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1.
Int J Mol Sci ; 22(1)2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33401508

RESUMO

Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.


Assuntos
Proteína 9 Associada à CRISPR , Edição de Genes/métodos , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Ativação Transcricional , Regulação da Expressão Gênica , Células HEK293 , Humanos , RNA Mensageiro , Regulação para Cima
3.
Nat Commun ; 12(1): 560, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495473

RESUMO

The squamous-columnar junction (SCJ) is a boundary consisting of precisely positioned transitional epithelium between the squamous and columnar epithelium. Transitional epithelium is a hotspot for precancerous lesions, and is therefore clinically important; however, the origins and physiological properties of transitional epithelium have not been fully elucidated. Here, by using mouse genetics, lineage tracing, and organoid culture, we examine the development of the SCJ in the mouse stomach, and thus define the unique features of transitional epithelium. We find that two transcription factors, encoded by Sox2 and Gata4, specify primitive transitional epithelium into squamous and columnar epithelium. The proximal-distal segregation of Sox2 and Gata4 expression establishes the boundary of the unspecified transitional epithelium between committed squamous and columnar epithelium. Mechanistically, Gata4-mediated expression of the morphogen Fgf10 in the distal stomach and Sox2-mediated Fgfr2 expression in the proximal stomach induce the intermediate regional activation of MAPK/ERK, which prevents the differentiation of transitional epithelial cells within the SCJ boundary. Our results have implications for tissue regeneration and tumorigenesis, which are related to the SCJ.


Assuntos
Células Epiteliais/metabolismo , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica , Junções Intercelulares/genética , Sistema de Sinalização das MAP Quinases/genética , Fatores de Transcrição SOXB1/genética , Animais , Células Cultivadas , Feminino , Fator de Transcrição GATA4/metabolismo , Mucosa Gástrica/metabolismo , Queratina-7/genética , Queratina-7/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição SOXB1/metabolismo
4.
Nat Commun ; 12(1): 28, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397924

RESUMO

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.


Assuntos
Núcleo Celular/metabolismo , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Drosophila/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Células-Tronco Neurais/metabolismo , Sinais de Localização Nuclear/metabolismo , Mutação Puntual/genética , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Fatores de Transcrição SOXB1/genética , Relação Estrutura-Atividade
5.
Viruses ; 13(1)2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33419104

RESUMO

Hearing loss, one of the most prevalent chronic health conditions, affects around half a billion people worldwide, including 34 million children. The World Health Organization estimates that the prevalence of disabling hearing loss will increase to over 900 million people by 2050. Many cases of congenital hearing loss are triggered by viral infections during different stages of pregnancy. However, the molecular mechanisms by which viruses induce hearing loss are not sufficiently explored, especially cases that are of embryonic origins. The present review first describes the cellular and molecular characteristics of the auditory system development at early stages of embryogenesis. These developmental hallmarks, which initiate upon axial specification of the otic placode as the primary root of the inner ear morphogenesis, involve the stage-specific regulation of several molecules and pathways, such as retinoic acid signaling, Sonic hedgehog, and Wnt. Different RNA and DNA viruses contributing to congenital and acquired hearing loss are then discussed in terms of their potential effects on the expression of molecules that control the formation of the auditory and vestibular compartments following otic vesicle differentiation. Among these viruses, cytomegalovirus and herpes simplex virus appear to have the most effect upon initial molecular determinants of inner ear development. Moreover, of the molecules governing the inner ear development at initial stages, SOX2, FGFR3, and CDKN1B are more affected by viruses causing either congenital or acquired hearing loss. Abnormalities in the function or expression of these molecules influence processes like cochlear development and production of inner ear hair and supporting cells. Nevertheless, because most of such virus-host interactions were studied in unrelated tissues, further validations are needed to confirm whether these viruses can mediate the same effects in physiologically relevant models simulating otic vesicle specification and growth.


