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1.
Anticancer Res ; 39(11): 6273-6282, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704857

RESUMO

BACKGROUND/AIM: We have yet to understand why JAK2-positive myeloproliferative neoplasms (MPN) patients manifest different phenotypes despite harboring JAK2 mutations and what drives secondary transformations. PATIENTS AND METHODS: Using targeted sequencing, we analyzed mutational status of 17 polycythemia vera (PV), 16 essential thrombocythemia (ET), 8 primary myelofibrosis (PMF) patients who tested positive for JAK by polymerase chain reaction. RESULTS: The somatic mutations in JAK2 influence the clinical behavior of the disease. We found that ASXL1 or EZH2 mutation acquisition after JAK2 leads to PV, while ASXL1 mutation acquisition before JAK2 leads to ET or PMF. Mutations in TP53, ASXL1, and splicing genes are associated with the prognosis of MPN. PMF was more frequently associated with splicing mutations, while PV was more closely related to mutations in chromatin modifiers. The presence of these mutations influenced hemogram at MPN diagnosis. CONCLUSION: Each subtype of MPN harbors distinct patterns of somatic mutations and acquisition order, while mutations in TP53, ASXL1, and splicing genes may be associated with the prognosis of MPN.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Genes p53 , Janus Quinase 2/genética , Mutação , Policitemia Vera/genética , Mielofibrose Primária/genética , Proteínas Repressoras/genética , Trombocitemia Essencial/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Prognóstico , Processamento de RNA
2.
Life Sci ; 237: 116945, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31605710

RESUMO

AIM: Over-expression of histone deacetylase 8 (HDAC8) has been demonstrated in breast cancer. But the underlying molecular mechanism of HDAC8 on the progression of breast cancer remains unknown. MicroRNAs (miRs) are proposed as important molecules in cancer progression by targeting specific oncogenes or tumor-suppressor genes. Our overall objective was to assess the miR-216b-5p role on HDAC8; and its impacts on breast cancer (BC) progression. MAIN METHODS: We acquired cancerous and noncancerous tissues from Iran Tumor Bank (I.T.B). The MDA-MB-231, MCF-7 and MCF-10A BC cell lines were also purchased. The tissue and cell line expression levels of miR-216b-5p and HDAC8 were determined by quantitative real-time PCR (qPCR). We next measured protein levels of HDAC8 by Western blotting assay. The cell cycle, cell proliferation, and colony formation assay were determined. Finally, we investigated the role of HDAC8 using a knockout vector; and confirmed the targeting of 3' untranslated region (3'-UTR) of HDAC8 through miR-216b-5p using a luciferase reporter assay. KEY FINDINGS: Our results demonstrated a significant decrease in miR-216b-5p, and remarkable increase in HDAC8 levels within human breast cancer tissues and cell lines. The lower levels of miR-216b-5p were negatively correlated with lymph node metastasis and advanced tumor size. The overexpression of miR-216b-5p in BC cell lines inhibited cellular proliferation and progression. HDAC8 was directly down-regulated by miR-216b-5p and knockout of HDAC8 showed the similar effects as miR-216b-5p overexpression. SIGNIFICANCE: Briefly, HDAC8 is an oncogene that accelerate breast cancer proliferation and progression and miR-216b-5p modulates those functions by binding to HDAC8 3'-UTR.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , MicroRNAs/genética , Proteínas Repressoras/metabolismo , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular , Regulação para Baixo , Feminino , Histona Desacetilases/genética , Humanos , Prognóstico , Proteínas Repressoras/genética , Células Tumorais Cultivadas
3.
Zhonghua Er Ke Za Zhi ; 57(10): 780-785, 2019 Oct 02.
Artigo em Chinês | MEDLINE | ID: mdl-31594065

