RESUMEN
In recent years, it has been demonstrated that immunotherapy is an effective strategy for the management of solid tumors. The origins of immunotherapy can be traced back to the work of William Coley, who elicited an immune response against sarcoma by injecting patients with a mixture of dead bacteria. Significant progress has been made since, with immune markers within the tumor now being used as predictors of cancer prognosis and manipulated to improve patient survival. While surgery remains central to the management of most patients with solid malignancies, it is important that surgeons consider the different immunotherapy strategies that can be employed to manage disease. Here, we highlight how the immune system influences tumorigenesis and bring attention to how current and future immunotherapies can serve as an adjunct to surgery.
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Vacunas contra el Cáncer/uso terapéutico , Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Humanos , PronósticoRESUMEN
The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα(+)) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located â¼1.7 kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy.
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Receptor alfa de Estrógeno/metabolismo , Genes myb , Factor B de Elongación Transcripcional Positiva/metabolismo , Transcripción Genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Estradiol/farmacología , Estrógenos/farmacología , Femenino , Células HEK293 , Humanos , Intrones , Fosforilación , Factor B de Elongación Transcripcional Positiva/antagonistas & inhibidores , Proto-Oncogenes Mas , ARN Polimerasa II/metabolismo , Elementos Reguladores de la TranscripciónRESUMEN
The Mix/Bix family of paired-like homeobox genes encode evolutionarily conserved, sequence specific, DNA-binding transcription factors that have been implicated in the co-ordination of gene expression, axis formation and cell fate determination during gastrulation in vertebrates. When mutated, these genes give rise to dramatic phenotypes in amphibians, zebrafish and mice, that can be traced back to defects in the formation and specification of mesoderm and endoderm. We review here the biochemical properties of the Mix/Bix proteins and summarise genetic, molecular and embryological studies of Mix/Bix function in mesendoderm development. We emphasise recent data generated using embryonic stem cell differentiation systems that have provided important new insights into Mix/Bix function and the biological roles of these proteins in regulating the earliest phases of vertebrate development.
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Endodermo/embriología , Endodermo/metabolismo , Genes Homeobox , Mesodermo/embriología , Mesodermo/metabolismo , Ambystoma mexicanum/embriología , Ambystoma mexicanum/genética , Ambystoma mexicanum/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Pollo , Células Madre Embrionarias/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteínas Smad/genética , Proteínas Smad/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genética , Xenopus laevis/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. METHODS: MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann-Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05). RESULTS: Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity. CONCLUSIONS: This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal/genética , Proteínas de Homeodominio/genética , Proteínas Proto-Oncogénicas c-myb/genética , Factores de Transcripción/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myb/metabolismo , ARN Interferente Pequeño , Células Tumorales Cultivadas , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Adult stem cells reside in most parts of the body where high tissue turn-over is evident. However there are vastly different demands on the number of cells that might be produced and no better examples of each extreme are the neurogenic zones of the brain, and the crypt compartments of the intestines. From a perspective of understanding the function of the transcription factor Myb, we have explored the biology of stem cell niches in both these radically different tissues. Each tissue has remarkable features, provide different in vivo and in vitro options for manipulation and open up novel insights into damage responses and diseases like cancer. A variety of studies using mouse models, conditional and hypomorphic Myb mutants, radiation induced damage and primary in vitro assays have advanced our understanding of both stem cell niches and has revealed a previously unrecognised role for Myb in the regulation of stem cells.
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Células Madre Adultas/metabolismo , Encéfalo/metabolismo , Epigénesis Genética , Mucosa Intestinal/metabolismo , Proteínas Proto-Oncogénicas c-myb/genética , Nicho de Células Madre/genética , Células Madre Adultas/citología , Animales , Encéfalo/citología , Ciclo Celular , Regulación del Desarrollo de la Expresión Génica , Intestinos/citología , Ratones , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-myb/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Rapid advances have been made in the understanding of how the highly proliferative gastrointestinal tract epithelium is regulated under homeostasis and disease. The identification of putative intestinal stem cell (ISC) genes and the ability to culture ISC capable of generating all four lineages plus the architecture of small intestinal (SI) crypts has been transformative. Here, we show that transcription factor Myb governs ISC gene expression, particularly Lgr5. Lgr5 is associated with cells that have the capacity to generate all cell lineages in SI organoid cultures and colorectal cancer cells, which overexpress Myb. Furthermore, Wnt signaling and Myb cooperate in maximal Lgr5 promoter activation while hypomorphic Myb (plt4/plt4) mice have decreased Lgr5 expression. After ionizing radiation (IR), ISC genes are elevated; but in plt4/plt4 mice, this response is substantially subdued. ISC genes bmi-1 and olfm4 are expressed at subnormal levels in plt4/plt4 mice, and bmi-1 is induced with IR to half the level in mutant mice. dcamkl-1 and olfm4 failed to recover after IR in both wild-type (wt) and mutant mice. Although not considered as an ISC gene, cyclinE1 is nevertheless used to assist cells in the emergence from a quiescent state (an expectation of ISC following IR) and is overexpressed after IR in wt mice but does not change from a very low base in plt4/plt4 mice. Self-renewal assays using organoid cultures and inducible Myb knockout studies further highlighted the dependence of ISC on Myb consistent with role in other stem cell-containing tissues.