Assuntos
Citomegalovirus/isolamento & purificação , Orelha Interna/embriologia , Orelha Interna/virologia , Perda Auditiva/virologia , Simplexvirus/isolamento & purificação , Animais , Diferenciação Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Citomegalovirus/patogenicidade , Perda Auditiva/congênito , Humanos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais , Simplexvirus/patogenicidade
6.
Anticancer Res ; 40(10): 5481-5487, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988870

RESUMO

BACKGROUND/AIM: γδ T cells mediate cytotoxicity against prostate cancer (PCa) cells in vitro; however, the clinical efficacy of γδ T cell-targeted immunotherapy for recurrent and metastatic PCa is unsatisfactory. We hypothesized that the resistance of recurrent and metastatic PCa to γδ T cells is related to the presence of prostate cancer stem cells (PCSCs), and we examined their relationship. MATERIALS AND METHODS: PCa spheres (prostaspheres) were generated from five PCa cell lines, and their susceptibility to cytotoxicity by γδ T cells was investigated. Expression of stemness-related markers was evaluated by qRT-PCR. RESULTS: Prostasphere-derived cancer cells were resistant to lysis by γδ T cells and expressed higher levels of several stemness markers, including CD133, NANOG, SOX2, and OCT4, than the parental PCa cell lines. CONCLUSION: Ex vivo-expanded γδ T cells are not effective against PCSCs.


Assuntos
Linfócitos Intraepiteliais/imunologia , Células-Tronco Neoplásicas/imunologia , Neoplasias da Próstata/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Antígeno AC133/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Masculino , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Fatores de Transcrição SOXB1/genética , Linfócitos T
7.
J Cancer Res Ther ; 16(4): 804-810, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32930122

RESUMO

Background: The promising improvement in the clinical outcome of lung cancer can be possibly achieved by identification of the molecular events that underlie its pathogenesis. Cancer stem cell (CSC) being one of the subsets of tumor majorly participates in drug resistance and treatment failure because of the moderate cell cycle, lower proliferation, and increased expression of DNA repair and anti-apoptosis genes. Although many putative CSC markers exist, a precise characterization for non-small cell lung cancer is of utmost importance due to increased mortality rate and lack of targeted therapies. Hence, the article focuses on the expression of stemness-associated markers, namely octamer-binding transcription factor 4 (OCT4), NANOG, and sex-determining region Y-box 2 (SOX2) in non-small cell lung cancer (NSCLC) patients. Methods: The expression of OCT4, NANOG, and SOX2 were evaluated in 32 histopathologically confirmed NSCLC tissues using real-time polymerase chain reaction. The obtained expression was correlated with clinical and pathological manifestations using the statistical test such as Student's t-test and Pearson correlation in varied statistical software. Results: Results showed a significantly higher expression of OCT4 and NANOG compared to SOX2 in the tumor tissues. When the expression of these markers was correlated with the clinical parameters, higher expression was seen in males, patients with age above 60 years, and in adenocarcinoma subtype. In correlation with the habit, higher expression of OCT4 and SOX2 was observed in habituated patients. Expression of NANOG and OCT4 was higher even in patients with poor differentiation. Conclusion: The expression and prognostic significance of CSC markers obviously vary depending on histological NSCLC subtype. Importantly, our findings suggest that OCT4, SOX2, and NANOG network together may be promising for ongoing targeted therapies in specific NSCLC subgroups.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Regulação Neoplásica da Expressão Gênica , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Células-Tronco Neoplásicas/metabolismo , Prognóstico
8.
PLoS One ; 15(9): e0238948, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915925

RESUMO

Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic.


