RESUMO
Glioblastoma is the most malignant brain tumor among adults. Despite multimodality treatment, it remains incurable, mainly because of its extensive heterogeneity and infiltration in the brain parenchyma. Recent evidence indicates dysregulation of the expression of the Promyelocytic Leukemia Protein (PML) in primary Glioblastoma samples. PML is implicated in various ways in cancer biology. In the brain, PML participates in the physiological migration of the neural progenitor cells, which have been hypothesized to serve as the cell of origin of Glioblastoma. The role of PML in Glioblastoma progression has recently gained attention due to its controversial effects in overall Glioblastoma evolution. In this work, we studied the role of PML in Glioblastoma pathophysiology using the U87MG cell line. We genetically modified the cells to conditionally overexpress the PML isoform IV and we focused on its dual role in tumor growth and invasive capacity. Furthermore, we targeted a PML action mediator, the Enhancer of Zeste Homolog 2 (EZH2), via the inhibitory drug DZNeP. We present a combined in vitro-in silico approach, that utilizes both 2D and 3D cultures and cancer-predictive computational algorithms, in order to differentiate and interpret the observed biological results. Our overall findings indicate that PML regulates growth and invasion through distinct cellular mechanisms. In particular, PML overexpression suppresses cell proliferation, while it maintains the invasive capacity of the U87MG Glioblastoma cells and, upon inhibition of the PML-EZH2 pathway, the invasion is drastically eliminated. Our in silico simulations suggest that the underlying mechanism of PML-driven Glioblastoma physiology regulates invasion by differential modulation of the cell-to-cell adhesive and diffusive capacity of the cells. Elucidating further the role of PML in Glioblastoma biology could set PML as a potential molecular biomarker of the tumor progression and its mediated pathway as a therapeutic target, aiming at inhibiting cell growth and potentially clonal evolution regarding their proliferative and/or invasive phenotype within the heterogeneous tumor mass.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteína da Leucemia Promielocítica/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Simulação por Computador , Humanos , Modelos Biológicos , Invasividade Neoplásica , Esferoides Celulares/patologiaRESUMO
Transcriptional silencing during mitosis is caused by inactivation of critical transcriptional regulators and/or chromatin condensation. Inheritance of gene expression patterns through cell division involves various bookmarking mechanisms. In this report, we have examined the mitotic and post-mitotic expression of the DRA major histocompatibility class II (MHCII) gene in different cell types. During mitosis the constitutively MHCII-expressing B lymphoblastoid cells showed sustained occupancy of the proximal promoter by the cognate enhanceosome and general transcription factors. In contrast, although mitotic epithelial cells were depleted of these proteins irrespectively of their MHCII transcriptional activity, a distal enhancer selectively recruited the PP2A phosphatase via NFY and maintained chromatin accessibility. Based on our data, we propose a novel chromatin anti-condensation role for this element in mitotic bookmarking and timing of post-mitotic transcriptional reactivation.
Assuntos
Cadeias alfa de HLA-DR/genética , Mitose/genética , Transcrição Gênica , Fator de Ligação a CCAAT/metabolismo , Ciclo Celular/genética , Linhagem Celular , Cromatina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Fase G1/genética , Regulação da Expressão Gênica , Humanos , Região de Controle de Locus Gênico , Regiões Promotoras Genéticas , Proteína Fosfatase 2/metabolismo , Fatores de Transcrição de Fator Regulador XRESUMO
Adjuvant Temozolomide is considered the front-line Glioblastoma chemotherapeutic treatment; yet not all patients respond. Latest trends in clinical trials usually refer to Doxorubicin; yet it can lead to severe side-effects if administered in high doses. While Glioblastoma prognosis remains poor, little is known about the combination of the two chemotherapeutics. Patient-derived spheroids were generated and treated with a range of Temozolomide/Doxorubicin concentrations either as monotherapy or in combination. Optical microscopy was used to monitor the growth pattern and cell death. Based on the monotherapy experiments, we developed a probabilistic mathematical framework in order to describe the drug-induced effect at the single-cell level and simulate drug doses in combination assuming probabilistic independence. Doxorubicin was found to be effective in doses even four orders of magnitude less than Temozolomide in monotherapy. The combination therapy doses tested in vitro were able to lead to irreversible growth inhibition at doses where monotherapy resulted in relapse. In our simulations, we assumed both drugs are anti-mitotic; Temozolomide has a growth-arrest effect, while Doxorubicin is able to cumulatively cause necrosis. Interestingly, under no mechanistic synergy assumption, the in silico predictions underestimate the in vitro results. In silico models allow the exploration of a variety of potential underlying hypotheses. The simulated-biological discrepancy at certain doses indicates a supra-additive response when both drugs are combined. Our results suggest a Temozolomide-Doxorubicin dual chemotherapeutic scheme to both disable proliferation and increase cytotoxicity against Glioblastoma.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Linhagem Celular Tumoral , Recidiva Local de Neoplasia , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismoRESUMO
The main reason why therapeutic schemes fail in Glioblastoma lies on its own peculiarities as a cancer and on our failure to fully decipher them. Fast tumor evolution, invasiveness and incomplete surgical resection contribute to disease recurrence, therapy resistance and high mortality. More faithful models must be developed to address Glioblastoma biology and better clinical guidance. Research studies are discussed in this review that: i) improve understanding and assessment of the growth mechanisms of Glioblastoma and ii) develop preclinical models (in vitro-in vivo-in silico) that mimic patient's tumor (phenocopying) in order to provide better prediction of response to therapies.