RESUMO

Objective: To summarize the clinical and genetic characteristics of focal epilepsy in children caused by GATOR1 complex gene variation. Methods: The clinical data, gene variation and treatment outcome of 12 children with focal epilepsy caused by GATOR1 complex gene variation admitted to Beijing Children's Hospital Affiliated to Capital Medical University from June 2016 to October 2018 were retrospectively analyzed. Results: There were 7 males and 5 females in 12 cases. The epilepsy onset age was 5.5 (3.0, 12.0) months, and from 11 days to 16 months of age. The epileptic seizure types were all focal motor seizures, and one case combined with epileptic spasms. The frequency of seizures in all patients was more than one time per day. Seven cases had frontal lobe epilepsy and two cases had lateral temporal lobe epilepsy. One case had a family history of febrile seizures and two had a family history of suspicious epilepsy. Epileptic form discharges were observed in 9 patients during the interictal phase by electroencephalograms (EEG), and all of them were focal discharges. Eight cases had clinical seizures detected by EEG, in 4 of whom the seizures were originated in frontal region. There were no abnormalities in brain magnetic resonance imaging in 11 cases whereas 1 case had malformation of cortical development of left frontal lobe. Eight patients had DEPDC5 gene variation, one had NPRL2 gene variation, three had NPRL3 gene variation. One case had de novo variation and the other 11 had hereditary variation. There were 11 types of gene variation, including 5 nonsense variations, 3 missense variations, 2 frame shift variations and 1 in frame deletion variation. There was no clear relationship between the clinical phenotype and the genotype. During the follow-up period from 6 months to 2 years and 6 months, 6 cases had seizure control, 3 of them were controlled by oxcarbazepine. The other 6 cases had drug-refractory epilepsy, 2 of them failed with vagus nerve stimulation and ketogenic dietary therapy as well, meanwhile combined with mental retardation. Conclusions: GATOR1 complex gene variation can lead to genetic focal epilepsy, which usually has early onset with frequent seizures. Most of the patients have focal epileptic form discharges on EEG, and there is usually no structural lesion in brain imaging. Most of the patients have hereditary loss-of-function variations. Approximately half of cases are drug-resistant epilepsy.


Assuntos
Epilepsias Parciais/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Grupo com Ancestrais do Continente Asiático/genética , Eletroencefalografia , Epilepsias Parciais/diagnóstico , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Retrospectivos , Convulsões/complicações , Convulsões/genética
4.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(4): 520-526, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31642229

RESUMO

OBJECTIVE: To investigate the effect of nuclear receptor Rev-erbß knockout on proliferation and migration ability of human hepatocellular carcinoma cell line HepG2. METHODS: -The Rev-erbß gene knockout HepG2 cell line was abtained by CRISPR/Cas9 genome editing technique with specific DNA modification of the target gene. The Rev-erbß gene targeting vectors were co-transfected into HepG2 cells. Through cloning and screening, the Rev-erbß gene knockout HepG2 cell line was constructed, PCR, sequencing and Western blot methods were carried out for the identification of the Rev-erbß gene knockout HepG2 cell line. The expression level of tumor migration and invasion-associated gene in Rev-erbß gene knockout cell was determined by real-time quantitative PCR (qRT-PCR) and was compared with normal cell as control.MTT, cell scratch and Transwell experiments were conducted in order to explore the effect of Rev-erbß gene on HepG2 cell's ability of proliferation, migration and invasion. RESULTS: A Rev-erbß gene knockout monoclonal cell line, which was identified by PCR, sequencing and Western blot, was successfully constructed and named HepG2 C5 (Rev-erbß -/-). qRT-PCR results showed that Rev-erbß knockout resulted in up-regulation of matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9) and extracellular matrix protein-1 (ECM1) gene expression (P < 0.05) and down-regulation of E-cadherin (CDH1) gene expression (P=0.05).Results of MTT, cell scratch and transwell experiments showed that HepG2 C5 had stronger proliferation, migration and invasion ability than control cells (P < 0.05). CONCLUSION: Rev-erbß gene knockout could change the expression of migration and adhesion-associated genes in HepG2 cell, and then affect the proliferation, migration and invasion ability of HepG2 cells.


Assuntos
Movimento Celular , Proliferação de Células , Invasividade Neoplásica , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Células Hep G2 , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo
5.
Se Pu ; 37(8): 887-896, 2019 Aug 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642260

