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1.
Hum Mol Genet ; 30(6): 467-484, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33693649

RESUMO

Isolated Microspherophakia (MSP) is an autosomal recessive disorder characterized by a smaller than normal spherical lens. Till date, LTBP2 is the only gene shown to cause MSP. We used homozygosity mapping and whole-exome sequencing and identified a homozygous mutation, c.1148C > T (p.Pro383Leu), in the WDR8 (or WRAP73) gene in two Indian MSP families. In vitro experiments showed that the missense mutation renders the protein unstable. WDR8 is a centriolar protein that has important roles in centrosomal assembly, spindle pole formation and ciliogenesis. Co-immunoprecipitation experiments from HeLa cells indicated that the mutation interferes with the interaction of WDR8 with its binding partners. In zebrafish, both morpholino-mediated knockdown and CRISPR/Cas knockout of wdr8 resulted in decreased eye and lens size. The lack of wdr8 affected cell cycle progression in the retinal cells, causing a reduction in cell numbers in the retina and lens. The reduction in eye size and the cell cycle defects were rescued by exogenous expression of the human wild-type WDR8. However, the human mutant WDR8 (p.Pro383Leu) was unable to rescue the eye defects, indicating that the missense mutation abrogates WDR8 protein function. Thus, our zebrafish results suggested that WDR8 is the causative gene for MSP in these Indian families.


Assuntos
Doenças da Córnea/patologia , Ectopia do Cristalino/patologia , Sequenciamento do Exoma/métodos , Exoma , Glaucoma/patologia , Iris/anormalidades , Mutação , Proteínas/genética , Adulto , Animais , Criança , Doenças da Córnea/etiologia , Doenças da Córnea/metabolismo , Ectopia do Cristalino/etiologia , Ectopia do Cristalino/metabolismo , Feminino , Glaucoma/etiologia , Glaucoma/metabolismo , Células HeLa , Humanos , Índia , Iris/metabolismo , Iris/patologia , Masculino , Linhagem , Proteínas/metabolismo , Adulto Jovem , Peixe-Zebra
2.
Nat Commun ; 10(1): 2861, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253795

RESUMO

Centromeres provide a pivotal function for faithful chromosome segregation. They serve as a foundation for the assembly of the kinetochore complex and spindle connection, which is essential for chromosome biorientation. Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment. Here, we reveal a previously undescribed mechanism named 'centromere disintegration' that drives chromosome misalignment in PLK1-inactivated cells. We find that PLK1 inhibition does not necessarily compromise metaphase establishment, but instead its maintenance. We demonstrate that this is caused by unlawful unwinding of DNA by BLM helicase at a specific centromere domain underneath kinetochores. Under bipolar spindle pulling, the distorted centromeres are promptly decompacted into DNA threadlike molecules, leading to centromere rupture and whole-chromosome arm splitting. Consequently, chromosome alignment collapses. Our study unveils an unexpected role of PLK1 as a chromosome guardian to maintain centromere integrity for chromosome biorientation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/fisiologia , Linhagem Celular , Pareamento Cromossômico/fisiologia , Humanos , Cinetocoros , Interferência de RNA , Timidina/farmacologia , Quinase 1 Polo-Like
4.
Sci Rep ; 8(1): 17120, 2018 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451952

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

5.
Nat Commun ; 9(1): 677, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29445165

RESUMO

Chromosome missegregation acts as one of the driving forces for chromosome instability and cancer development. Here, we find that in human cancer cells, HeLa and U2OS, depletion of 53BP1 (p53-binding protein 1) exacerbates chromosome non-disjunction resulting from a new type of sister-chromatid intertwinement, which is distinct from FANCD2-associated ultrafine DNA bridges (UFBs) induced by replication stress. Importantly, the sister DNA intertwinements trigger gross chromosomal rearrangements through a distinct process, named sister-chromatid rupture and bridging. In contrast to conventional anaphase bridge-breakage models, we demonstrate that chromatid axes of the intertwined sister-chromatids rupture prior to the breakage of the DNA bridges. Consequently, the ruptured sister arms remain tethered and cause signature chromosome rearrangements, including whole-arm (Robertsonian-like) translocation/deletion and isochromosome formation. Therefore, our study reveals a hitherto unreported chromatid damage phenomenon mediated by sister DNA intertwinements that may help to explain the development of complex karyotypes in tumour cells.


Assuntos
Cromátides/genética , Quebras de DNA , Troca de Cromátide Irmã/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Instabilidade Cromossômica , Aberrações Cromossômicas , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Humanos , Neoplasias/genética , Neoplasias/patologia , Fase S/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
6.
Hum Mol Genet ; 26(6): 1104-1114, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28087737

RESUMO

Anencephaly (APH) is characterized by the absence of brain tissues and cranium. During primary neurulation stage of the embryo, the rostral part of the neural pore fails to close, leading to APH. APH shows a heterogeneous etiology, ranging from environmental to genetic causes. The autosomal recessive inheritance of APH has been reported in several populations. In this study, we employed whole-exome sequencing and identified a homozygous missense mutation c.1522C > A (p.Pro508Thr) in the TRIM36 gene as the cause of autosomal recessive APH in an Indian family. The TRIM36 gene is expressed in the developing brain, suggesting a role in neurogenesis. In silico analysis showed that proline at codon position 508 is highly conserved in 26 vertebrate species, and the mutation is predicted to affect the conformation of the B30.2/SPRY domain of TRIM36. Both in vitro and in vivo results showed that the mutation renders the TRIM36 protein less stable. TRIM36 is known to associate with microtubules. Transient expression of the mutant TRIM36 in HeLa and LN229 cells resulted in microtubule disruption, disorganized spindles, loosely arranged chromosomes, multiple spindles, abnormal cytokinesis, reduced cell proliferation and increased apoptosis as compared with cells transfected with its wild-type counterpart. The siRNA knock down of TRIM36 in HeLa and LN229 cells also led to reduced cell proliferation and increased apoptosis. We suggest that microtubule disruption and disorganized spindles mediated by mutant TRIM36 affect neural cell proliferation during neural tube formation, leading to APH.