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Genes myb , Yeyuno/citología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Células Madre/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Ciclina E/genética , Ciclina E/metabolismo , Rayos gamma , Regulación Neoplásica de la Expresión Génica , Humanos , Yeyuno/metabolismo , Yeyuno/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myb/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/citología , Activación Transcripcional , Vía de Señalización WntRESUMEN
The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse embryonic stem cell (ESC) differentiation. We show that three independently derived ESC lines constitutively expressing Mixl1 (Mixl1(C) ESCs) differentiate into embryoid bodies (EBs) containing a higher proportion of E-cadherin (E-Cad)(+) cells. Our analysis also shows that this differentiation occurs at the expense of hematopoietic mesoderm differentiation, with Mixl1(C) ESCs expressing only low levels of Flk1 and failing to develop hemoglobinized cells. Immunohistochemistry and immunofluorescence studies revealed that Mixl1(C) EBs have extensive areas containing cells with an epithelial morphology that express E-Cad, FoxA2, and Sox17, consistent with enhanced endoderm formation. Luciferase reporter transfection experiments indicate that Mixl1 can transactivate the Gsc, Sox17, and E-Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. Taken together, these studies suggest that high levels of Mixl1 preferentially allocate cells to the endoderm during ESC differentiation.
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Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Proteínas de Homeodominio/fisiología , Mesodermo/citología , Mesodermo/metabolismo , Animales , Células 3T3 BALB , Western Blotting , Cadherinas/genética , Cadherinas/metabolismo , Diferenciación Celular/genética , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Endodermo/citología , Citometría de Flujo , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Ratones , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismoRESUMEN
BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the USA. Over 80% of CRC develop from adenomatous polyps. Hence, early treatment and prevention of adenomas would lead to a significant decrease of disease burden for CRC. MYB is a transcription factor that is overexpressed in both precancerous adenomatous polyps and colorectal cancer, and hence an ideal immunotherapeutic target. We have developed a cancer vaccine, TetMYB, that targets MYB and aim to evaluate its efficacy in the prophylactic and therapeutic management of adenomatous polyps. MATERIAL AND METHODS: Six- to eight-week-old Apcmin/+ (Familial Adenomatous Polyposis model) and Apc580S (sporadic model) C57BL/6 mice were used. The Apcmin/+ mice are carried a germline mutation of one Apc allele whereas the Apc580S model has an inducible silencing of one Apc allele, when exposed to tamoxifen, via the Cre-Lox recombination enzyme system. In the prophylactic treatment group, Apcmin/+ and Apc580S C57BL/6 mice were vaccinated and surveyed for clinical signs of distress. Number of adenoma and survival were measured. In the therapeutic cohort, Apc580S C57BL/6 mice were given tamoxifen-laced food to activate Cre-Lox recombinase mediated silencing of one Apc allele and thus inducing adenoma development. Following adenoma detection, mice were vaccinated with TetMYB and treated with anti-PD-1 antibody and were analyzed for overall survival. RESULTS: In both the prophylactic and therapeutic setting, mice vaccinated with TetMYB had a significantly improved outcome, with the vaccinated Apcmin/+ mice having a median survival benefit of 70 days (p = 0.008) and the vaccinated Apc580S mice having a mean survival benefit of 134 days (p = 0.01) over the unvaccinated mice. In the prophylactic cohort, immunofluorescence confirmed a stronger cytotoxic CD8+ T cell infiltrate in the vaccinated group, implying an anti-tumor immune response. In the therapeutic cohort, vaccinated Apc580S mice showed significantly reduced adenoma progression rate compared to the unvaccinated mice (p = 0.0005). CONCLUSION: TetMYB vaccine has shown benefit in a prophylactic and therapeutic setting in the management of colonic adenoma in a murine model. This will form the basis for a future clinical trial to prevent and treat colonic adenomatous polyps.