RESUMO

Speckle type BTB/POZ protein (SPOP) is one of the most frequently mutated protein in prostate cancer. In this study, proteomics and metabolomics were integrated to study the effects of SPOP mutation on metabolism. First, LNCaP control (CON), SPOP wild-type (SPOP_WT), and SPOP mutation (SPOP_Y87N and SPOP_F133L) cells were subjected to a metabolomics study. The metabolomics data of LNCaP CON, SPOP_WT, SPOP_Y87N, and SPOP_F133L cells were evaluated by partial least squares-discriminant analysis (PLS-DA). Four groups could be clearly differentiated with an explanation ability of R2X=0.512, R2Y=0.616 and predictive ability of Q2=0.475. Totally, 36 differential metabolites were defined with variable importance for the projection (VIP) value > 1. Then, the 36 metabolites were subjected to one-way ANOVA analysis. Fumaric acid, malic acid, citric acid, aspartic acid, and asparagine were increased in LNCaP SPOP mutation cells compared to that in LNCaP SPOP_WT cells. Using a proteomics study, 909 differential proteins were found in LNCaP SPOP_Y87N and SPOP_F133L cells. MetaboAnalyst 3.0 was used to enrich metabolic pathways by using differential metabolites. KOBAS 3.0 was used to enrich metabolic pathways by using differential proteins. Both metabolomics and proteomics analysis showed that the tricarboxylic acid (TCA) cycle and aminoacyl-tRNA biosynthesis were significantly changed. To validate these findings, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was performed in Du145 SPOP knock-out cells. The results indicated that the TCA cycle was activated in Du145 SPOP knock-out cells. Collectively, this study found that SPOP mutation significantly promoted TCA cycle in prostate cancer cells.


Assuntos
Domínio BTB-POZ , Metabolômica , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Proteômica , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Humanos , Masculino , Redes e Vias Metabólicas , Mutação
6.
J Agric Food Chem ; 67(42): 11815-11824, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31550160

RESUMO

Yan73 is a teinturier Vitis vinifera variety with red berry flesh, but the molecular mechanisms underlying its flesh coloration remain unclear. We analyzed the flavonoid metabolic and transcriptome profiles of Yan73 berry red and white flesh using HPLC-ESI-MS/MS and RNA-sequencing technologies. Anthocyanins are the main flavonoids responsible for Yan73 berry flesh color, and the coloration is coordinately regulated by the VvMYBA1 transcriptional activator and VvMYBC2-L1 transcriptional repressor. Furthermore, yeast one- and two-hybrid, dual luciferase, and bimolecular fluorescence complementation assays suggested that VvMYBA1 positively regulates Yan73 berry flesh color via interactions with VvWDR1 and the activation of the VvCHI3, VvOMT, and VvGST4 promoters, whereas VvMYBC2-L1 negatively regulates Yan73 berry flesh color, possibly by competing with the R2R3-MYB transcriptional activators for bHLH partners or by repressing VvOMT and VvGST4 expression. Our findings provide new insights into the molecular mechanisms regulating grape flesh color.


Assuntos
Frutas/química , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas Repressoras/genética , Vitis/genética , Cor , Frutas/genética , Frutas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vitis/química , Vitis/metabolismo
7.
Arch Virol ; 164(12): 2953-2961, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31552532

RESUMO

Human papillomavirus genotype 16 (HPV16) is the most frequent high-risk HPV (HR-HPV) identified in cervical precursor lesions and cervical cancer (CC) worldwide. The oncogenic potential of HPV16 is partly dependent on the lineage involved in the infection and the presence of clinically relevant mutations. In this report, we present the distribution of HR-HPV and the mutational profile and intra-host variability of HPV16 lineages, based on analysis of the long control region (LCR) and the E6 gene in samples with normal cytology (n = 39), squamous intraepithelial lesions (n = 25), and CC (n = 39). HR-HPV genotyping was performed using multiplex real-time PCR. HPV16 lineage assignments and mutation frequencies were determined by conventional PCR and Sanger DNA sequencing, and intra-patient viral populations were analyzed using next-generation sequencing (NGS). The most frequent HR-HPV type was HPV16, followed by HPV31 and HPV18. The frequency of HPV16 sublineages was A1/A2 > D2 > D3 and B1. Moreover, the most frequent mutations, both in samples from this study and in the available sequences from Mexican isolates in the GenBank database were LCR-G7518A, which is involved in carcinogenesis, and E6-T350G (producing L83V), associated with persistence of infection. Otherwise, deep sequencing revealed high conservation of viral lineages and mutations, independently of the stages studied. In conclusion, the high frequency and stability of these molecular markers, as well as the circulating viral lineages, could be related to the incidence of CC associated with HPV16. Hence, they deserve a broader analysis to determine the risk of specific populations for progression of the disease.