Assuntos
Glioblastoma , Humanos , Glioblastoma/patologia , Glioblastoma/terapiaRESUMO
Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with the suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed the induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth, and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Proteína da Leucemia Promielocítica/genética , Fator A de Crescimento do Endotélio Vascular , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Fatores de Transcrição/genética , Movimento CelularRESUMO
Intra-thymic T cell development is coordinated by the regulatory actions of SATB1 genome organizer. In this report, we show that SATB1 is involved in the regulation of transcription and splicing, both of which displayed deregulation in Satb1 knockout murine thymocytes. More importantly, we characterized a novel SATB1 protein isoform and described its distinct biophysical behavior, implicating potential functional differences compared to the commonly studied isoform. SATB1 utilized its prion-like domains to transition through liquid-like states to aggregated structures. This behavior was dependent on protein concentration as well as phosphorylation and interaction with nuclear RNA. Notably, the long SATB1 isoform was more prone to aggregate following phase separation. Thus, the tight regulation of SATB1 isoforms expression levels alongside with protein post-translational modifications, are imperative for SATB1's mode of action in T cell development. Our data indicate that deregulation of these processes may also be linked to disorders such as cancer.
RESUMO
Sall1 is a multi-zinc finger transcription factor that regulates kidney organogenesis. It is considered to be a transcriptional repressor, preferentially localized on heterochromatin. Mutations or deletions of the human SALL1 gene are associated with the Townes-Brocks syndrome. Despite its high expression, no function was yet assigned for Sall1 in embryonic stem (ES) cells. In the present study, we show that Sall1 is expressed in a differentiation-dependent manner and physically interacts with Nanog and Sox2, two components of the core pluripotency network. Genome-wide mapping of Sall1-binding loci has identified 591 genes, 80% of which are also targeted by Nanog. A large proportion of these genes are related to self-renewal and differentiation. Sall1 positively regulates and synergizes with Nanog for gene transcriptional regulation. In addition, our data show that Sall1 suppresses the ectodermal and mesodermal differentiation. Specifically, the induction of the gastrulation markers T brachyury, Goosecoid, and Dkk1 and the neuroectodermal markers Otx2 and Hand1 was inhibited by Sall1 overexpression during embryoid body differentiation. These data demonstrate a novel role for Sall1 as a member of the transcriptional network that regulates stem cell pluripotency.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Diferenciação Celular/fisiologia , Cromatina/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Proteína Homeobox Nanog , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/fisiologiaRESUMO
The promyelocytic leukemia protein (PML) is the core organizer of cognate nuclear bodies (PML-NBs). Through physical interaction or modification of diverse protein clients, PML-NBs regulate a multitude of - often antithetical- biological processes such as antiviral and stress response, inhibition of cell proliferation and autophagy, and promotion of apoptosis or senescence. Although PML was originally recognized as a tumor-suppressive factor, more recent studies have revealed a "double-faced" agent role for PML. Indeed, PML displayed tumor cell pro-survival and pro-migratory functions via inhibition of migration suppressing molecules or promotion of transforming growth factor beta (TGF-ß) mediated Epithelial-Mesenchymal Transition (EMT) that may promote cancer cell dissemination. In this line, PML was found to correlate with poor patient prognosis in distinct tumor contexts. Furthermore, in the last decade, a number of publications have implicated PML in the physiology of normal or cancer stem cells (CSCs). Promyelocytic leukemia protein activates fatty acid oxidation (FAO), a metabolic mechanism required for the asymmetric divisions and maintenance of hematopoietic stem cells (HSCs). In embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PML is required for maintenance of the naïve and acquisition of the induced pluripotency state, respectively. Correspondingly, PML ablation causes significant morphological gene expression and lineage choice changes. In this review, we focus on the mechanisms orchestrated by PML and PML-NBs in cancer and healthy stem cells, from cell physiology to the regulation of chromatin dynamics.
