correlation between BPTF and c-MYC mRNA and protein levels, together with an anti-correlation between BPTF and NF-κB pathway activity. Our results indicate that BPTF is a relevant therapeutic target in B-cell lymphomas and that, upon its inhibition, cells acquire distinct oncogenic dependencies.


Assuntos
Antígenos Nucleares/genética , Linfoma de Células B/genética , Proteínas do Tecido Nervoso/genética , Vício Oncogênico/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genética , Animais , Antígenos Nucleares/metabolismo , Linfócitos B/metabolismo , Carcinogênese/genética , Montagem e Desmontagem da Cromatina/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma de Células B/metabolismo , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo
3.
J Biol Chem ; 295(13): 4114-4123, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32047112

RESUMO

Ether-a-go-go (EAG) potassium selective channels are major regulators of neuronal excitability and cancer progression. EAG channels contain a Per-Arnt-Sim (PAS) domain in their intracellular N-terminal region. The PAS domain is structurally similar to the PAS domains in non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, it is not known whether the PAS domain can regulate EAG channel function via ligand binding. Here, using surface plasmon resonance, tryptophan fluorescence, and analysis of EAG currents recorded in Xenopus laevis oocytes, we show that a small molecule chlorpromazine (CH), widely used as an antipsychotic medication, binds to the isolated PAS domain of EAG channels and inhibits currents from these channels. Mutant EAG channels that lack the PAS domain show significantly lower inhibition by CH, suggesting that CH affects currents from EAG channels directly through the binding to the PAS domain. Our study lends support to the hypothesis that there are previously unaccounted steps in EAG channel gating that could be activated by ligand binding to the PAS domain. This has broad implications for understanding gating mechanisms of EAG and related ERG and ELK K+ channels and places the PAS domain as a new target for drug discovery in EAG and related channels. Up-regulation of EAG channel activity is linked to cancer and neurological disorders. Our study raises the possibility of repurposing the antipsychotic drug chlorpromazine for treatment of neurological disorders and cancer.


Assuntos
Clorpromazina/farmacologia , Canal de Potássio ERG1/genética , Canais de Potássio Éter-A-Go-Go/genética , Neurônios/efeitos dos fármacos , Sequência de Aminoácidos/genética , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos Nucleares/química , Antígenos Nucleares/genética , Sítios de Ligação/efeitos dos fármacos , Excitabilidade Cortical/efeitos dos fármacos , Excitabilidade Cortical/genética , Canal de Potássio ERG1/química , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Ligantes , Neurônios/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Domínios Proteicos/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Xenopus laevis/genética
4.
Exp Neurol ; 326: 113178, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31926165

RESUMO

Physical exercise can reduce the cognitive decline associated with traumatic brain injury, yet little is known about the optimal administration schedules. Here, different protocols of voluntary wheel running were evaluated for their effects on object recognition memory (ORM), neuroprotection (NeuN+ cells), microglial reactivity (Iba1 staining) and neurogenesis (DCX+ cells) after controlled cortical impact injury (CCI). CCI-lesioned rats were divided into a sedentary group and three exercise groups: early discontinued exercise (3 weeks of exercise initiated 4 days post-injury, followed by 4 weeks in a sedentary state); delayed exercise (3 weeks of exercise initiated 4 weeks post-injury), and early continuous exercise (7 weeks of exercise starting 4 days post-injury). The deficits induced by CCI in a 24 h ORM test were reversed in the delayed exercise group and reduced in the early discontinued and early continuous groups. The early discontinued protocol also reduced the loss of NeuN+ cells in the hilus, while attenuated microglial reactivity was found in the dorsal hippocampus of both the early exercising groups. Running at the end of the experiment increased the number of DCX+ cells in the early continuous and delayed groups, and an inverted U-shaped relationship was found between the mean daily exercise time and the amount of neurogenesis. Thus, exercise had benefits on memory both when it was commenced soon and later after injury, although the neural mechanisms implicated differed. Accordingly, the effects of exercise on memory and neurogenesis appear to not only depend on the specific temporal schedule but also, they may be influenced by the amount of daily exercise.