Assuntos
Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Sequências Repetidas Terminais , Neoplasias do Colo do Útero/virologia , Adulto , Sequência de Bases , Feminino , Regulação Viral da Expressão Gênica , Papillomavirus Humano 16/classificação , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 16/metabolismo , Humanos , México , Mutação , Proteínas Oncogênicas Virais/metabolismo , Filogenia , Proteínas Repressoras/metabolismo , Estudos Retrospectivos
8.
Chem Biol Interact ; 313: 108818, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494106

RESUMO

Diabetic nephropathy (DN) is a common complication of diabetes that remains the major cause of end-stage renal disease (ESRD). Forkhead box P1 (FOXP1) is a member of FOX family involved in the progression of diabetes. However, the pathogenic role of FOXP1 in DN remains unclear. This study was aimed to explore the effects of FOXP1 on glomerular mesangial cells (MCs) in response to high glucose (HG) stimulation. We found that HG stimulation markedly inhibited the FOXP1 expression in MCs in dose-and time-dependent manner. CCK-8 assay proved that FOXP1 overexpression attenuated HG-induced cell proliferation in MCs. FOXP1 exhibited anti-oxidative activity in HG-induced MCs, as proved by the decreased production of ROS and expressions of ROS producing enzymes, NADPH oxidase (NOX) 2 and NOX4. Besides, FOXP1 suppressed the expression and secretion of extracellular matrix (ECM) proteins including collagen IV (Col IV) and fibronectin (FN). Furthermore, FOXP1 overexpression significantly prevented HG-induced activation of Akt/mTOR signaling in MCs, and Akt activator blocked FOXP1-mediated cell proliferation, ROS production and ECM accumulation in MCs. Collectively, FOXP1 prevented HG-induced proliferation, oxidative stress, and ECM accumulation in MCs via inhibiting the activation of Akt/mTOR signaling pathway. The findings suggested that FOXP1 might be a therapeutic target for the treatment of DN.


Assuntos
Matriz Extracelular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Células Mesangiais/metabolismo , Proteínas Repressoras/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Relação Dose-Resposta a Droga , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/metabolismo , Fatores de Transcrição Forkhead/genética , Glucose/administração & dosagem , Glucose/farmacologia , Humanos , Células Mesangiais/efeitos dos fármacos , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Serina-Treonina Quinases TOR/metabolismo
9.
BMC Infect Dis ; 19(1): 802, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31510934

RESUMO

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is a serious global health problem. Persistence of the virus occurs as a result of stability of the replication intermediate comprising covalently closed circular DNA (cccDNA). Development of drugs that are capable of disabling this cccDNA is vital. METHODS: To investigate an epigenetic approach to inactivating viral DNA, we engineered transcriptional repressors that comprise an HBV DNA-binding domain of transcription activator like effectors (TALEs) and a fused Krüppel Associated Box (KRAB). These repressor TALEs (rTALEs) targeted the viral surface open reading frame and were placed under transcription control of constitutively active or liver-specific promoters. RESULTS: Evaluation in cultured cells and following hydrodynamic injection of mice revealed that the rTALEs significantly inhibited production of markers of HBV replication without evidence of hepatotoxicity. Increased methylation of HBV DNA at CpG island II showed that the rTALEs caused intended epigenetic modification. CONCLUSIONS: Epigenetic modification of HBV DNA is a new and effective means of inactivating the virus in vivo. The approach has therapeutic potential and avoids potentially problematic unintended mutagenesis of gene editing.


Assuntos
DNA Viral/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/genética , Hepatite B/terapia , Hepatite B/virologia , Proteínas Repressoras/metabolismo , Replicação Viral/genética , Animais , Linhagem Celular , Ilhas de CpG , Metilação de DNA , DNA Circular/genética , DNA Viral/biossíntese , Epigênese Genética , Feminino , Fígado/metabolismo , Fígado/virologia , Camundongos , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética
10.
Nucleic Acids Res ; 47(18): 9696-9707, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400115

RESUMO

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk-, with the complexes failing to associate with chromosomes.


Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Escherichia coli/genética , Proteínas Repressoras/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos/química , Cromossomos/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Ligação Proteica , Proteínas Repressoras/química
11.
Gene ; 717: 143998, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31381951

RESUMO

Eid1 is a member of the EID protein family, which regulates differentiation, transcription and acetyltransferase activity. Accumulating evidence suggests that Eid1 is relevant to neurological disorder, but the main function of Eid1 is still unclear, especially in the brain. To better understand this issue, we generated Eid1-knockout (Eid1-KO) mice and profiled its gene expression changes in the brain by RNA sequencing. This study identified 2531 genes differentially expressed in Eid1-KO mice compared with the wild-type, then qRT-PCR verification demonstrated that the transcriptomic data are reliable. By protein-protein interaction cluster analysis, 'regulation of cell proliferation' were unexpectedly discovered as important Eid1 functions. We then isolated neural progenitor cells (NPCs) and showed that the number of neurospheres and the proliferation rate of Eid1-KO NPCs were obviously lower than that in the control group, furthermore, CCK-8 and immunofluorescence assay clearly demonstrated that the Eid1-KO NPCs showed significantly less cell proliferation than the control group. To the best of our knowledge, this is the first comprehensive report of the Eid1-KO transcriptome of mice brain. Our analysis and experimental data provide a foundation for further studies on understanding function of Eid1 in the brain.


Assuntos
Encéfalo/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Proliferação de Células/genética , Feminino , Perfilação da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Gravidez , Mapas de Interação de Proteínas , Análise de Sequência de RNA
12.
BMC Med Genet ; 20(1): 134, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31382906

RESUMO

BACKGROUND: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene syndrome caused by partial 4p deletion highly variable in size in individual patients. The core WHS phenotype is defined by the association of growth delay, typical facial characteristics, intellectual disability and seizures. The WHS critical region (WHSCR) has been narrowed down and NSD2 falls within this 200 kb region. Only four patients with NSD2 variants have been documented with phenotypic features in detail. CASE PRESENTATION: Herein, we report the case of a 12-year-old boy with developmental delay. He had dysmorphic facial features including wide-spaced eyes, prominent nasal bridge continuing to forehead, abnormal teething and micrognathia. He also had mild clinodactyly of both hands. Using whole-exome sequencing, we identified a pathogenic mutation in NSD2 [c.4029_4030insAA, p.Glu1344Lysfs*49] isolated from peripheral blood DNA. Sanger confirmation of this variant revealed it as a de novo truncating variant in the family. CONCLUSION: Here, we reported a boy with de novo truncating variant in NSD2 with atypical clinical features comparing with 4p16.3 deletion related WHS. Our finding further supported the pathogenesis of truncating variants in NSD2 and delineated the possible symptom spectrum caused by these variants.


Assuntos
Predisposição Genética para Doença/genética , Histona-Lisina N-Metiltransferase/genética , Fenótipo , Proteínas Repressoras/genética , Síndrome de Wolf-Hirschhorn/genética , Sequência de Bases , Criança , Cromossomos Humanos Par 4 , DNA/sangue , Deficiências do Desenvolvimento/genética , Humanos , Deficiência Intelectual/genética , Masculino , Convulsões/genética , Sequenciamento Completo do Exoma , Síndrome de Wolf-Hirschhorn/fisiopatologia
13.
Nucleic Acids Res ; 47(16): 8913-8925, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392336

RESUMO

The development of synthetic biological systems requires modular biomolecular components to flexibly alter response pathways. In previous studies, we have established a module-swapping design principle to engineer allosteric response and DNA recognition properties among regulators in the LacI family, in which the engineered regulators served as effective components for implementing new cellular behavior. Here we introduced this protein engineering strategy to two regulators in the TetR family: TetR (UniProt Accession ID: P04483) and MphR (Q9EVJ6). The TetR DNA-binding module and the MphR ligand-binding module were used to create the TetR-MphR. This resulting hybrid regulator possesses DNA-binding properties of TetR and ligand response properties of MphR, which is able to control gene expression in response to a molecular signal in cells. Furthermore, we studied molecular interactions between the TetR DNA-binding module and MphR ligand-binding module by using mutant analysis. Together, we demonstrated that TetR family regulators contain discrete and functional modules that can be used to build biological components with novel properties. This work highlights the utility of rational design as a means of creating modular parts for cell engineering and introduces new possibilities in rewiring cellular response pathways.