Assuntos
Neoplasias , Proteína da Leucemia Promielocítica , Fatores de Transcrição , Autofagia , Humanos , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Major Glioblastoma's hallmarks include proliferation, invasion and heterogeneity. Biological 3D tumor spheroid models can serve as intermediate systems between traditional 2D cell culture and complex in vivo models. Tumor spheroids have been shown to more accurately reproduce the spatial organization and microenvironmental factors of in vivo micro-tumors, such as relevant gradients of nutrients and other molecular agents, while they maintain cell-to-cell and cell-to-matrix interactions. In vitro 3D assays are useful to monitor these properties. Here, we test the suitability of the well-known T98â¯G Glioblastoma cell line in such a 3D assay. The doubling time and death rate parameters of T98â¯G are estimated, as well as their spheroidal growth-expansion curves with and without the presence of basement membrane substrate. The T98â¯G invasive profile is characterized by collective morphology and proliferation-associated invasion. We show that the T98â¯G secondary GB cell line exhibits both invasive and proliferative capabilities in 3D and thus, can serve as control cell line for the 3D in vitro study of primary GB cell cultures.
Assuntos
Glioblastoma , Modelos Biológicos , Esferoides Celulares , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologiaRESUMO
The multitasking promyelocytic leukemia (PML) protein was originally recognized as a tumor-suppressive factor, but more recent evidence has implicated PML in tumor cell prosurvival actions and poor patient prognosis in specific cancer settings. Here, we report that inducible PMLIV expression inhibits cell proliferation as well as self-renewal and impairs cell cycle progression of breast cancer cell lines in a reversible manner. Transcriptomic profiling identified a large number of PML-deregulated genes associated with various cell processes. Among them, cell cycle- and division-related genes and their cognitive regulators are highly ranked. In this study, we focused on previously unknown PML targets, namely the Forkhead transcription factors. PML suppresses the Forkhead box subclass M1 (FOXM1) transcription factor at both the RNA and protein levels, along with many of its gene targets. We show that FOXM1 interacts with PMLIV primarily via its DNA-binding domain and dynamically colocalizes in PML nuclear bodies. In parallel, PML modulates the activity of Forkhead box O3 (FOXO3), a factor opposing certain FOXM1 activities, to promote cell survival and stress resistance. Thus, PMLIV affects the balance of FOXO3 and FOXM1 transcriptional programs by acting on discrete gene subsets to favor both growth inhibition and survival. Interestingly, PMLIV-specific knockdown mimicked ectopic expression vis-à-vis loss of proliferative ability and self-renewal, but also led to loss of survival ability as shown by increased apoptosis. We propose that divergent or similar effects on cell physiology may be elicited by high or low PMLIV levels dictated by other concurrent genetic or epigenetic cancer cell states that may additionally account for its disparate effects in various cancer types.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fatores de Transcrição Forkhead/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Western Blotting , Neoplasias da Mama/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Imunofluorescência , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Fatores de Transcrição Forkhead/genética , Células HEK293 , Humanos , Imunoprecipitação , Células MCF-7 , Análise de Sequência com Séries de Oligonucleotídeos , Proteína da Leucemia Promielocítica/genéticaRESUMO
The deacetylase inhibitor Trichostatin A (TSA) induces the transcription of the Major Histocompatibility Class II (MHC II) DRA gene in a way independent of the master coactivator CIITA. To analyze the molecular mechanisms by which this epigenetic regulator stimulates MHC II expression, we used chromatin immunoprecipitation (ChIP) assays to monitor the alterations in histone modifications that correlate with DRA transcription after TSA treatment. We found that a dramatic increase in promoter linked histone acetylation is followed by an increase in Histone H3 lysine 4 methylation and a decrease of lysine 9 methylation. Fluorescence recovery after photobleaching (FRAP) experiments showed that TSA increases the mobility of HDAC while decreasing the mobility of the class II enhanceosome factor RFX5. These data, in combination with ChIP experiments, indicate that the TSA-mediated induction of DRA transcription involves HDAC relocation and enhanceosome stabilization. In order to gain a genome-wide view of the genes responding to inhibition of deacetylases, we compared the transcriptome of B cells before and after TSA treatment using Affymetrix microarrays. This analysis showed that in addition to the DRA gene, the entire MHC II family and the adjacent histone cluster that are located in chromosome 6p21-22 locus are strongly induced by TSA. A complex pattern of gene reprogramming by TSA involves immune recognition, antiviral, apoptotic and inflammatory pathways and extends the rationale for using Histone Deacetylase Inhibitors (HDACi) to modulate the immune response.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Genes MHC da Classe II , Inibidores de Histona Desacetilases , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Ativação Transcricional , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/genética , Cadeias alfa de HLA-DR , Células HeLa , Humanos , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/metabolismoRESUMO
Breast cancer and Glioblastoma brain cancer are severe malignancies with poor prognosis. In this study, primary Glioblastoma and secondary breast cancer spheroids are formed and treated with the well-known Temozolomide and Doxorubicin chemotherapeutics, respectively. High resolution imaging of both primary and secondary cancer cell spheroids is possible using a custom multi-angle Light Sheet Fluorescence Microscope. Such a technique is successful in realizing preclinical drug screening, while enables the discrimination among physiologic tumor parameters.