Assuntos
Anencefalia/epidemiologia , Anencefalia/genética , Proteínas de Transporte/genética , Mutação/genética , Anencefalia/fisiopatologia , Exoma/genética , Feminino , Feto , Homozigoto , Humanos , Índia/epidemiologia , Masculino , Linhagem
7.
Sci Rep ; 5: 17621, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26639757

RESUMO

The ESRRA gene encodes a transcription factor and regulates several genes, such as WNT11 and OPN, involved in tumorigenesis. It is upregulated in several cancers, including OSCC. We have previously shown that the tumor suppressor miR-125a targets ESRRA, and its downregulation causes upregulation of ESRRA in OSCC. Upregulation of ESRRA in the absence of downregulation of miR-125a in a subset of OSCC samples suggests the involvement of an alternative mechanism. Using TaqMan(®) copy number assay, here we report for the first time that the genomic amplification of ESRRA causes its upregulation in a subset of OSCC samples. Ectopic overexpression of ESRRA led to accelerated cell proliferation, anchorage-independent cell growth and invasion, and inhibited apoptosis. Whereas, knockdown of ESRRA expression by siRNA led to reduced cell proliferation, anchorage-independent cell growth and invasion, and accelerated apoptosis. Furthermore, the delivery of a synthetic biostable ESRRA siRNA to OSCC cells resulted in regression of xenografts in nude mice. Thus, the genomic amplification of ESRRA is another novel mechanism for its upregulation in OSCC. Based on our in vitro and in vivo experiments, we suggest that targeting ESRRA by siRNA could be a novel therapeutic strategy for OSCC and other cancers.


Assuntos
Carcinoma de Células Escamosas/genética , Amplificação de Genes , Neoplasias Bucais/genética , Receptores de Estrogênio/genética , Regulação para Cima/genética , Apoptose/genética , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genômica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Bucais/patologia , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo , Transcrição Gênica , Receptor ERRalfa Relacionado ao Estrogênio
8.
J Biol Chem ; 289(46): 32276-32290, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25266720

RESUMO

Estrogen-related receptor α (ESRRA) functions as a transcription factor and regulates the expression of several genes, such as WNT11 and OPN. Up-regulation of ESRRA has been reported in several cancers. However, the mechanism underlying its up-regulation is unclear. Furthermore, the reports regarding the role and regulation of ESRRA in oral squamous cell carcinoma (OSCC) are completely lacking. Here, we show that tumor suppressor miR-125a directly binds to the 3'UTR of ESRRA and represses its expression. Overexpression of miR-125a in OSCC cells drastically reduced the level of ESRRA, decreased cell proliferation, and increased apoptosis. Conversely, the delivery of an miR-125a inhibitor to these cells drastically increased the level of ESRRA, increased cell proliferation, and decreased apoptosis. miR-125a-mediated down-regulation of ESRRA impaired anchorage-independent colony formation and invasion of OSCC cells. Reduced cell proliferation and increased apoptosis of OSCC cells were dependent on the presence of the 3'UTR in ESRRA. The delivery of an miR-125a mimic to OSCC cells resulted in marked regression of xenografts in nude mice, whereas the delivery of an miR-125a inhibitor to OSCC cells resulted in a significant increase of xenografts and abrogated the tumor suppressor function of miR-125a. We observed an inverse correlation between the expression levels of miR-125a and ESRRA in OSCC samples. In summary, up-regulation of ESRRA due to down-regulation of miR-125a is not only a novel mechanism for its up-regulation in OSCC, but decreasing the level of ESRRA by using a synthetic miR-125a mimic may have an important role in therapeutic intervention of OSCC and other cancers.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Neoplasias Bucais/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Azacitidina/análogos & derivados , Azacitidina/química , Linhagem Celular Tumoral , Proliferação de Células , Decitabina , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Invasividade Neoplásica , Transplante de Neoplasias , Plasmídeos/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos
9.
Nucleic Acids Res ; 42(10): 6243-55, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24748662

RESUMO

The TSC2 gene, mutated in patients with tuberous sclerosis complex (TSC), encodes a 200 kDa protein TSC2 (tuberin). The importance of TSC2 in the regulation of cell growth and proliferation is irrefutable. TSC2 in complex with TSC1 negatively regulates the mTOR complex 1 (mTORC1) via RHEB in the PI3K-AKT-mTOR pathway and in turn regulates cell proliferation. It shows nuclear as well as cytoplasmic localization. However, its nuclear function remains elusive. In order to identify the nuclear function of TSC2, a whole-genome expression profiling of TSC2 overexpressing cells was performed, and the results showed differential regulation of 266 genes. Interestingly, transcription was found to be the most populated functional category. EREG (Epiregulin), a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in TSC lesions, making its regulatory aspects intriguing. Using the luciferase reporter, ChIP and EMSA techniques, we show that TSC2 binds to the EREG promoter between -352 bp and -303 bp and negatively regulates its expression. This is the first evidence for the role of TSC2 as a transcription factor and of TSC2 binding to the promoter of any gene.


Assuntos
Fator de Crescimento Epidérmico/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/biossíntese , Epirregulina , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Anotação de Sequência Molecular , Complexos Multiproteicos/metabolismo , Sinais de Localização Nuclear , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/química
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