Assuntos
DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Regulação Alostérica , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Rinsho Ketsueki ; 60(6): 680-690, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31281161

RESUMO

Recent progress in whole genome sequencing has identified recurrent somatic mutations in the additional sex combs like 1 (ASXL1) gene in a variety of hematological disorders and even in premalignant conditions. However, the molecular mechanisms regarding the contribution of ASXL1 mutation to the pathogenesis of premalignant conditions remain largely unelucidated. Thus, we investigated the biological effects of mutant Asxl1 using newly-generated knock-in (KI) mice. Heterozygous mutant KI mice developed phenotypes resembling human low-risk myelodysplastic syndromes (MDS), and some of them developed an MDS/myeloproliferative neoplasm-like disease after a long latency. The H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in KI hematopoietic stem cells (HSCs), suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutant Asxl1. The mutant Asxl1 failed to interact with Bmi1, although wild type ASXL1 protein did not. When p16Ink4a expression was depleted in Asxl1 KI mice, the HSC pool was restored, and apoptosis was ameliorated in HSCs. These findings demonstrate that the loss of protein interaction between mutant Asxl1 and Bmi1 affected the activity of Prc1. The subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in low-risk MDS-like phenotypes in heterozygous Asxl1 KI mice.


Assuntos
Mutação , Síndromes Mielodisplásicas/genética , Proteínas Repressoras/genética , Animais , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Células-Tronco Hematopoéticas , Histonas/metabolismo , Camundongos , Fenótipo , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Ubiquitinação
15.
Nucleic Acids Res ; 47(16): 8548-8562, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31276581

RESUMO

Cockayne syndrome is an accelerated aging disorder, caused by mutations in the CSA or CSB genes. In CSB-deficient cells, poly (ADP ribose) polymerase (PARP) is persistently activated by unrepaired DNA damage and consumes and depletes cellular nicotinamide adenine dinucleotide, which leads to mitochondrial dysfunction. Here, the distribution of poly (ADP ribose) (PAR) was determined in CSB-deficient cells using ADPr-ChAP (ADP ribose-chromatin affinity purification), and the results show striking enrichment of PAR at transcription start sites, depletion of heterochromatin and downregulation of H3K9me3-specific methyltransferases SUV39H1 and SETDB1. Induced-expression of SETDB1 in CSB-deficient cells downregulated PAR and normalized mitochondrial function. The results suggest that defects in CSB are strongly associated with loss of heterochromatin, downregulation of SETDB1, increased PAR in highly-transcribed regions, and mitochondrial dysfunction.


Assuntos
Senescência Celular/genética , Síndrome de Cockayne/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Histonas/genética , Mitocôndrias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Metiltransferases de Proteína/genética , Fatores de Transcrição/genética , Linhagem Celular Transformada , Cromatina/química , Cromatina/metabolismo , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/patologia , Mutação , NAD/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Metiltransferases de Proteína/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Genética
16.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(7): 672-675, 2019 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-31302908

RESUMO

OBJECTIVE: To explore the genetic basis for three patients with development delay and to correlate their clinical phenotypes with genetic findings. METHODS: The karyotypes of the probands and their parents were analyzed by conventional G-banding. Chromosomal microarray analysis (CMA) was used to detect microdeletion and microduplication. RESULTS: No kartotypic abnormality was detected in the patients and their parents. CMA analysis identified a de novo 3.10 Mb deletion on chromosome 15q24.1q24.2 in case 1, a de novo 3.14 Mb deletion at 15q24.1q24.2 in case 2, and a 3.13 Mb deletion at 15q24.1q24.2 in case 3. All deletions have encompassed the CPLX3,SEMA7A and SIN3A genes. CONCLUSION: The three patients were diagnosed with 15q24 microdeletion syndrome. CPLX3,SEMA7A and SIN3A may be the key genes responsible for this syndrome.


Assuntos
Transtornos Cromossômicos/genética , Deficiência Intelectual/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Antígenos CD/genética , Criança , Deleção Cromossômica , Cromossomos Humanos Par 15/genética , Proteínas Ligadas por GPI/genética , Humanos , Proteínas Repressoras/genética , Semaforinas/genética
17.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(7): 720-723, 2019 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-31302920

RESUMO

OBJECTIVE: To explore the genetic cause of a neonate with congenital dysplasia, growth retardation through clinical evaluation, laboratory tests and next generation sequencing (NGS). METHODS: Peripheral blood samples were obtained from the child and his parents. Whole genomic DNA was extracted and subjected to NGS. Suspected mutation was predicted by bioinformatic tools and validated by Sanger sequencing. RESULTS: The child was found to carry a c.556G>A (p.E186K) mutation of the HDAC8 gene on the X chromosome, which was predicted to be pathogenic by Bioinformatic analysis. CONCLUSION: The patient was diagnosed as Cornelia de Lange syndrome 5 caused by the c.556G>A mutation of the HDAC8 gene.