Assuntos
Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Neoplasias , Esferoides Celulares , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , HumanosRESUMO
Nuclear Factor Y (NF-Y) was first described as one of the CCAAT binding factors. Although CCAAT motifs were found to be present in various genes, NF-Y attracted a lot of interest early on, due to its role in Major Histocompatibility Complex (MHC) gene regulation. MHC genes are crucial in immune response and show peculiar expression patterns. Among other conserved elements on MHC promoters, an NF-Y binding CCAAT box was found to contribute to MHC transcriptional regulation. NF-Y along with other DNA binding factors assembles in a stereospecific manner to form a multiprotein scaffold, the MHC enhanceosome, which is necessary but not sufficient to drive transcription. Transcriptional activation is achieved by the recruitment of yet another factor, the class II transcriptional activator (CIITA). In this review, we briefly discuss basic findings on MHCII transcription regulation and we highlight NF-Y different modes of function in MHCII gene activation. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.
Assuntos
Fator de Ligação a CCAAT/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Transcrição Gênica/imunologia , Ativação Transcricional/imunologia , Animais , Fator de Ligação a CCAAT/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Transcrição Gênica/genética , Ativação Transcricional/genéticaRESUMO
Promyelocytic leukemia protein (PML), the main constituent of PML nuclear bodies, regulates various physiological processes in different cell types. However, little is known about its functions in embryonic stem cells (ESC). Here, we report that PML contributes to ESC self-renewal maintenance by controlling cell-cycle progression and sustaining the expression of crucial pluripotency factors. Transcriptomic analysis and gain- or loss-of-function approaches showed that PML-deficient ESC exhibit morphological, metabolic, and growth properties distinct to naive and closer to the primed pluripotent state. During differentiation of embryoid bodies, PML influences cell-fate decisions between mesoderm and endoderm by controlling the expression of Tbx3. PML loss compromises the reprogramming ability of embryonic fibroblasts to induced pluripotent stem cells by inhibiting the transforming growth factor ß pathway at the very early stages. Collectively, these results designate PML as a member of the regulatory network for ESC naive pluripotency and somatic cell reprogramming.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Ectoderma/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Proteína da Leucemia Promielocítica/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Transcriptoma , Fator de Crescimento Transformador beta/metabolismoRESUMO
Over the past years, microRNAs (miRNAs) have emerged as crucial factors that regulate self-renewal and differentiation of embryonic stem cells (ESCs). Although much is known about their role in maintaining ESC pluripotency, the mechanisms by which they affect cell fate decisions remain poorly understood. By performing deep sequencing to profile miRNA expression in mouse ESCs (mESCs) and differentiated embryoid bodies (EBs), we identified four differentially expressed miRNAs. Among them, miR-191 and miR-16-1 are highly expressed in ESCs and repress Smad2, the most essential mediator of Activin-Nodal signaling, resulting in the inhibition of mesendoderm formation. miR-23a, which is also down-regulated in the differentiated state, suppresses differentiation toward the endoderm and ectoderm lineages. We further identified miR-421 as a differentiation-associated regulator through the direct repression of the core pluripotency transcription factor Oct4 and the bone morphogenetic protein (BMP)-signaling components, Smad5 and Id2. Collectively, our findings uncover a regulatory network between the studied miRNAs and both branches of TGF-ß/BMP-signaling pathways, revealing their importance for ESC lineage decisions.
