RESUMEN
Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been implicated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5- Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells. [BMB Reports 2020; 53(11): 576-581].
Asunto(s)
Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Apoptosis/genética , Azepinas/farmacología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Inmunoprecipitación de Cromatina/métodos , Eucromatina/genética , Expresión Génica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Antígenos de Histocompatibilidad/fisiología , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/fisiología , Histonas/genética , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Unión al GTP Monoméricas/fisiología , Proteómica , Quinazolinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/genética , Transcriptoma/genéticaRESUMEN
BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a highly malignant epithelial tumor of the biliary tree with poor prognosis. In the current study, we present evidence that the histone-lysine methyltransferase G9a is up-regulated in human CCA and that G9a enhances CCA cell growth and invasiveness through regulation of the Hippo pathway kinase large tumor suppressor 2 (LATS2) and yes-associated protein (YAP) signaling pathway. APPROACH AND RESULTS: Kaplan-Meier survival analysis revealed that high G9a expression is associated with poor prognosis of CCA patients. In experimental systems, depletion of G9a by small interfering RNA/short hairpin RNA or inhibition of G9a by specific pharmacological inhibitors (UNC0642 and UNC0631) significantly inhibited human CCA cell growth in vitro and in severe combined immunodeficient mice. Increased G9a expression was also observed in mouse CCA induced by hydrodynamic tail vein injection of notch intracellular domain (NICD) and myr-Akt. Administration of the G9a inhibitor UNC0642 to NICD/Akt-injected mice reduced the growth of CCA, in vivo. These findings suggest that G9a inhibition may represent an effective therapeutic strategy for the treatment of CCA. Mechanistically, our data show that G9a-derived dimethylated H3K9 (H3K9me2) silenced the expression of the Hippo pathway kinase LATS2, and this effect led to subsequent activation of oncogenic YAP. Consequently, G9a depletion or inhibition reduced the level of H3K9me2 and restored the expression of LATS2 leading to YAP inhibition. CONCLUSIONS: Our findings provide evidence for an important role of G9a in cholangiocarcinogenesis through regulation of LATS2-YAP signaling and suggest that this pathway may represent a potential therapeutic target for CCA treatment.
Asunto(s)
Neoplasias de los Conductos Biliares/etiología , Proteínas de Ciclo Celular/fisiología , Colangiocarcinoma/etiología , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Factores de Transcripción/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Neoplasias de los Conductos Biliares/patología , Línea Celular Tumoral , Colangiocarcinoma/patología , Vía de Señalización Hippo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Ratones , Transducción de Señal/fisiologíaRESUMEN
Epigenetic dysregulation, which leads to the alteration of gene expression in the brain, is suggested as one of the key pathophysiological bases of ageing and neurodegeneration. Here we found that, in the late-stage familial Alzheimer's disease (FAD) mouse model, repressive histone H3 dimethylation at lysine 9 (H3K9me2) and euchromatic histone methyltransferases EHMT1 and EHMT2 were significantly elevated in the prefrontal cortex, a key cognitive region affected in Alzheimer's disease. Elevated levels of H3K9me2 were also detected in the prefrontal cortex region of post-mortem tissues from human patients with Alzheimer's disease. Concomitantly, H3K9me2 at glutamate receptors was increased in prefrontal cortex of aged FAD mice, which was linked to the diminished transcription, expression and function of AMPA and NMDA receptors. Treatment of FAD mice with specific EHMT1/2 inhibitors reversed histone hyper-methylation and led to the recovery of glutamate receptor expression and excitatory synaptic function in prefrontal cortex and hippocampus. Chromatin immunoprecipitation-sequencing (ChIP-seq) data indicated that FAD mice exhibited genome-wide increase of H3K9me2 enrichment at genes involved in neuronal signalling (including glutamate receptors), which was reversed by EHMT1/2 inhibition. Moreover, the impaired recognition memory, working memory, and spatial memory in aged FAD mice were rescued by the treatment with EHMT1/2 inhibitors. These results suggest that disrupted epigenetic regulation of glutamate receptor transcription underlies the synaptic and cognitive deficits in Alzheimer's disease, and targeting histone methylation enzymes may represent a novel therapeutic strategy for this prevalent neurodegenerative disorder.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Animales , Deleción Cromosómica , Cognición/fisiología , Trastornos del Conocimiento/genética , Disfunción Cognitiva/metabolismo , Metilación de ADN/genética , Modelos Animales de Enfermedad , Epigénesis Genética/genética , Hipocampo/metabolismo , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Lisina/genética , Trastornos de la Memoria/genética , Metilación , Ratones , Ratones Transgénicos , Corteza Prefrontal/metabolismo , Sinapsis/metabolismoRESUMEN