Assuntos
Desenvolvimento Embrionário , Equidae/embriologia , Equidae/genética , Cavalos/fisiologia , Oócitos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Núcleo Celular/fisiologia , Clonagem de Organismos/veterinária , Citoplasma/fisiologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Espécies em Perigo de Extinção , Equidae/metabolismo , Feminino , Perfilação da Expressão Gênica , Cavalos/genética , Técnicas In Vitro , Masculino , Técnicas de Transferência Nuclear/veterinária , Fatores de Transcrição SOXB1/genética , Injeções de Esperma Intracitoplásmicas/veterinária , Sus scrofa
9.
Proc Natl Acad Sci U S A ; 117(32): 19287-19298, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723825

RESUMO

Retinal ganglion cell axons forming the optic nerve (ON) emerge unmyelinated from the eye and become myelinated after passage through the optic nerve lamina region (ONLR), a transitional area containing a vascular plexus. The ONLR has a number of unusual characteristics: it inhibits intraocular myelination, enables postnatal ON myelination of growing axons, modulates the fluid pressure differences between eye and brain, and is the primary lesion site in the age-related disease open angle glaucoma (OAG). We demonstrate that the human and rodent ONLR possesses a mitotically active, age-depletable neural progenitor cell (NPC) niche, with unique characteristics and culture requirements. These NPCs generate both forms of macroglia: astrocytes and oligodendrocytes, and can form neurospheres in culture. Using reporter mice with SOX2-driven, inducible gene expression, we show that ONLR-NPCs generate macroglial cells for the anterior ON. Early ONLR-NPC loss results in regional dysfunction and hypomyelination. In adulthood, ONLR-NPCs may enable glial replacement and remyelination. ONLR-NPC depletion may help explain why ON diseases such as OAG progress in severity during aging.


Assuntos
Neurônios/citologia , Nervo Óptico/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Animais , Astrócitos , Axônios/metabolismo , Diferenciação Celular , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/fisiopatologia , Humanos , Camundongos , Bainha de Mielina/metabolismo , Neuroglia , Neurônios/metabolismo , Oligodendroglia , Nervo Óptico/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/metabolismo
10.
Prostate ; 80(13): 1134-1144, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32628304

RESUMO

BACKGROUND: Although androgen deprivation therapy (ADT) is the initial treatment strategy for prostate cancer (PCa), recurrent castration-resistant prostate cancer (CRPC) eventually ensues. In this study, cancer-derived immunoglobulin G (CIgG) is found to be induced after ADT, identifying CIgG as a potential CRPC driver gene. METHODS: The expression of CIgG and its clinical significance in PCa tissue was analyzed by The Cancer Genome Atlas database and immunohistochemistry. Subsequently, the sequence features of prostate cell line VHDJH rearrangements were analyzed. We also assessed the effect of CIgG on the migratory, invasive and proliferative abilities of PCa cells in vitro and vivo. Suspended microsphere, colony formation and drug-resistant assays were performed using PC3 cells with high CIgG expression (CIgGhigh ) and low CIgG expression (CIgG-/low ), and A nonobese diabetic/severe combined immunodeficiency mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. The SOX2-CIgG signaling pathway was validated by immunohistochemistry, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, Western blot, luciferase, and chromatin immunoprecipitation assays and bioinformatics analyses. Finally, we investigated the effect of RP215 inhibition on the progression of PCa in vivo using a Babl/c nude mouse xenograft model. RESULTS: CIgG is frequently expressed in PCa and associated with clinicopathological characteristics, moreover, CIgG transcripts with unique patterns of VHDJH rearrangements are found in PCa cells. Functional analyses identified that CIgG was induced by ADT and upregulated by SOX2 (SRY (sex determining region Y)-box 2) in PCa, promoting the development of PCa. In addition, our findings underscore a novel role of CIgG signaling in the maintenance of stemness and the progression of cancer through mitogen activated protein kinase/extracellular-signal-regulated kinase and AKT in PCa. In vivo experiments further demonstrated that depleting CIgG significantly suppressed the growth of PCa cell xenografts. Furthermore, a CIgG monoclonal antibody named RP215 exhibits tumor inhibitory effect as well. CONCLUSION: Our data suggests that CIgG could be a driver of PCa development, and that targeting the SOX2-CIgG axis may therefore inhibit PCa development after ADT.