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Adenoma/terapia , Vacunas contra el Cáncer/uso terapéutico , Neoplasias del Colon/terapia , Neoplasias Experimentales , Adenoma/diagnóstico , Animales , Neoplasias del Colon/diagnóstico , Colonoscopía/métodos , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: MYB is a transcription factor that is overexpressed in colorectal cancer (CRC) and also a driver mutation in adenoid cystic carcinoma (AdCC). Therefore, the MYB protein is an ideal target to vaccinate against to aid recruitment of tumour infiltrating lymphocytes (TILs) against these tumours. The Peter MacCallum Cancer Centre (Melbourne, Australia) has engineered a DNA vaccine, TetMYB, based on the pVAX1 plasmid vector carrying a fusion construct consisting of the universal tetanus toxin T-cell epitopes flanking an inactivated MYB gene. METHODS: This prospective first-in-human phase I single-arm multi-centre clinical trial involves patients with metastatic CRC or AdCC. Stage 1 will evaluate the safety profile of escalating doses of TetMYB vaccine, given sequentially and in combination with an anti-PD-1 inhibitory antibody, to determine the maximum tolerated dose (MTD). Stage 2 will assess the MTD in an expanded cohort. The calculated sample size is 32 patients: 12 in Stage 1 and 20 in Stage 2. The expected total duration of the trial is 3 years with 15 months of recruitment followed by a minimum of 18 months follow-up. DISCUSSION: MYB transcription factor is aberrantly overexpressed in a range of epithelial cancers, not limited to the above tumour types. Based on promising pre-clinical data of vaccine-induced tumour clearance and establishment of anti-tumour memory, we are embarking on this first-in-human trial. If successful, the results from this trial will allow progression to a Phase II trial and validation of this breakthrough immunotherapeutic approach, not only in CRC and AdCC, but other MYB over-expressing cancers. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT03287427. Registered: September 19, 2017.
RESUMEN
Transcriptional activity of the TATA-binding protein (TBP) is controlled by a variety of proteins. The BTAF1 protein (formerly known as TAF(II)170/TAF-172 and the human ortholog of Saccharomyces cerevisiae Mot1p) and the NC2 complex composed of NC2alpha (DRAP1) and NC2beta (Dr1) are able to bind to TBP directly and regulate RNA polymerase II transcription both positively and negatively. Here, we present evidence that the NC2alpha subunit interacts with BTAF1. In contrast, the NC2beta subunit is not able to associate with BTAF1 and seems to interfere with the BTAF1-TBP interaction. Addition of NC2alpha or the NC2 complex can stimulate the ability of BTAF1 to interact with TBP. This function is dependent on the presence of ATP in cell extracts but does not involve the ATPase activity of BTAF1 nor phosphorylation of NC2alpha. Together, our results constitute the first evidence of the physical cooperation between BTAF1 and NC2alpha in TBP regulation and provide a framework to understand transcription functions of NC2alpha and NC2beta in vivo.
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Proteínas Represoras/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/metabolismo , Adenosina Trifosfato/metabolismo , ADN/genética , ADN/metabolismo , Humanos , Técnicas In Vitro , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Proteína de Unión a TATA-Box/genética , Factor de Transcripción TFIID/genética , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , Tirosina/químicaRESUMEN
The presence of tumor immune infiltrating cells (TILs), particularly CD8(+) T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8(+) T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8(+) and CD45RO(+) -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), ß-catenin and MYB. High CD8(+) TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8(+) TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies.
RESUMEN
Colorectal cancer (CRC) is a genetically heterogeneous disease that develops and progresses through several distinct pathways characterized by genomic instability. In recent years, it has emerged that inherent plasticity in some populations of CRC cells can contribute to heterogeneity in differentiation state, metastatic potential, therapeutic response, and disease relapse. Such plasticity is thought to arise through interactions between aberrant signaling events, including persistent activation of the APC/ß-catenin and KRAS/BRAF/ERK pathways, and the tumor microenvironment. Here, we highlight key concepts and evidence relating to the role of epithelial-mesenchymal plasticity as a driver of CRC progression and stratification of the disease into distinct molecular and clinicopathological subsets.