In the first half of the 20th century, the major histocompatibility complex (MHC) of the laboratory mouse, the H-2 complex, was defined by a combination of serology and genetics. In the second half of the 20th century, its human counterpart, the human leukocyte antigen (HLA) complex was similarly defined and shown to mediate rejection of allogeneic kidney grafts. The clinical relevance of the transplantation antigens created the field of transplant immunology, which aimed to reduce graft rejection by HLA matching of transplant donors and recipients, and to use immunosuppressive drugs to prevent and treat rejection. Because tissue transplantation is not a natural phenomenon, the relevance of the MHC for immunology and immune defense against microbial pathogens was frequently questioned. In the 1970s, the general observation that cytotoxic T-cell responses to viral infection required recognition of both a viral antigen and a transplantation antigen argued for the immunological importance of the MHC. Proving this point was not achieved until close to the end of the 20th century. This required detailed biochemical and structural analysis of the transplantation antigens, the viral antigens, and the T-cell receptors that recognized them. This century of research culminated in 1996 with the three-dimensional crystallographic structure of the complex of these three components. In this complex is MAC, the very first HLA antigen to be detected and now more formally known as HLA-A*02:01.
Asunto(s)
Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/fisiología , Trasplante de Tejidos , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Moléculas de Adhesión Celular , Femenino , Rechazo de Injerto/inmunología , Supervivencia de Injerto/inmunología , Antígenos H-2/química , Antígenos de Histocompatibilidad/historia , Historia del Siglo XX , Humanos , Ratones , Proteínas de Neoplasias/química , Proteínas de Neoplasias/inmunología , Embarazo , Trasplante de Piel , Solubilidad , Linfocitos T Citotóxicos/inmunología , Microglobulina beta-2/inmunologíaRESUMEN
Epigenetic mechanisms, including histone post-translational modifications, are central regulators of cell cycle control. The euchromatic G9a histone methyltransferase (G9a HMT) is a key enzyme catalyzing histone H3 methylation on lysines 9 and 27, and its dysregulation has been linked to uncontrolled proliferation of tumor cells. Here, we have investigated the effect of G9a HMT silencing on cell proliferation of microvascular endothelial cells, a process necessary to sustain tumor growth through the formation of the vascular capillary network. Inhibition of G9a HMT activity in human microvascular endothelial cells (HMEC-1) was performed either pharmacologically, by treatment of cells with BIX-01294 or chaetocin, or transcriptionally, using shRNA. Cell viability and proliferation were examined using the resazurin reduction assay, flow cytometry and immunostaining of phosphorylated checkpoint kinase 1 (pSer317Chk1). Expression of cell cycle- and redox homeostasis-related genes was determined by quantitative PCR. Reactive oxygen species production was measured by oxidation of the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate and the cell's total antioxidant capacity by using the ABTS assay. Inhibition of G9a HMT activity by BIX-01294 treatment or by shRNA attenuated the proliferation of HMEC-1, nuclear localization of phosphorylated Chk1, and induced cell cycle arrest in G1 phase. Transcriptional analysis demonstrated increased gene expression of the cyclin-dependent kinase (CDK) inhibitor p21, and also of Rb1, in BIX-01294 treated cells. Decreased proliferation rate was accompanied by enhanced antioxidant potential of HMEC-1 cells, as demonstrated by reduced production of reactive oxygen species, increased total antioxidant capacity and expression of the antioxidant enzymes catalase and superoxide dismutase 1. Collectively, our results demonstrate of the central role of G9a HMT in the promotion of endothelial cells proliferation, and suggest that endothelial G9a HMT may be a target in the treatment of vascular proliferative disorders and tumor neovascularization.