Assuntos
Imunoglobulina G/imunologia , Neoplasias de Próstata Resistentes à Castração/imunologia , Fatores de Transcrição SOXB1/imunologia , Animais , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Células PC-3 , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Transdução de Sinais/imunologia , Análise Serial de Tecidos
11.
Anim Sci J ; 91(1): e13408, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32578338

RESUMO

We examined the allelic expression and positioning of two pluripotency-associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4- and 8-cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi-allelically increased from 45% at the 4-cell stage to 60% at the 8-cell stage. Moreover, in 8-cell embryos, SOX2 was expressed bi-allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4- to 8-cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4- or 8-cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency-associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.


Assuntos
Blastômeros/citologia , Blastômeros/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Expressão Gênica , Suínos/embriologia , Suínos/genética , Alelos , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Fertilização In Vitro , Hibridização in Situ Fluorescente , Técnicas de Maturação in Vitro de Oócitos , Técnicas In Vitro , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(24): 13552-13561, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482884

RESUMO

Precise control of organ growth and patterning is executed through a balanced regulation of progenitor self-renewal and differentiation. In the auditory sensory epithelium-the organ of Corti-progenitor cells exit the cell cycle in a coordinated wave between E12.5 and E14.5 before the initiation of sensory receptor cell differentiation, making it a unique system for studying the molecular mechanisms controlling the switch between proliferation and differentiation. Here we identify the Yap/Tead complex as a key regulator of the self-renewal gene network in organ of Corti progenitor cells. We show that Tead transcription factors bind directly to the putative regulatory elements of many stemness- and cell cycle-related genes. We also show that the Tead coactivator protein, Yap, is degraded specifically in the Sox2-positive domain of the cochlear duct, resulting in down-regulation of Tead gene targets. Further, conditional loss of the Yap gene in the inner ear results in the formation of significantly smaller auditory and vestibular sensory epithelia, while conditional overexpression of a constitutively active version of Yap, Yap5SA, is sufficient to prevent cell cycle exit and to prolong sensory tissue growth. We also show that viral gene delivery of Yap5SA in the postnatal inner ear sensory epithelia in vivo drives cell cycle reentry after hair cell loss. Taken together, these data highlight the key role of the Yap/Tead transcription factor complex in maintaining inner ear progenitors during development, and suggest new strategies to induce sensory cell regeneration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Autorrenovação Celular , Órgão Espiral/embriologia , Órgão Espiral/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ciclo Celular , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas , Camundongos , Órgão Espiral/citologia , Ligação Proteica , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/genética
13.
Nat Commun ; 11(1): 2124, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358507

RESUMO

Penile squamous cell carcinoma (PSCC) accounts for over 95% of penile malignancies and causes significant mortality and morbidity in developing countries. Molecular mechanisms and therapies of PSCC are understudied, owing to scarcity of laboratory models. Herein, we describe a genetically engineered mouse model of PSCC, by co-deletion of Smad4 and Apc in the androgen-responsive epithelium of the penis. Mouse PSCC fosters an immunosuppressive microenvironment with myeloid-derived suppressor cells (MDSCs) as a dominant population. Preclinical trials in the model demonstrate synergistic efficacy of immune checkpoint blockade with the MDSC-diminishing drugs cabozantinib or celecoxib. A critical clinical problem of PSCC is chemoresistance to cisplatin, which is induced by Pten deficiency on the backdrop of Smad4/Apc co-deletion. Drug screen studies informed by targeted proteomics identify a few potential therapeutic strategies for PSCC. Our studies have established what we believe to be essential resources for studying PSCC biology and developing therapeutic strategies.


