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1.
PLoS Genet ; 12(6): e1006129, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27352031

RESUMO

Mutations in the human MECP2 gene cause Rett syndrome (RTT), a severe neurodevelopmental disorder that predominantly affects girls. Despite decades of work, the molecular function of MeCP2 is not fully understood. Here we report a systematic identification of MeCP2-interacting proteins in the mouse brain. In addition to transcription regulators, we found that MeCP2 physically interacts with several modulators of RNA splicing, including LEDGF and DHX9. These interactions are disrupted by RTT causing mutations, suggesting that they may play a role in RTT pathogenesis. Consistent with the idea, deep RNA sequencing revealed misregulation of hundreds of splicing events in the cortex of Mecp2 knockout mice. To reveal the functional consequence of altered RNA splicing due to the loss of MeCP2, we focused on the regulation of the splicing of the flip/flop exon of Gria2 and other AMPAR genes. We found a significant splicing shift in the flip/flop exon toward the flop inclusion, leading to a faster decay in the AMPAR gated current and altered synaptic transmission. In summary, our study identified direct physical interaction between MeCP2 and splicing factors, a novel MeCP2 target gene, and established functional connection between a specific RNA splicing change and synaptic phenotypes in RTT mice. These results not only help our understanding of the molecular function of MeCP2, but also reveal potential drug targets for future therapies.


Assuntos
Processamento Alternativo/genética , Síndrome de Rett/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Éxons/genética , Feminino , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Knockout , Mutação/genética , Fenótipo , RNA/genética
2.
Nucleic Acids Res ; 40(14): 6725-40, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22561374

RESUMO

The transcription factor (TF) SOX2 is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition to its normal stem cell function, SOX2 over-expression is associated with cancer development. The ability to selectively target this and other oncogenic TFs in cells, however, remains a significant challenge due to the 'undruggable' characteristics of these molecules. Here, we employ a zinc finger (ZF)-based artificial TF (ATF) approach to selectively suppress SOX2 gene expression in cancer cells. We engineered four different proteins each composed of 6ZF arrays designed to bind 18 bp sites in the SOX2 promoter and enhancer region, which controls SOX2 methylation. The 6ZF domains were linked to the Kruppel Associated Box (SKD) repressor domain. Three engineered proteins were able to bind their endogenous target sites and effectively suppress SOX2 expression (up to 95% repression efficiencies) in breast cancer cells. Targeted down-regulation of SOX2 expression resulted in decreased tumor cell proliferation and colony formation in these cells. Furthermore, induced expression of an ATF in a mouse model inhibited breast cancer cell growth. Collectively, these findings demonstrate the effectiveness and therapeutic potential of engineered ATFs to mediate potent and long-lasting down-regulation of oncogenic TF expression in cancer cells.


Assuntos
Neoplasias da Mama/terapia , Inativação Gênica , Fatores de Transcrição SOXB1/antagonistas & inibidores , Fatores de Transcrição/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , DNA/metabolismo , Feminino , Humanos , Camundongos , Engenharia de Proteínas , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Biol Chem ; 287(35): 29873-86, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22782891

RESUMO

Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Experimentais/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/biossíntese , Dedos de Zinco , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/farmacologia , Serpinas/biossíntese , Tetraspaninas/genética , Tetraspaninas/metabolismo , Fatores de Transcrição/genética , Transcriptoma
4.
Breast Cancer Res ; 13(5): R94, 2011 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-21952072