Asunto(s)
Proliferación Celular/fisiología , Células Endoteliales/fisiología , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Microvasos/citología , Azepinas/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Homeostasis , Humanos , Oxidación-Reducción , Quinazolinas/farmacología , ARN Interferente Pequeño/genéticaRESUMEN
Inflammation and impaired secretion by lacrimal and salivary glands are hallmarks of the autoimmune disease, Sjögren's Syndrome. These changes in the lacrimal gland promote dryness and inflammation of the ocular surface, causing pain, irritation and corneal damage. The changes that initiate and sustain autoimmune inflammation in the lacrimal gland are not well-established. Here we demonstrate that interferon-γ (IFN-γ) is significantly elevated in lacrimal gland and tears of the male NOD mouse, a model of autoimmune dacryoadenitis which exhibits many ocular characteristics of Sjögren's Syndrome, by 12 weeks of age early in lacrimal gland inflammation. Working either with primary cultured lacrimal gland acinar cells from BALB/c mice and/or rabbits, in vitro IFN-γ treatment for 48 hr decreased expression of Rab3D concurrent with increased expression of cathepsin S. Although total cellular cathepsin S activity was not commensurately increased, IFN-γ treated lacrimal gland acinar cells showed a significant increase in carbachol-stimulated secretion of cathepsin S similar to the lacrimal gland in disease. In vitro IFN-γ treatment did not increase the expression of most components of major histocompatibility complex (MHC) class II-mediated antigen presentation although antigen presentation was slightly but significantly stimulated in primary cultured lacrimal gland acinar cells. However, exposure of cultured human corneal epithelial cells to IFN-γ more robustly increased expression and activity of cathepsin S in parallel with increased expression and function of MHC class II-mediated antigen presentation. We propose that early elevations in IFN-γ contribute to specific features of ocular disease pathology in Sjögren's Syndrome.
Asunto(s)
Presentación de Antígeno/efectos de los fármacos , Catepsinas/metabolismo , Córnea/efectos de los fármacos , Interferón gamma/farmacología , Aparato Lagrimal/efectos de los fármacos , Animales , Células Cultivadas , Córnea/metabolismo , Córnea/patología , Antígenos de Histocompatibilidad/metabolismo , Antígenos de Histocompatibilidad/fisiología , Humanos , Interferón gamma/metabolismo , Interferón gamma/fisiología , Aparato Lagrimal/metabolismo , Aparato Lagrimal/patología , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Conejos , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patologíaRESUMEN
Recent evidence points to a role of chromatin in regulation of alternative pre-mRNA splicing (AS). In order to identify novel chromatin regulators of AS, we screened an RNAi library of chromatin proteins using a cell-based high-throughput in vivo assay. We identified a set of chromatin proteins that regulate AS. Using simultaneous genome-wide expression and AS analysis, we demonstrate distinct and non-overlapping functions of these chromatin modifiers on transcription and AS. Detailed mechanistic characterization of one dual function chromatin modifier, the H3K9 methyltransferase EHMT2 (G9a), identified VEGFA as a major chromatin-mediated AS target. Silencing of EHMT2, or its heterodimer partner EHMT1, affects AS by promoting exclusion of VEGFA exon 6a, but does not alter total VEGFA mRNA levels. The epigenetic regulatory mechanism of AS by EHMT2 involves an adaptor system consisting of the chromatin modulator HP1γ, which binds methylated H3K9 and recruits splicing regulator SRSF1. The epigenetic regulation of VEGFA is physiologically relevant since EHMT2 is transcriptionally induced in response to hypoxia and triggers concomitant changes in AS of VEGFA. These results characterize a novel epigenetic regulatory mechanism of AS and they demonstrate separate roles of epigenetic modifiers in transcription and alternative splicing.