Assuntos
Lesões Encefálicas Traumáticas/psicologia , Memória , Condicionamento Físico Animal/métodos , Condicionamento Físico Animal/psicologia , Reconhecimento Psicológico , Animais , Antígenos Nucleares/genética , Lesões Encefálicas Traumáticas/patologia , Proteínas de Ligação ao Cálcio/genética , Giro Denteado/patologia , Hipocampo/patologia , Masculino , Proteínas dos Microfilamentos/genética , Microglia/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Neurogênese , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neuroproteção , Desempenho Psicomotor , Ratos , Ratos Sprague-Dawley , Corrida , Fatores de Tempo
5.
Front Med ; 14(1): 60-67, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31104301

RESUMO

Bromodomain PHD-finger transcription factor (BPTF) is the largest subunit of the nucleosome remodeling factor and plays an important role in chromatin remodeling for gene activation through its association with histone acetylation or methylation. BPTF is also involved in oncogene transcription in diverse progressions of cancers. Despite clinical trials for inhibitors of bromodomain and extra-terminal family proteins in human cancers, no potent and selective inhibitor targeting the BPTF bromodomain has been discovered. In this study, we identified a potential inhibitor, namely, C620-0696, by computational docking modeling to target bromodomain. Results of biolayer interferometry revealed that compound C620-0696 exhibited high binding affinity to the BPTF bromodomain. Moreover, C620-0696 was cytotoxic in BPTF with a high expression of non-small-cell lung cancer (NSCLC) cells. It suppressed the expression of the BPTF target gene c-MYC, which is known as an oncogenic transcriptional regulator in various cancers. C620-0696 also partially inhibited the migration and colony formation of NSCLC cells owing to apoptosis induction and cell cycle blockage. Thus, our study presents an effective strategy to target a bromodomain factor-mediated tumorigenesis in cancers with small molecules, supporting further exploration of the use of these inhibitors in oncology.


Assuntos
Antígenos Nucleares/metabolismo , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Células A549 , Acetilação , Antígenos Nucleares/genética , Apoptose , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Simulação de Acoplamento Molecular , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética
6.
Mol Cell ; 76(5): 767-783.e11, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31540874

RESUMO

Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and pre-rRNA processing. Here, we report that a human nucleolus consists of several dozen FC/DFC units, each containing 2-3 transcriptionally active rDNAs at the FC/DFC border. Pre-rRNA processing factors, such as fibrillarin (FBL), form 18-24 clusters that further assemble into the DFC surrounding the FC. Mechanistically, the 5' end of nascent 47S pre-rRNA binds co-transcriptionally to the RNA-binding domain of FBL. FBL diffuses to the DFC, where local self-association via its glycine- and arginine-rich (GAR) domain forms phase-separated clusters to immobilize FBL-interacting pre-rRNA, thus promoting directional traffic of nascent pre-rRNA while facilitating pre-rRNA processing and DFC formation. These results unveil FC/DFC ultrastructures in nucleoli and suggest a conceptual framework for considering nascent RNA sorting using multivalent interactions of their binding proteins.


Assuntos
Nucléolo Celular/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Transporte Ativo do Núcleo Celular , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Precursores de RNA/genética , Precursores de RNA/ultraestrutura , RNA Ribossômico/genética , RNA Ribossômico/ultraestrutura
7.
Med Sci Monit ; 25: 6955-6964, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31558691