RESUMO

INTRODUCTION: Tumor-initiating cells (TIC) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. The isolation of TICs has been limited by the scarcity of this population in the tissue of origin and because the molecular signatures that characterize these cells are not well understood. Herein, we describe the generation of TIC-like cell lines by ectopic expression of the OCT4 transcription factor (TF) in primary breast cell preparations. METHODS: OCT4 cDNA was over-expressed in four different primary human mammary epithelial (HMEC) breast cell preparations from reduction mammoplasty donors. OCT4-transduced breast cells (OTBCs) generated colonies (frequency ~0.01%) in self-renewal conditions (feeder cultures in human embryonic stem cell media). Differentiation assays, immunofluorescence, immunohistochemistry, and flow cytometry were performed to investigate the cell of origin of OTBCs. Serial dilutions of OTBCs were injected in nude mice to address their tumorigenic capabilities. Gene expression microarrays were performed in OTBCs, and the role of downstream targets of OCT4 in maintaining self-renewal was investigated by knock-down experiments. RESULTS: OTBCs overcame senescence, overexpressed telomerase, and down-regulated p16INK4A. In differentiation conditions, OTBCs generated populations of both myoepithelial and luminal cells at low frequency, suggesting that the cell of origin of some OTBCs was a bi-potent stem cell. Injection of OTBCs in nude mice generated poorly differentiated breast carcinomas with colonization capabilities. Gene expression microarrays of OTBC lines revealed a gene signature that was over-represented in the claudin-low molecular subtype of breast cancer. Lastly, siRNA-mediated knockdown of OCT4 or downstream embryonic targets of OCT4, such as NANOG and ZIC1, suppressed the ability of OTBCs to self-renew. CONCLUSIONS: Transduction of OCT4 in normal breast preparations led to the generation of cell lines possessing tumor-initiating and colonization capabilities. These cells developed high-grade, poorly differentiated breast carcinomas in nude mice. Genome-wide analysis of OTBCs outlined an embryonic TF circuitry that could be operative in TICs, resulting in up-regulation of oncogenes and loss of tumor suppressive functions. These OTBCs represent a patient-specific model system for the discovery of novel oncogenic targets in claudin-low tumors.


Assuntos
Transformação Celular Neoplásica/patologia , Glândulas Mamárias Humanas/patologia , Fator 3 de Transcrição de Octâmero/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma/genética , Carcinoma/patologia , Diferenciação Celular/genética , Linhagem da Célula , Transformação Celular Neoplásica/genética , Senescência Celular/genética , Células Epiteliais , Feminino , Regulação da Expressão Gênica , Genes Supressores de Tumor , Proteínas de Homeodomínio/genética , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Mesoderma/fisiologia , Camundongos , Camundongos Nus , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno , Fatores de Transcrição SOXB1/genética , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética , Células Tumorais Cultivadas
5.
Tuberculosis (Edinb) ; 87(6): 489-97, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17881291

RESUMO

Mycobacterium tuberculosis CDC 1551, a highly immunogenic outbreak strain, previously reported to have unique surface distribution of capsular polysaccharide, was used to generate novel monoclonal antibodies (mabs) to surface mycobacterial targets. Two immunoglobulin G1 (IgG1) mAbs, 16a1 and 16a6 were generated. The mAbs originated from the same B cell, bound strongly to whole cell M. tuberculosis CDC1551 and to its cell wall, membrane and cytosol fractions recognizing a 90kDa protein. Immunoprecipitation using mAb 16a1 isolated a protein with amino acid peptide sequences matching MPT51 from the cytosol. This immunogenic protein of unknown function was previously reported only in culture filtrates of M. tuberculosis. Our findings suggest for the first time that this protein is found within the M. tuberculosis cell.


Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Mycobacterium tuberculosis/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Aderência Bacteriana/imunologia , Proteínas de Bactérias/imunologia , Citosol/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Imunoeletrônica , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestrutura
6.
Epigenetics ; 7(4): 350-60, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22419067

RESUMO

An obstacle in the treatment of human diseases such as cancer is the inability to selectively and effectively target historically undruggable targets such as transcription factors. Here, we employ a novel technology using artificial transcription factors (ATFs) to epigenetically target gene expression in cancer cells. We show that site-specific DNA methylation and long-term stable repression of the tumor suppressor Maspin and the oncogene SOX2 can be achieved in breast cancer cells via zinc-finger ATFs targeting DNA methyltransferase 3a (DNMT3a) to the promoters of these genes. Using this approach, we show Maspin and SOX2 downregulation is more significant as compared with transient knockdown, which is also accompanied by stable phenotypic reprogramming of the cancer cell. These findings indicate that multimodular Zinc Finger Proteins linked to epigenetic editing domains can be used as novel cell resources to selectively and heritably alter gene expression patterns to stably reprogram cell fate.


Assuntos
Neoplasias da Mama/metabolismo , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Sequência de Bases , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Feminino , Técnicas de Silenciamento de Genes , Genes Neoplásicos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Serpinas/genética , Serpinas/metabolismo , Transfecção , Dedos de Zinco
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