Asunto(s)
Empalme Alternativo , Epigénesis Genética , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Factor A de Crecimiento Endotelial Vascular/genética , Hipoxia de la Célula , Línea Celular Tumoral , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Microscopía Fluorescente , Proteínas Nucleares/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We report that H3K9 HMTase G9a activates transcription of the cell cycle regulatory gene, p21, in p53-null H1299 cells. Positive regulation of p21 by G9a is independent of its HMTase activity. We demonstrate that G9a upregulates p21 via interaction with PCAF, and provide evidence that the activating complex is recruited to the p21 promoter upon DNA damage-inducing agent etoposide treatment. Our study suggests that G9a decreases proliferation and cell viability by increasing the level of p21-mediated apoptosis. Our results suggest that G9a functions as a coactivator for p21 transcription, and directs cells to undergo apoptosis.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Activación Transcripcional , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Células HEK293 , Humanos , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba , Factores de Transcripción p300-CBP/metabolismoRESUMEN
G9a/GLP and Polycomb Repressive Complex 2 (PRC2) are two major epigenetic silencing machineries, which in particular methylate histone H3 on lysines 9 and 27 (H3K9 and H3K27), respectively. Although evidence of a crosstalk between H3K9 and H3K27 methylations has started to emerge, their actual interplay remains elusive. Here, we show that PRC2 and G9a/GLP interact physically and functionally. Moreover, combining different genome-wide approaches, we demonstrate that Ezh2 and G9a/GLP share an important number of common genomic targets, encoding developmental and neuronal regulators. Furthermore, we show that G9a enzymatic activity modulates PRC2 genomic recruitment to a subset of its target genes. Taken together, our findings demonstrate an unanticipated interplay between two main histone lysine methylation mechanisms, which cooperate to maintain silencing of a subset of developmental genes.
Asunto(s)
Silenciador del Gen , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Histonas/metabolismo , Complejo Represivo Polycomb 2/fisiología , Proteína Potenciadora del Homólogo Zeste 2 , Regulación de la Expresión Génica , Células HeLa , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación , Regiones Promotoras GenéticasRESUMEN
Major histocompatibility complex (MHC) appears to be a suitable tool for solving various tasks of popu- lationgenetics. Information on genetic basis of immunity facilitates understanding of evolutionary his- tory and assessment of current state and prospects of studied population/species survival. On the one hand, MHC variability is maintained through pathogen dependent mechanisms, i.e., directional selection of individuals resistant to diseases, that are present in the environment and balancing selection which gives advantage to those individuals carrying unusual or rare alleles of MHC genes. On the other hand, MHC genes have an influence on reproduction efficiency of individuals. Because of MHC polygeny, its studying requires an application of methods that introduce additional stages between amplification of a certain gene segment and its sequencing. In the article, different tech- niques of allele separation are considered, as well as a simplified version of MHC variability analysis based on the examination of microsatellite loci. Despite the high information value of MHC, it is still not used in zoological studies as often as it deserves. Using as an example predatory mammals of Felidae family which contains quite a few threatened species, we show that a majority of studies on MHC in wild cats is descriptive ones and only few of them deal with genes comparative analysis. The rise of interest to the studies of major histocompatibility complex in non-model species may help not only in solving the fundamental problems of evolution and phylogenetic structure of the family but also in planning the measures for conservation of rare and endangered species exposed to various anthropogenic stresses.