RESUMO

BACKGROUND PAS domain containing repressor 1 (PASD1), the cancer-testis antigen (CTA), has been reported to be aberrantly expressed in various cancer tissues and cancer cell lines; however, normal PASD1 expression can be detected in normal tissue, excluding testicular tissue. Moreover, PASD1 is reported to be abnormally expressed in various malignant tumors. However, it remains unclear whether PASD1 participates in tumorigenesis of glioma. MATERIAL AND METHODS PASD1 expression was detected by immunohistochemistry in 155 glioma tissue specimens in this study. Furthermore, the relationship of PASD1 expression with clinicopathological features in glioma cases was statistically analyzed. In addition, PASD1 was knocked down by small interference RNA (shRNA) in glioma cell line (LN229), so as to assess the potential to use it as the target for treating glioma. RESULTS Our findings suggested that PASD1 expression in glioma patients was extremely upregulated compared with that in normal tissue samples and cell lines. Moreover, PASD1 expression was found to be markedly correlated with gender, The World Health Organization grade and p53 expression; in addition, high PASD1 expression indicated poor prognosis for glioma patients. Additionally, downregulation of PASD1 inhibited the proliferation ability of cells and resulted in cell arrest at the G2/M phase, which was achieved through accelerating apoptosis. Furthermore, our results indicated that PASD1 downregulation could upregulate some apoptosis-modulating proteins at the same time it downregulated some cycle-regulating proteins. CONCLUSIONS Taken together, our findings demonstrated that PASD1, an oncogene, can potentially serve as an independent prognostic factor for glioma patients.


Assuntos
Antígenos de Neoplasias/metabolismo , Antígenos Nucleares/metabolismo , Glioma/metabolismo , Adulto , Idoso , Antígenos de Neoplasias/genética , Antígenos Nucleares/genética , Apoptose/genética , Neoplasias Encefálicas/genética , Carcinogênese/genética , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Glioma/genética , Glioma/patologia , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores Sexuais , Transcriptoma/genética
8.
Nat Commun ; 10(1): 2983, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278301

RESUMO

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development.


Assuntos
Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Matriz Extracelular/metabolismo , Feminino , Adesões Focais/genética , Humanos , Microscopia Intravital , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Processamento de RNA/genética , RNA Interferente Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Transdução de Sinais/genética , Análise de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade
9.
Nat Commun ; 10(1): 2208, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101817

RESUMO

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.


Assuntos
Antígenos Nucleares/metabolismo , Polaridade Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/isolamento & purificação , Células CACO-2 , Cristalografia por Raios X , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/isolamento & purificação , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
10.
BMC Res Notes ; 12(1): 225, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987672

RESUMO

OBJECTIVE: Delivery of constructs for silencing or over-expressing genes or their modified versions is a crucial step for studying neuronal cell biology. Therefore, efficient transfection is important for the success of these experimental techniques especially in post-mitotic cells like neurons. In this study, we have assessed the transfection rate, using a previously established protocol, in both primary cortical cultures and neuroblastoma cell lines. Transfection efficiencies in these preparations have not been systematically determined before. RESULTS: Transfection efficiencies obtained herein were (10-12%) for neuroblastoma, (5-12%) for primary astrocytes and (1.3-6%) for primary neurons. We also report on cell-type specific transfection efficiency of neurons and astrocytes within primary cortical cultures when applying cell-type selective transfection protocols. Previous estimations described in primary cortical or hippocampal cultures were either based on general observations or on data derived from unspecified number of biological and/or technical replicates. Also to the best of our knowledge, transfection efficiency of pure primary neuronal cultures or astrocytes cultured in the context of pure or mixed (neurons/astrocytes) population cultures have not been previously determined. The transfection strategy used herein represents a convenient, and a straightforward tool for targeted cell transfection that can be utilized in a variety of in vitro applications.


Assuntos
Astrócitos/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Plasmídeos/metabolismo , Transfecção/métodos , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Expressão Gênica , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lipídeos/química , Lipídeos/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Especificidade de Órgãos , Plasmídeos/química , Cultura Primária de Células
11.
Nat Commun ; 10(1): 1686, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975996

RESUMO

Cohesin is a multiprotein ring that is responsible for cohesion of sister chromatids and formation of DNA loops to regulate gene expression. Genomic analyses have identified that the cohesin subunit STAG2 is frequently inactivated by mutations in cancer. However, the reason STAG2 mutations are selected during tumorigenesis and strategies for therapeutically targeting mutant cancer cells are largely unknown. Here we show that STAG2 is essential for DNA replication fork progression, whereby STAG2 inactivation in non-transformed cells leads to replication fork stalling and collapse with disruption of interaction between the cohesin ring and the replication machinery as well as failure to establish SMC3 acetylation. As a consequence, STAG2 mutation confers synthetic lethality with DNA double-strand break repair genes and increased sensitivity to select cytotoxic chemotherapeutic agents and PARP or ATR inhibitors. These studies identify a critical role for STAG2 in replication fork procession and elucidate a potential therapeutic strategy for cohesin-mutant cancers.