Assuntos
Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , MicroRNAs/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Proteína 2 Inibidora de Diferenciação/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína Smad2/metabolismo , Proteína Smad5/metabolismoRESUMO
Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture.
Assuntos
Imageamento Tridimensional/métodos , Imagem Molecular/métodos , Imagem Óptica/métodos , Tomografia Óptica/métodos , Animais , Desenho de Equipamento , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Reprodutibilidade dos TestesRESUMO
We show here that steroid receptor coactivator 1 (SRC-1) is a coactivator of MHC class II genes that stimulates their interferon gamma (IFNgamma) and class II transactivator (CIITA)-mediated expression. SRC-1 interacts physically with the N-terminal activation domain of CIITA through two regions: one central [extending from amino acids (aa) 360-839] that contains the nuclear receptors binding region and one C-terminal (aa 1138-1441) that contains the activation domain 2. Using chromatin immunoprecipitation assays we show that SRC-1 recruitment on the class II promoter is enhanced upon IFNgamma stimulation. Most importantly, SRC-1 relieves the inhibitory action of estrogens on the IFNgamma-mediated induction of class II genes in transient transfection assays. We provide evidence that inhibition by estradiol is due to multiple events such as slightly reduced recruitment of CIITA and SRC-1 and severely inhibited assembly of the preinitiation complex.
Assuntos
Interferon gama/fisiologia , Esteroides/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Neoplasias da Mama , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Primers do DNA , Genes MHC da Classe II , Genes Reporter , Células HeLa , Antígenos de Histocompatibilidade Classe II/imunologia , Histona Acetiltransferases , Humanos , Luciferases/genética , Camundongos , Coativador 1 de Receptor Nuclear , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Transcrição/genética , TransfecçãoRESUMO
Pluripotency of embryonic stem cells (ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal transducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors (cancer stem cells), provides a common conceptual and research framework for basic and applied stem cell biology. In this review, we highlight current results on biomarkers, gene signatures, signaling pathways and epigenetic regulators that are common in embryonic and cancer stem cells. We discuss their role in determining the cell phenotype and finally, their potential use to design next generation biological and pharmaceutical approaches for regenerative medicine and cancer therapies.
RESUMO
Cell adhesion molecules (CAMs) of the immunoglobulin superfamily (IgSF) exhibit restricted spatial and temporal expression profiles requiring a tight regulatory program during development. The rodent glycoprotein TAG-1 and its orthologs TAX-1 in the human and axonin-1 in chick are cell adhesion molecules belonging to the contactin/F3 subgroup of the IgSF. TAG-1 is expressed in restricted subsets of central and peripheral neurons, not only during development but also in adulthood, and is implicated in neurite outgrowth, axon guidance and fasciculation, as well as neuronal migration. In an attempt to identify the regulatory elements that guide the neuronal expression of TAG-1, we have isolated genomic clones containing 4 kb of the TAX-1 upstream sequence and used them to drive the expression of the LacZ reporter gene in transgenic mice. We demonstrate that this sequence includes elements not only sufficient to restrict expression to the nervous system, but also to recapitulate to a great extent the endogenous pattern of the TAG-1 expression in the developing CNS.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Envelhecimento/genética , Animais , Animais Recém-Nascidos , Sequência de Bases/genética , Adesão Celular/genética , Contactina 2 , Feto , Genes Reporter/genética , Humanos , Óperon Lac/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Sistema Nervoso/embriologia , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Neurônios/citologia , RNA Mensageiro/metabolismo , Transgenes/genéticaRESUMO
Major histocompatibility complexes class II are responsible for the antigen presentation that shapes the repertoire of the adaptive immune responses. All members of the MHCII family of genes are controlled by the same set of conserved transcription factors and promoter elements, resulting in coordinated transcription. We report the role of a previously unidentified AT-hook motif of the MHCII regulatory factor RFX5, and show that this is involved in regulating the transcription of the HLA-DQ, but not HLA-DR, MHCII isotype. Furthermore, PRMT6, an arginine methyltransferase known to methylate AT-hook motifs, downregulates the expression of HLA-DQ, but not HLA-DR, in an AT-hook-dependent manner. This can provide a fine-tuning mechanism for isotype-specific transcriptional regulation, where a post-translational modification modulates the relative levels of the MHCII isotypes.