RESUMEN
UNLABELLED: Cyclin E1 is essential for the reentry of quiescent cells into the cell cycle. When hypomorphic mutant Myb mice (Myb(Plt4)) were examined, it was noted that Cyclin E1 (Ccne1) expression was reduced. Furthermore, the induction of Ccne1 in recovering intestinal epithelia following radiation-induced damage was ablated in Myb-mutant mice. These data prompted us to investigate whether Myb directly regulated Ccne1 and to examine whether elevated Myb in colorectal cancer is responsible for Cyclin E1-driven tumor growth. Here, it was found that Myb/MYB and Ccne1/CCNE1 expressions were coupled in both mouse and human adenomas. In addition, the low molecular weight Cyclin E1 was the predominant form in intestinal crypts and adenomatous polyposis coli (Apc)-mutant adenomas. Chromatin immunoprecipitation (ChIP) analysis confirmed that Myb bound directly to the Ccne1 promoter and regulated its endogenous expression. In contrast, Myb(Plt4) served as a dominant-negative factor that inhibited wild-type Myb and this was not apparently compensated for by the transcription factor E2F1 in intestinal epithelial cells. Myb(Plt4/Plt4) mice died prematurely on an Apc(Min/) (+) background associated with hematopoietic defects, including a myelodysplasia; nevertheless, Apc(Min/) (+) mice were protected from intestinal tumorigenesis when crossed to Myb(Plt4/) (+) mice. Knockdown of CCNE1 transcript in murine colorectal cancer cells stabilized chromosome ploidy and decreased tumor formation. These data suggest that Cyclin E1 expression is Myb dependent in normal and transformed intestinal epithelial cells, consistent with a cell-cycle progression and chromosome instability role in cancer. IMPLICATIONS: This study demonstrates that Myb regulates Cyclin E1 expression in normal gastrointestinal tract epithelial cells and is required during intestinal tumorigenesis.
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Carcinogénesis/metabolismo , Neoplasias Colorrectales/metabolismo , Ciclina E/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Oncogénicas v-myb/genética , Proteínas Oncogénicas/metabolismo , Adenoma/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Inmunoprecipitación de Cromatina , Inestabilidad Cromosómica , Cromosomas/ultraestructura , Progresión de la Enfermedad , Femenino , Hematopoyesis , Humanos , Inmunoprecipitación , Mucosa Intestinal/metabolismo , Neoplasias Intestinales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Ploidias , Regiones Promotoras GenéticasRESUMEN
MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.
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Proteínas de Unión al ADN/genética , Genes myb/genética , VIH-1/genética , Subunidad p50 de NF-kappa B/genética , Proteínas de Unión al ADN/metabolismo , VIH-1/patogenicidad , Humanos , Intrones/genética , Secuencias Invertidas Repetidas/genética , Mutación , Subunidad p50 de NF-kappa B/metabolismo , ARN Viral/genéticaRESUMEN
Regulation of RNA polymerase II (pol II) transcription is a highly dynamic process requiring the coordinated interaction of an array of regulatory proteins. Central to this process is the TATA-binding protein (TBP), the key component of the multiprotein complex TFIID. Interaction of TBP with core promoters nucleates the assembly of the preinitiation complex and subsequent recruitment of pol II. Despite recent advances in our understanding of the dynamic nature of the pol II transcription apparatus, the dynamics of TBP function on pol II promoters has remained largely unexplored. Human BTAF1 (TAF(II)170/TAF-172) and its yeast ortholog, Mot1p, are evolutionarily conserved members of the SNF2-like family of ATPase proteins. Genetic identification of Mot1p as a repressor of pol II transcription was supported by findings that Mot1p and BTAF1 could dissociate TBP from TATA DNA complexes using the energy of ATP hydrolysis. Recent data have revealed new aspects of BTAF1 and Mot1p as positive regulators of TBP function in the pol II system and have described new observations relating to their molecular mechanism of action. We review these data in the context of previous findings with particular attention paid to how human BTAF1 and Mot1p may dynamically regulate TBP function on pol II promoters in cells.
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ARN Polimerasa II/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/metabolismo , Transcripción Genética , Adenosina Trifosfatasas , Animales , ADN Helicasas/genética , ADN Helicasas/metabolismo , Humanos , Modelos Biológicos , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Proteína de Unión a TATA-Box/genética , Factor de Transcripción TFIID/genéticaRESUMEN
We investigated the role of canonical WNT signaling in mesoderm and hematopoietic development from human embryonic stem cells (hESCs) using a recombinant human protein-based differentiation medium (APEL). In contrast to prior studies using less defined culture conditions, we found that WNT3A alone was a poor inducer of mesoderm. However, WNT3A synergized with BMP4 to accelerate mesoderm formation, increase embryoid body size, and increase the number of hematopoietic blast colonies. Interestingly, inclusion of WNT3A or a GSK3 inhibitor in methylcellulose colony-forming assays at 4 days of differentiation abrogated blast colony formation but supported the generation of mesospheres that expressed genes associated with mesenchymal lineages. Mesospheres differentiated into cells with characteristics of bone, fat, and smooth muscle. These studies identify distinct effects for WNT3A, supporting the formation of hematopoietic or mesenchymal lineages from human embryonic stem cells, depending upon differentiation stage at the time of exposure.