Assuntos
Antígenos Nucleares/metabolismo , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Neoplasias/genética , Mutações Sintéticas Letais , Antígenos Nucleares/genética , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Cromátides/metabolismo , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Inativação de Genes , Humanos , Mutagênese , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Reparo de DNA por Recombinação
12.
Mol Cancer ; 18(1): 45, 2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30922402

RESUMO

BACKGROUND: Circular RNAs (circRNAs) are a novel type of noncoding RNAs and play important roles in tumorigenesis, including gastric cancer (GC). However, the functions of most circRNAs remain poorly understood. In our study, we aimed to investigate the functions of a new circRNA circ-DONSON in GC progression. METHODS: The expression of circ-DONSON in gastric cancer tissues and adjacent normal tissues was analyzed by bioinformatics method, qRT-PCR, Northern blotting and in situ hybridization (ISH). The effects of circ-DONSON on GC cell proliferation, apoptosis, migration and invasion were measured by using CCK8, colony formation, EdU, immunofluorescence (IF), FACS and Transwell assays. qRT-PCR and Western blotting were utilized to validate how circ-DONSON regulates SOX4 expression. ChIP, DNA fluorescence in situ hybridization (DNA-FISH) and DNA accessibility assays were used to investigate how circ-DONSON regulates SOX4 transcription. The interaction between circ-DONSON and NURF complex was evaluated by mass spectrum, RNA immunoprecipitation (RIP), pulldown and EMSA assays. Xenograft mouse model was used to analyze the effect of circ-DONSON on GC growth in vivo. RESULTS: Elevated expression of circ-DONSON was observed in GC tissues and positively associated with advanced TNM stage and unfavorable prognosis. Silencing of circ-DONSON significantly suppressed the proliferation, migration and invasion of GC cells while promoting apoptosis. circ-DONSON was localized in the nucleus, recruited the NURF complex to SOX4 promoter and initiated its transcription. Silencing of the NURF complex subunit SNF2L, BPTF or RBBP4 similarly attenuated GC cell growth and increased apoptosis. circ-DONSON knockdown inhibited GC growth in vivo. CONCLUSION: circ-DONSON promotes GC progression through recruiting the NURF complex to initiate SOX4 expression.


Assuntos
Antígenos Nucleares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , RNA/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Fatores de Transcrição SOXC/metabolismo , Neoplasias Gástricas/patologia , Fatores de Transcrição/metabolismo , Animais , Antígenos Nucleares/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Prognóstico , RNA Circular , Proteína 4 de Ligação ao Retinoblastoma/genética , Fatores de Transcrição SOXC/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Taxa de Sobrevida , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
In Vivo ; 33(2): 441-445, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30804123

RESUMO

BACKGROUND/AIM: Cerebral ischemia is a major cause of abnormal brain development. In a cerebral ischemia model, periventricular leukomalacia (PVL), white matter lesion and a decrease in the number of subcortical neurons were observed. The aim of this study was to investigate the effect of hypoxia on neurogenesis and cell survival. MATERIALS AND METHODS: In seven-day postnatal rats, the right carotid artery was ligated. The rats were incubated either in a regular normoxic chamber (control group) or in a hypoxic chamber (PVL group, 8% 02 and 92% N2 at 37°C) for 2 h. Nestin- and NeuN-positive neurons were detected by immunohistochemistry. RESULTS: The densities of nestin-immunoreactivity (IR) cells in the cerebral parietal cortex and subventricular zone were increased with hypoxia. NeuN-IR cells in the cerebral cortex were significantly decreased in the PVL group. CONCLUSION: Perinatal white matter injury induced neurogenesis, while the survival of neurons was decreased in the cerebral cortex.