Asunto(s)
Especies en Peligro de Extinción , Variación Genética/fisiología , Antígenos de Histocompatibilidad/fisiología , Complejo Mayor de Histocompatibilidad/fisiología , Animales , Gatos , Especificidad de la EspecieRESUMEN
The classical pathway complement regulator C4b-binding protein (C4BP) is composed of two polypeptides (α- and ß-chains), which form three plasma oligomers with different subunit compositions (α7ß1, α7ß0, and α6ß1). We show in this article that the C4BP α7ß0 isoform (hereafter called C4BP[ß(-)] [C4BP lacking the ß-chain]), overexpressed under acute-phase conditions, induces a semimature, tolerogenic state on human monocyte-derived dendritic cells (DCs) activated by a proinflammatory stimulus. C4BP isoforms containing ß-chain (α7ß1 and α6ß1; C4BP[ß(+)]) neither interfered with the normal maturation of DCs nor competed with C4BP(ß(-)) activity on these cells. Immature DCs (iDCs) treated with C4BP(ß(-)) retained high endocytic activity, but, upon LPS treatment, they did not upregulate surface expression of CD83, CD80, and CD86. Transcriptional profiling of these semimature DCs revealed that treatment with C4BP(ß(-)) prevented the induction of IDO and BIC-1, whereas TGF-ß1 expression was maintained to the level of iDCs. C4BP(ß(-))-treated DCs were also unable to release proinflammatory Th1 cytokines (IL-12, TNF-α, IFN-γ, IL-6, IL-8) and, conversely, increased IL-10 secretion. They prevented surface CCR7 overexpression and, accordingly, displayed reduced chemotaxis, being morphologically indistinguishable from iDCs. Moreover, C4BP(ß(-))-treated DCs failed to enhance allogeneic T cell proliferation, impairing IFN-γ production in these cells and, conversely, promoting CD4(+)CD127(low/neg)CD25(high)Foxp3(+) T cells. Deletion mutant analysis revealed that the complement control protein-6 domain of the α-chain is necessary for the tolerogenic activity of C4BP(ß(-)). Our data demonstrate a novel anti-inflammatory and immunomodulatory function of the complement regulator C4BP, suggesting a relevant role of the acute-phase C4BP(ß(-)) isoform in a number of pathophysiological conditions and potential applications in autoimmunity and transplantation.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Diferenciación Celular/inmunología , Proteína de Unión al Complemento C4b/fisiología , Células Dendríticas/química , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad/fisiología , Diferenciación Celular/genética , Proteína de Unión al Complemento C4b/química , Proteína de Unión al Complemento C4b/genética , Células Dendríticas/patología , Células HEK293 , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/genética , Humanos , Tolerancia Inmunológica/genética , Inflamación/genética , Inflamación/inmunología , Inflamación/prevención & control , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologíaRESUMEN
Histone H3 lysine-9 methyltransferase G9a/EHMT2/KMT1C is a key corepressor of gene expression. However, activation of a limited number of genes by G9a (independent of its catalytic activity) has also been observed, although the precise molecular mechanisms are unknown. By using RNAi in combination with gene expression microarray analysis, we found that G9a functions as a positive and a negative transcriptional coregulator for discrete subsets of genes that are regulated by the hormone-activated Glucocorticoid Receptor (GR). G9a was recruited to GR-binding sites (but not to the gene body) of its target genes and interacted with GR, suggesting recruitment of G9a by GR. In contrast to its corepressor function, positive regulation of gene expression by G9a involved G9a-mediated enhanced recruitment of coactivators CARM1 and p300 to GR target genes. Further supporting a role for G9a as a molecular scaffold for its coactivator function, the G9a-specific methyltransferase inhibitor UNC0646 did not affect G9a coactivator function but selectively decreased G9a corepressor function for endogenous target genes. Overall, G9a functioned as a coactivator for hormone-activated genes and as a corepressor in support of hormone-induced gene repression, suggesting that the positive or negative actions of G9a are determined by the gene-specific regulatory environment and chromatin architecture. These findings indicate distinct mechanisms of G9a coactivator vs. corepressor functions in transcriptional regulation and provide insight into the molecular mechanisms of G9a coactivator function. Our results also suggest a physiological role of G9a in fine tuning the set of genes that respond to glucocorticoids.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Receptores de Glucocorticoides/metabolismo , Transactivadores/metabolismo , Biocatálisis , Humanos , Receptores de Glucocorticoides/genética , Transcripción GenéticaRESUMEN
Breast cancers are highly heterogeneous but can be grouped into subtypes based on several criteria, including level of expression of certain markers. Claudin-low breast cancer (CLBC) is associated with early metastasis and resistance to chemotherapy, while gene profiling indicates it is characterized by the expression of markers of epithelial-mesenchymal transition (EMT) - a phenotypic conversion linked with metastasis. Although the epigenetic program controlling the phenotypic and cellular plasticity of EMT remains unclear, one contributor may be methylation of the E-cadherin promoter, resulting in decreased E-cadherin expression, a hallmark of EMT. Indeed, reduced E-cadherin often occurs in CLBC and may contribute to the early metastasis and poor patient survival associated with this disease. Here, we have determined that methylation of histone H3 on lysine 9 (H3K9me2) is critical for promoter DNA methylation of E-cadherin in three TGF-ß-induced EMT model cell lines, as well as in CLBC cell lines. Further, Snail interacted with G9a, a major euchromatin methyltransferase responsible for H3K9me2, and recruited G9a and DNA methyltransferases to the E-cadherin promoter for DNA methylation. Knockdown of G9a restored E-cadherin expression by suppressing H3K9me2 and blocking DNA methylation. This resulted in inhibition of cell migration and invasion in vitro and suppression of tumor growth and lung colonization in in vivo models of CLBC metastasis. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also paves a way for the development of new treatment strategies for CLBC.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Cadherinas/biosíntesis , Regulación Neoplásica de la Expresión Génica/genética , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Proteínas de Neoplasias/fisiología , Factores de Transcripción/fisiología , Adenocarcinoma/patología , Adulto , Anciano , Animales , Neoplasias de la Mama/patología , Cadherinas/genética , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/trasplante , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Antígenos de Histocompatibilidad/genética , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Metilación , Ratones , Ratones Endogámicos ICR , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/fisiología , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genéticaRESUMEN
The major histocompatibility complex (MHC) has been known to play a critical role in immune recognition since the 1950s. It was a surprise, then, in the 1970s when the first report appeared indicating MHC might also function in social signaling. Since this seminal discovery, MHC signaling has been found throughout vertebrates and its known functions have expanded beyond mate choice to include a suite of behaviors from kin-biased cooperation, parent-progeny recognition to pregnancy block. The widespread occurrence of MHC in social signaling has revealed conserved behavioral-genetic mechanisms that span vertebrates and includes humans. The identity of the signal's chemical constituents and the receptors responsible for the perception of the signal have remained elusive, but recent advances have enabled the identification of the key components of the behavioral circuit. In this chapter we organize recent findings from the literature and discuss them in relation to four nonmutually exclusive models wherein MHC functions as a signal of (i) individuality, (ii) relatedness, (iii) genetic compatibility and (iv) quality. We also synthesize current mechanistic studies, showing how knowledge about the molecular basis of MHC signaling can lead to elegant and informative experimental manipulations. Finally, we discuss current evidence relating to the primordial functions of the MHC, including the possibility that its role in social signaling may be ancestral to its central role in adaptive immunity.
Asunto(s)
Antígenos de Histocompatibilidad/fisiología , Complejo Mayor de Histocompatibilidad/fisiología , Modelos Inmunológicos , Transducción de Señal/fisiología , Animales , Femenino , Humanos , Masculino , EmbarazoRESUMEN
Histone methyltransferase G9a has been understood primarily as a corepressor of gene expression, but we showed previously that G9a positively regulates nuclear receptor-mediated transcription in reporter gene assays. Here, we show that endogenous G9a contributes to the estradiol (E(2))-dependent induction of some endogenous target genes of estrogen receptor (ER)α in MCF-7 breast cancer cells while simultaneously limiting the E(2)-induced expression of other ERα target genes. Thus, G9a has a dual and selective role as a coregulator for ERα target genes. The ERα binding regions associated with the pS2 gene, which requires G9a for E(2)-induced expression, are transiently occupied by G9a at 15 min after beginning E(2) treatment, suggesting that G9a coactivator function is by direct interaction with ERα target genes. Transient reporter gene assays with deletion mutants of G9a demonstrated that domains previously associated with the corepressor functions of G9a (C-terminal methyltransferase domain, ankyrin repeat domain, and cysteine-rich domain) were unnecessary for G9a coactivator function in ERα-mediated transcription. In contrast, the N-terminal domain of G9a was necessary and sufficient for enhancement of ERα-mediated transcription and for E(2)-induced occupancy of G9a on ERα binding sites associated with endogenous target genes of ERα. In addition to a previously identified activation domain, this region contains a previously uncharacterized ligand-dependent ERα binding function, indicating how G9a is recruited to the target genes. Therefore, the coactivator and corepressor functions of G9a involve different G9a domains and different molecular mechanisms.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Animales , Células COS , Línea Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Inmunoprecipitación de Cromatina , Estrógenos/metabolismo , Glutatión Transferasa/metabolismo , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Humanos , Metilación , Ratones , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/metabolismo , Transcripción GenéticaRESUMEN
Lysine 9 of histone 3 (H3K9) can be mono-, di-, or trimethylated, inducing distinct effects on gene expression and chromatin compaction. H3K9 methylation can be mediated by several histone methyltransferases (HKMTs) that possess mono-, di-, or trimethylation activities. Here we provide evidence that a subset of each of the main H3K9 HKMTs, G9a/KMT1C, GLP/KMT1D, SETDB1/KMT1E, and Suv39h1/KMT1A, coexist in the same megacomplex. Moreover, in Suv39h or G9a null cells, the remaining HKMTs are destabilized at the protein level, indicating that the integrity of these HKMTs is interdependent. The four HKMTs are recruited to major satellite repeats, a known Suv39h1 genomic target, but also to multiple G9a target genes. Moreover, we report a functional cooperation between the four H3K9 HKMTs in the regulation of known G9a target genes. Altogether, our data identify a H3K9 methylation multimeric complex.