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Cuerpos Embrioides/citología , Hematopoyesis , Células Madre Mesenquimatosas/citología , Proteína Wnt3A/metabolismo , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Linaje de la Célula , Células Cultivadas , Cuerpos Embrioides/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Células Madre Mesenquimatosas/metabolismo , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Vía de Señalización Wnt , Proteína Wnt3A/antagonistas & inhibidores , Proteína Wnt3A/genéticaRESUMEN
The Mixl1 homeodomain protein plays a key role in mesendoderm patterning during embryogenesis, but its target genes remain to be identified. We compared gene expression in differentiating heterozygous Mixl1(GFP/w) and homozygous null Mixl1(GFP/Hygro) mouse embryonic stem cells to identify potential downstream transcriptional targets of Mixl1. Candidate Mixl1 regulated genes whose expression was reduced in GFP+ cells isolated from differentiating Mixl1(GFP/Hygro) embryoid bodies included Pdgfrα and Flk1. Mixl1 bound to ATTA sequences located in the Pdgfrα and Flk1 promoters and chromatin immunoprecipitation assays confirmed Mixl1 occupancy of these promoters in vivo. Furthermore, Mixl1 transactivated the Pdgfrα and Flk1 promoters through ATTA sequences in a DNA binding dependent manner. These data support the hypothesis that Mixl1 directly regulates Pdgfrα and Flk1 gene expression and strengthens the position of Mixl1 as a key regulator of mesendoderm development during mammalian gastrulation.
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Diferenciación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Endodermo/citología , Endodermo/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/química , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Activación Transcripcional/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity.
Asunto(s)
Proteínas Fetales/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de Dominio T Box/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Células HEK293 , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Mapeo de Interacción de Proteínas , Transcripción GenéticaRESUMEN
MYB oncogene upregulation is associated with estrogen receptor (ER)-positive breast cancer, but disease requirements for MYB function in vivo have not been explored. In this study, we provide evidence of a critical requirement for MYB functions in models of human and murine breast cancer. In human breast cancer, we found that MYB expression was critical for tumor cell growth both in vitro and in vivo in xenograft settings. In transgenic knockout mice, tissue-specific deletion of the murine MYB gene caused a transient defect in mammary gland development that was reflected in delayed ductal branching and defective apical bud formation. In mouse mammary tumor virus (MMTV)-NEU mice where tumors are initiated by activation of HER2, MYB deletion was sufficient to abolish tumor formation. In the more aggressive MMTV-PyMT model system, MYB deletion delayed tumorigenesis significantly. Together, the findings in these transgenic knockout models implied that MYB was critical during an early window in mammary development when it was essential for tumor initiation, even though MYB loss did not exert a lasting impact upon normal mammary function. Two important MYB-target genes that promote cell survival, BCL2 and GRP78/BIP, were each elevated compared with nontransformed mammary epithelial cells, thereby promoting survival as confirmed in colony formation assays in vitro. Taken together, our findings establish a role for MYB at the hub of ER- and HER2-dependent pathways in mammary carcinogenesis.
Asunto(s)
Neoplasias Mamarias Experimentales/etiología , Proteínas Oncogénicas v-myb/fisiología , Animales , Antígenos Virales de Tumores/genética , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Receptor alfa de Estrógeno/análisis , Etilnitrosourea , Femenino , Neoplasias Mamarias Experimentales/química , Virus del Tumor Mamario del Ratón/genética , Ratones , Receptor ErbB-2/análisisRESUMEN
Slain1 was originally identified as a novel stem cell-associated gene in transcriptional profiling experiments comparing mouse and human embryonic stem cells (ESCs) and their immediate differentiated progeny. In order to obtain further insight into the potential function of Slain1, we examined the expression of beta-galactosidase in a gene-trap mouse line in which a beta-geo reporter gene was inserted into the second intron of Slain1. In early stage embryos (E7.5), the Slain1-betageo fusion protein was expressed within the entire epiblast, but by E9.5 became restricted to the developing nervous system and gastrointestinal tract. In later stage embryos (E11.5 - E13.5), expression was predominantly within the developing nervous system. Lower level expression was also observed in the developing limb buds, in the condensing mesenchyme, along the apical epidermal ridge and, at later stages, within the digital zones. These observations suggest that Slain1 may play a role in the development of the nervous system, as well as in the morphogenesis of several embryonic structures.