Assuntos
Córtex Cerebral/metabolismo , Hipóxia-Isquemia Encefálica/genética , Neurogênese/genética , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Antígenos Nucleares/genética , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Proliferação de Células/genética , Córtex Cerebral/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hipóxia-Isquemia Encefálica/patologia , Imuno-Histoquímica , Ligadura , Proteínas do Tecido Nervoso/genética , Nestina/genética , Neurônios/patologia , Ratos , Substância Branca/lesões , Substância Branca/metabolismo , Substância Branca/patologia
14.
J Hum Genet ; 64(5): 487-492, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30765867

RESUMO

We herein report two individuals with novel nonsense mutations in STAG2 on Xq25, encoding stromal antigen 2, a component of the cohesion complex. A male fetus (Case 1) clinically presented with holoprosencephaly, cleft palate and lip, blepharophimosis, nasal bone absence, and hypolastic left heart by ultrasonography at 15 gestational weeks. Another female patient (Case 2) showed a distinct phenotype with white matter hypoplasia, cleft palate, developmental delay (DD), and intellectual disability (ID) at 7 years. Whole-exome sequencing identified de novo nonsense mutations in STAG2: c.3097C>T, p.(Arg1033*) in Case 1 and c.2229G>A, p.(Trp743*) in Case 2. X-inactivation was highly skewed in Case 2. To date, only 10 STAG2 pathogenic variants (four nonsense, four missense, and two frameshift) have been reported in patients with multiple congenital anomalies, ID, and DD. Although Case 2 showed similar clinical features to the reported female patients with STAG2 abnormalities, Case 1 showed an extremely severe phenotype, which could be explained by the first detected truncating variant in males.


Assuntos
Anormalidades Múltiplas/genética , Antígenos Nucleares/genética , Códon sem Sentido , Mutação de Sentido Incorreto , Anormalidades Múltiplas/patologia , Criança , Feminino , Humanos , Masculino , Fatores Sexuais
15.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626680

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates episome persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence through its carboxy-terminal domain to mediate DNA replication. LANA simultaneously binds mitotic chromosomes and TR DNA to segregate virus genomes to daughter cell nuclei. Amino-terminal LANA attaches to chromosomes by binding histones H2A/H2B, and carboxy-terminal LANA contributes to mitotic-chromosome binding. Although amino- and carboxy-terminal LANA are essential for episome persistence, they are not sufficient, since deletion of all internal LANA sequence renders LANA highly deficient for episome maintenance. Internal LANA sequence upstream of the internal repeat elements contributes to episome segregation and persistence. Here, we investigate this region with a panel of LANA deletion mutants. Mutants retained the ability to associate with mitotic chromosomes and bind TR DNA. In contrast to prior results, deletion of most of this sequence did not reduce LANA's ability to mediate DNA replication. Deletions of upstream sequence within the region compromised segregation of TR DNA to daughter cells, as assessed by retention of green fluorescent protein (GFP) expression from a replication-deficient TR plasmid. However, deletion of this upstream sequence did not reduce episome maintenance. In contrast, deletions that included an 80-amino-acid sequence immediately downstream resulted in highly deficient episome persistence. LANA with this downstream sequence deleted maintained the ability to replicate and segregate TR DNA, suggesting a unique role for the residues. Therefore, this work identifies adjacent LANA regions with distinct roles in episome segregation and persistence.IMPORTANCE KSHV LANA mediates episomal persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence to mediate DNA replication and tethers KSHV DNA to mitotic chromosomes to segregate genomes to daughter cell nuclei. Here, we investigate LANA sequence upstream of the internal repeat elements that contributes to episome segregation and persistence. Mutants with deletions within this sequence maintained the ability to bind mitotic chromosomes or bind and replicate TR DNA. Deletion of upstream sequence within the region reduced segregation of TR DNA to daughter cells, but not episome maintenance. In contrast, mutants with deletions of 80 amino acids immediately downstream were highly deficient for episome persistence yet maintained the ability to replicate and segregate TR DNA, the two principal components of episome persistence, suggesting another role for the residues. In summary, this work identifies adjacent LANA sequence with distinct roles in episome segregation and persistence.