Assuntos
Carcinoma de Células Escamosas/terapia , Imunoterapia/métodos , Neoplasias Penianas/terapia , Animais , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular , Cisplatino/farmacologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/metabolismo , Neoplasias Penianas/metabolismo , Proteômica , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Análise Serial de Tecidos , Transcriptoma/genética
14.
Mol Cell ; 78(3): 506-521.e6, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32386543

RESUMO

Higher-order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we find that large (>7.3 kb) DNA methylation nadirs (termed "grand canyons") can form long loops connecting anchor loci that may be dozens of megabases (Mb) apart, as well as inter-chromosomal links. The interacting loci cover a total of ∼3.5 Mb of the human genome. The strongest interactions are associated with repressive marks made by the Polycomb complex and are diminished upon EZH2 inhibitor treatment. The data are suggestive of the formation of these loops by interactions between repressive elements in the loci, forming a genomic subcompartment, rather than by cohesion/CTCF-mediated extrusion. Interestingly, unlike previously characterized subcompartments, these interactions are present only in particular cell types, such as stem and progenitor cells. Our work reveals that H3K27me3-marked large DNA methylation grand canyons represent a set of very-long-range loops associated with cellular identity.


Assuntos
Cromatina/química , Cromatina/genética , Metilação de DNA , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Hibridização in Situ Fluorescente , Lisina/genética , Lisina/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição SOXB1/genética , Proteína de Homoeobox de Baixa Estatura/genética , Fatores de Transcrição/genética
15.
Indian J Cancer ; 57(2): 166-171, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32445320

RESUMO

Background: Despite the advances in screening during the past decades, colorectal cancer (CRC) still is a leading cause of cancer deaths worldwide. Therefore, the development of new diagnostic methods is necessary. Aim: The aim of this study was to compare methylation changes of SRY-Box 21 (SOX21) gene promoter in tumor tissues and their normal adjacent mucosa in patients with CRC and to examine the relationship between the methylation levels and demographic/clinicopathological factors. Materials and methods: A total of 41 CRC patients participated in the present study. After the extraction of DNA and bisulfite treatment of the samples, the methylation levels were determined by using the MethyLight method. Statistical analysis: Two-sided Mann-Whitney U test was used to compare the median level of methylation in tumor tissues and their adjacent normal mucosa. Results: The methylation rates in tumor tissue samples were significantly higher compared to their adjacent normal mucosa (P < 0.0001). No association between demographic/clinicopathological factors and methylation status observed in tumor tissues. A receiver operating characteristics curve was constructed and tissue samples exhibited a sensitivity of 80.5% and specificity of 97.6% for SOX21 promoter methylation. Conclusion: The results of this study indicated the high potential of SOX21 gene promoter methylation as a candidate noninvasive diagnostic biomarker in stool and plasma of colorectal cancer patients. However, further studies with larger sample sizes are required to evaluate the specific role of SOX21 methylation as a biomarker for early detection of CRC.


Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , Fatores de Transcrição SOXB1/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Detecção Precoce de Câncer , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição SOXB1/metabolismo
16.
Sci Rep ; 10(1): 6751, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317665

RESUMO

SOX3 is a transcription factor expressed within the developing and adult nervous system where it mostly functions to help maintain neural precursors. Sox3 is also expressed in other locations, notably within the spermatogonial stem/progenitor cell population in postnatal testis. Independent studies have shown that Sox3 null mice exhibit a spermatogenic block as young adults, the mechanism of which remains poorly understood. Using a panel of spermatogonial cell marker genes, we demonstrate that Sox3 is expressed within the committed progenitor fraction of the undifferentiated spermatogonial pool. Additionally, we use a Sox3 null mouse model to define a potential role for this factor in progenitor cell function. We demonstrate that Sox3 expression is required for transition of undifferentiated cells from a GFRα1+ self-renewing state to the NGN3 + transit-amplifying compartment. Critically, using chromatin immunoprecipitation, we demonstrate that SOX3 binds to a highly conserved region in the Ngn3 promoter region in vivo, indicating that Ngn3 is a direct target of SOX3. Together these studies indicate that SOX3 functions as a pro-commitment factor in spermatogonial stem/progenitor cells.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética , Espermatogônias/metabolismo , Testículo/metabolismo , Células-Tronco Germinativas Adultas/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Ligação Proteica , Fatores de Transcrição SOXB1/deficiência , Transdução de Sinais , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/crescimento & desenvolvimento , Testículo/citologia , Testículo/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Biochem Pharmacol ; 177: 113984, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32311348