Assuntos
Síndrome de Lange/genética , Histona Desacetilases/genética , Proteínas Repressoras/genética , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Masculino , Mutação
18.
Nat Commun ; 10(1): 2835, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249377

RESUMO

During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here, we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Proteínas Repressoras/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos/embriologia , Camundongos/metabolismo , Camundongos Endogâmicos ICR , Camundongos Knockout , Células-Tronco Neurais/química , Células-Tronco Neurais/citologia , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Célula Única
19.
Leukemia ; 33(8): 1835-1850, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31209280

RESUMO

Outcomes for patients with chronic myeloid leukemia (CML) have substantially improved due to advances in drug development and rational treatment intervention strategies. Despite these significant advances there are still unanswered questions on patient management regarding how to more reliably predict treatment failure at the time of diagnosis and how to select frontline tyrosine kinase inhibitor (TKI) therapy for optimal outcome. The BCR-ABL1 transcript level at diagnosis has no established prognostic impact and cannot guide frontline TKI selection. BCR-ABL1 mutations are detected in ~50% of TKI resistant patients but are rarely responsible for primary resistance. Other resistance mechanisms are largely uncharacterized and there are no other routine molecular testing strategies to facilitate the evaluation and further stratification of TKI resistance. Advances in next-generation sequencing technology has aided the management of a growing number of other malignancies, enabling the incorporation of somatic mutation profiles in diagnosis, classification, and prognostication. A largely unexplored area in CML research is whether expanded genomic analysis at diagnosis, resistance, and disease transformation can enhance patient management decisions, as has occurred for other cancers. The aim of this article is to review publications that reported mutated cancer-associated genes in CML patients at various disease phases. We discuss the frequency and type of such variants at initial diagnosis and at the time of treatment failure and transformation. Current limitations in the evaluation of mutants and recommendations for future reporting are outlined. The collective evaluation of mutational studies over more than a decade suggests a limited set of cancer-associated genes are indeed recurrently mutated in CML and some at a relatively high frequency. Genomic studies have the potential to lay the foundation for improved diagnostic risk classification according to clinical and genomic risk, and to enable more precise early identification of TKI resistance.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Genes Neoplásicos , Hematopoese , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/etiologia , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Repressoras/genética , Medição de Risco
20.
PLoS Pathog ; 15(6): e1007866, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31188899

RESUMO

The gastric lamina propria of mice that have been experimentally infected with the pathobiont Helicobacter pylori hosts a dense network of myeloid cells that includes BATF3-dependent CD103+ dendritic cells (DCs). We show here that CD103+ DCs are strictly required for gastric Th1 responses to H. pylori and for H. pylori infection control. A similar dependence of type 1 immunity on CD103+ DCs is observed in a Mycobacterium bovis BCG infection model, and in a syngeneic colon cancer model. Strikingly, we find that not only the expansion and/or recruitment of Th1 cells, but also of peripherally induced, neuropilin-negative regulatory T-cells to sites of infection requires BATF3-dependent DCs. A shared feature of the examined models is the strongly reduced production of the chemokines and CXCR3 ligands CXCL9, 10 and 11 in BATF3-deficient mice. The results implicate BATF3-dependent DCs in the recruitment of CXCR3+ effector and regulatory T-cells to target tissues and in their local expansion.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Células Dendríticas/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Mycobacterium bovis/imunologia , Proteínas Repressoras/imunologia , Linfócitos T Reguladores/imunologia , Tuberculose/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular Tumoral , Quimiocinas CXC/genética , Quimiocinas CXC/imunologia , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Camundongos , Camundongos Knockout , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Proteínas Repressoras/genética , Linfócitos T Reguladores/microbiologia , Linfócitos T Reguladores/patologia , Tuberculose/genética , Tuberculose/patologia
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