Assuntos
Antígenos Virais/genética , Herpesvirus Humano 8/genética , Proteínas Nucleares/genética , Plasmídeos/genética , Sarcoma de Kaposi/virologia , Antígenos Nucleares/genética , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/virologia , Cromossomos/genética , Replicação do DNA/genética , DNA Viral/genética , Genoma Viral/genética , Células HEK293 , Humanos , Mitose/genética , Sequências Repetidas Terminais/genética , Proteínas Virais/genética , Latência Viral/genética , Replicação Viral/genética
16.
Mol Genet Genomic Med ; 7(2): e00501, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30447054

RESUMO

BACKGROUND: The cohesin complex is a multi-subunit protein complex which regulates sister chromatid cohesion and separation during cellular division. In addition, this evolutionarily conserved protein complex plays an integral role in DNA replication, DNA repair, and the regulation of transcription. The core complex is composed of four subunits: RAD21, SMC1A, SMC3, and STAG1/2. Mutations in these proteins have been implicated in human developmental disorders collectively termed "cohesinopathies." METHODS: Using clinical exome sequencing, we have previously identified three female cases with heterozygous STAG2 mutations and overlapping syndromic phenotypes. Subsequently, a familial missense variant was identified in five male family members. RESULTS: We now present the case of a 4-year-old male with developmental delay, failure to thrive, short stature, and polydactyly with a likely pathogenic STAG2 de novo missense hemizygous variant, c.3027A>T, p.Lys1009Asn. Furthermore, we compare the phenotypes of the four previously reported STAG2 variants with our case. CONCLUSION: We conclude that mutations in STAG2 cause a novel constellation of sex-specific cohesinopathy-related phenotypes and are furthermore, essential for neurodevelopment, human growth, and behavioral development.


Assuntos
Antígenos Nucleares/genética , Deficiências do Desenvolvimento/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Transtornos do Crescimento/genética , Fenótipo , Polidactilia/genética , Pré-Escolar , Deficiências do Desenvolvimento/patologia , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Transtornos do Crescimento/patologia , Humanos , Masculino , Mutação de Sentido Incorreto , Polidactilia/patologia , Síndrome
17.
Mol Cell ; 73(2): 212-223.e7, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30554942

RESUMO

Cohesin subunits are frequently mutated in cancer, but how they function as tumor suppressors is unknown. Cohesin mediates sister chromatid cohesion, but this is not always perturbed in cancer cells. Here, we identify a previously unknown role for cohesin. We find that cohesin is required to repress transcription at DNA double-strand breaks (DSBs). Notably, cohesin represses transcription at DSBs throughout interphase, indicating that this is distinct from its known role in mediating DNA repair through sister chromatid cohesion. We identified a cancer-associated SA2 mutation that supports sister chromatid cohesion but is unable to repress transcription at DSBs. We further show that failure to repress transcription at DSBs leads to large-scale genome rearrangements. Cancer samples lacking SA2 display mutational patterns consistent with loss of this pathway. These findings uncover a new function for cohesin that provides insights into its frequent loss in cancer.


Assuntos
Neoplasias Ósseas/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Interfase , Osteossarcoma/genética , Transcrição Genética , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Reparo do DNA , Regulação para Baixo , Fase G1 , Fase G2 , Regulação Neoplásica da Expressão Gênica , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
J Proteome Res ; 18(3): 1064-1077, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30585729

RESUMO

The Ku heterodimer, composed of Ku70 and Ku80, is best characterized for its role in repairing double-stranded DNA breaks but is also known to participate in other regulatory processes. Despite our understanding of Ku protein interplay during DNA repair, the extent of Ku's protein interactions in other processes has never been fully determined. Using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS) with wild-type Ku70, we identified candidate proteins that interact with the Ku heterodimer in HEK293 cells, in the absence of exogenously induced DNA damage. BioID analysis identified approximately 250 nuclear proteins, appearing in at least two replicates, including known Ku-interacting factors such as MRE11A, WRN, and NCOA6. Meanwhile, AP-MS analysis identified approximately 50 candidate proteins. Of the novel protein interactors identified, many were involved in functions already suspected to involve Ku such as transcriptional regulation, DNA replication, and DNA repair, while several others suggest that Ku may be involved in additional functions such as RNA metabolism, chromatin-remodeling, and microtubule dynamics. Using a combination of BioID and AP-MS, this is the first report that comprehensively characterizes the Ku protein interaction landscape, revealing new cellular processes and protein complexes involving the Ku complex.