RESUMO

Pluripotent stem cells are have therapeutic applications in regenerative medicine and drug discovery. However, the differentiation of stem cells in vitro hinders their large-scale production and clinical applications. The maintenance of cell pluripotency relies on a complex network of transcription factors; of these, octamer-binding transcription factor-4 (Oct4) plays a key role. This study aimed to construct an Oct4 gene promoter-driven firefly luciferase reporter and screen small-molecule compounds could maintain cell self-renewal and pluripotency. The results showed that ethyl-p-methoxycinnamate (EPMC) enhance the promoter activity of the Oct4 gene, increased the expression of Oct4 at both mRNA and protein levels, and significantly promoted the colony formation of P19 cells. These findings suggesting that EPMC could reinforce the self-renewal capacity of P19 cells. The pluripotency markers Oct4, SRY-related high-mobility-group-box protein-2, and Nanog were expressed at higher levels in EPMC-induced colonies. EPMC could promote teratoma formation and differentiation potential of P19 cells in vivo. It also enhanced self-renewal and pluripotency of human umbilical cord mesenchymal stem cells and mouse embryonic stem cells. Moreover, it significantly activated the nuclear factor kappa B (NF-κB) signaling pathway via the myeloid differentiation factor 88-dependent pathway. The expression level of Oct4 decreased after blocking the NF-κB signaling pathway, suggesting that EPMC promoted the expression of Oct4 partially through the NF-κB signaling pathway. This study indicated that EPMC could maintain self-renewal and pluripotency of stem cells.


Assuntos
Autorrenovação Celular/efeitos dos fármacos , Cinamatos/farmacologia , NF-kappa B/genética , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/agonistas , NF-kappa B/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/agonistas , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/genética
18.
Sci Rep ; 10(1): 5195, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251342

RESUMO

Pluripotency maintenance requires transcription factors (TFs) that induce genes necessary to preserve the undifferentiated state and repress others involved in differentiation. Recent observations support that the heterogeneous distribution of TFs in the nucleus impacts on gene expression. Thus, it is essential to explore how TFs dynamically organize to fully understand their role in transcription regulation. Here, we examine the distribution of pluripotency TFs Oct4 and Sox2 in the nucleus of embryonic stem (ES) cells and inquire whether their organization changes during early differentiation stages preceding their downregulation. Using ES cells expressing Oct4-YPet or Sox2-YPet, we show that Oct4 and Sox2 partition between nucleoplasm and a few chromatin-dense foci which restructure after inducing differentiation by 2i/LIF withdrawal. Fluorescence correlation spectroscopy showed distinct changes in Oct4 and Sox2 dynamics after differentiation induction. Specifically, we detected an impairment of Oct4-chromatin interactions whereas Sox2 only showed slight variations in its short-lived, and probably more unspecific, interactions with chromatin. Our results reveal that differentiation cues trigger early changes of Oct4 and Sox2 nuclear distributions that also include modifications in TF-chromatin interactions. This dynamical reorganization precedes Oct4 and Sox2 downregulation and may contribute to modulate their function at early differentiation stages.


Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Células-Tronco Embrionárias/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transcrição Genética , Animais , Ciclo Celular , Diferenciação Celular , Núcleo Celular/ultraestrutura , Células Cultivadas , Doxiciclina/farmacologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Reporter , Camundongos , Microscopia de Fluorescência , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/citologia , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1/genética , Transfecção
19.
Neoplasma ; 67(3): 519-527, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32122144

RESUMO

Krüppel-like factor 8 (KLF8) regulates critical gene transcription associated with different types of cancer. A novel paradigm in tumor biology suggests that the initiation and progression of osteosarcoma (OS) are driven by osteosarcoma stem cell-like cells (OSCs), but the role and underlying mechanisms of KLF8 in OSCs are poorly elucidated. In this study, an obviously increased level of KLF8 is shown in 9 out of 10 primary OS tissues and is associated with the poor progression-free interval. Significantly, KLF8 expression in CD133+ OSCs is higher than that in CD133- counterparts. By knocking down KLF8 in CD133+ OSCs, we show that si-KLF8-OSCs can hardly form compact spheres. In the meantime, infection with si-KLF8 in CD133+ OSCs results in the downregulation of OCT4 and SOX2; increased adriamycin (ADM) sensitivity; and decreased tumorigenic potential in vivo. Mechanisms study demonstrates that KLF8 directly binds the miR-429 promoter region and regulates its expression transcriptionally. Furthermore, we indicate that miR-429 directly targets SOX2 to mediate cancer stem cell-like features in CD133+ OSCs. In the clinic, miR-429 levels are negatively associated with KLF8 levels in OS, suggesting that an elevated KLF8/miR-429 ratio may have clinical value as a predictive biomarker. In conclusion, targeting the KLF8-miR-429-SOX2 signaling pathway may provide an effective therapeutic approach to suppress the initiation and progression of OS.


Assuntos
Neoplasias Ósseas/patologia , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/citologia , Osteossarcoma/patologia , Fatores de Transcrição SOXB1/genética , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Osteossarcoma/genética , Fenótipo , Regiões Promotoras Genéticas , Transdução de Sinais
20.
Gene ; 739: 144518, 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32119915

RESUMO

Glioblastomas (GBMs) are primary brain tumors with extremely bad prognosis and therefore; discovery of novel regulators of their pathology is of immense importance. LncRNAs (long noncoding RNAs) regulate nuclear structure, embryonic pluripotency, cell differentiation, development and carcinogenesis. Many lncRNAs have weak evolutionary conservation; however, a nuclear lncRNA, MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), is exceptionally conserved and is among the most abundant lncRNAs in benign tissues. The majority of cell culture studies and clinico-epidemiological studies demonstrated that MALAT1 acts a tumor promoter in GBMs and inhibition of MALAT1 suppressed tumor growth in various preclinical models of GBM. MALAT1 involves in stemness of GBM cells by regulating SOX2, nestin and members of WNT pathway. MALAT1 induces protective autophagy and suppresses apoptosis in GBM cells via sponging miRNA-101 and increases temozolomide chemoresistance via enhancing epithelial-mesenchymal transition, suppressing miR-203 and promoting thymidilate synthase. Moreover, knockdown of MALAT1 expression enhances blood-brain tumor barrier permeability via miR-140, which may provide a double benefit of MALAT1 suppression by increasing the delivery of chemotherapy agents into the GBM tissues. On the other hand, there also exist some cell culture and animal studies showing that MALAT1 acts as a tumor suppressor in GBMs by suppression of ERK/MAPK and MMP2 signaling and by repression of miR-155 with subsequent increase of FBXW7. Whether protective or detrimental, MALAT1 seems to be an important component of GBM pathogenesis and hence; novels studies are needed in versatile models, including many different primary GBM cultures, orthotopic and xenogreft in vivo models and transgenic mice.


Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/terapia , Glioblastoma/diagnóstico , Glioblastoma/terapia , Humanos , MicroRNAs/metabolismo , Nestina/genética , Nestina/metabolismo , Prognóstico , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Via de Sinalização Wnt/genética
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