Assuntos
Antígenos Nucleares/genética , Reparo do DNA/genética , Autoantígeno Ku/genética , Proteínas Nucleares/genética , Antígenos Nucleares/química , Biotina/química , Dano ao DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Autoantígeno Ku/química , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/química , Coativadores de Receptor Nuclear/genética , Multimerização Proteica/genética , Helicase da Síndrome de Werner/genética
19.
Nucleic Acids Res ; 47(4): e24, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30590765

RESUMO

Chimeric RNAs generated by cis-splicing between adjacent genes (cis-SAGe) are increasingly recognized as a widespread phenomenon. These chimeric messenger RNAs are present in normal human cells, and are also detected in various cancers. The mechanisms for how this group of chimeras is formed are not yet clear, in part due to the lack of a tractable system for their experimental investigation. Here we developed a fast, easy and versatile cell-based reporter system to identify regulators of cis-SAGe. The reporter, consisting of four main cassettes, simultaneously measures the effects of a candidate regulator on cis-SAGe and canonical splicing. Using this cell-based assay, we screened 102 candidate factors involved in RNA pol II cleavage and termination, elongation, splicing, alternative splicing and R-loop formation. We discovered that two factors, SRRM1 and SF3B1, affect not only cis-SAGe chimeras, but also other types of chimeric RNAs in a genome-wide fashion. This system can be used for studying trans-acting factors and cis-acting sequence elements and factors, as well as for screening small molecule inhibitors.


Assuntos
Antígenos Nucleares/genética , Regulação da Expressão Gênica/genética , Proteínas Associadas à Matriz Nuclear/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Regulação Neoplásica da Expressão Gênica/genética , Fusão Gênica/genética , Genes Reporter/genética , Genoma Humano/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Clivagem do RNA/genética , RNA Polimerase II/genética
20.
Biochem Cell Biol ; 97(4): 415-422, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30481052

RESUMO

A previous study by our group indicted that overexpression of bromodomain PHD-finger transcription factor (BPTF) occurs in lung adenocarcinoma, and is closely associated with advanced clinical stage, higher numbers of metastatic lymph nodes, the occurrence of distant metastasis, low histological grade, and poor prognosis. Down-regulation of BPTF inhibited lung adenocarcinoma cell proliferation and promoted lung adenocarcinoma cell apoptosis. The purpose of this study is to identify valuable microRNAs (miRNAs) that target BPTF to modulate lung adenocarcinoma cell proliferation. In our results, we found that miR-3666 was notably reduced in lung adenocarcinoma tissues and cell lines. Using an miR-3666 mimic, we discovered that cell proliferation, migration, and invasiveness were suppressed by miR-3666 overexpression, but these were all enhanced when the expression of miR-3666 was reduced. Moreover, bioinformatics analysis using the TargetScan database and miRanda software suggested a putative target site in BPTF 3'-UTR. Furthermore, using a luciferase reporter assay, we verified that miR-3666 directly targets the 3'-UTR of BPTF. Using Western blot we discovered that overexpression of miR-3666 negatively regulates the protein expression of BPTF. Finally, we identified that the PI3K-AKT and epilthelial-mesenchymal transition (EMT) signaling pathways were inhibited by miR-3666 overexpression in lung cancer cells. In conclusion, our data indicate that miR-3666 could play an essential role in cell proliferation, migration, and invasiveness by targeting BPTF and partly inhibiting the PI3K-AKT and EMT signaling pathways in human lung cancers.


Assuntos
Antígenos Nucleares/genética , Movimento Celular/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Antígenos Nucleares/metabolismo , Proliferação de Células/genética , Biologia Computacional , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas
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