Asunto(s)
Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Histonas/metabolismo , Metiltransferasas/fisiología , Proteína Metiltransferasas/fisiología , Proteínas Represoras/fisiología , ADN Satélite/metabolismo , Estabilidad de Enzimas , Regulación de la Expresión Génica , Células HeLa , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteína Metiltransferasas/genética , Proteína Metiltransferasas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The tumor suppressor p53 is regulated by numerous post-translational modifications. Lysine methylation has recently emerged as a key post-translational modification that alters the activity of p53. Here, we describe a novel lysine methylation site in p53 that is carried out by two homologous histone methyltransferases, G9a and Glp. G9a and Glp specifically methylate p53 at Lys(373), resulting mainly in dimethylation. During DNA damage, the overall level of p53 modified at Lys(373)me2 does not increase, despite the dramatic increase in total p53, indicating that Lys(373)me2 correlates with inactive p53. Further, reduction of G9a and/or Glp levels leads to a larger population of apoptotic cells. Examination of the Oncomine data base shows that G9a and Glp are overexpressed in various cancers compared with corresponding normal tissues, suggesting that they are putative oncogenes. These data reveal a new methylation site within p53 mediated by the methylases G9a and Glp and indicate that G9a is a potential inhibitory target for cancer treatment.
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Regulación de la Expresión Génica , Antígenos de Histocompatibilidad/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Lisina/química , Proteínas/fisiología , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/fisiología , Autoantígenos , Daño del ADN , Metilación de ADN , Proteínas de la Matriz de Golgi , Antígenos de Histocompatibilidad/genética , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación , Neoplasias/metabolismo , Péptidos/química , Procesamiento Proteico-Postraduccional , Proteínas/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/químicaRESUMEN
The co-receptors CD4 and CD8 are important in the activation of T cells primarily because of their ability to interact with the proteins of the MHC enhancing recognition of the MHC-peptide complex by the T cell receptor (TCR). An antigen-presenting cell presents a small number of antigenic peptides on its MHC molecules, in the presence of a much larger number of endogenous, mostly nonstimulatory, peptides. Recent work has demonstrated that these endogenous MHC-peptide complexes have an important role in modulating the sensitivity of the TCR. But the role of the endogenous nonstimulatory MHC-peptide complexes differs in MHC class I and class II-restricted T cells. This chapter discusses the data on the role of CD4 or CD8 co-receptors in T cell activation at the immunological synapse, and the role of non stimulatory MHC-peptide complexes in aiding antigen recognition.
Asunto(s)
Antígenos CD4/fisiología , Antígenos CD8/fisiología , Sinapsis Inmunológicas/fisiología , Animales , Presentación de Antígeno , Autoantígenos/inmunología , Transferencia Resonante de Energía de Fluorescencia , Antígenos de Histocompatibilidad/fisiología , Humanos , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/fisiologíaRESUMEN
The ecotropic viral integration site-1 (EVI-1) is a nuclear transcription factor and has an essential function in the proliferation/maintenance of haematopoietic stem cells. Aberrant expression of EVI-1 has been frequently found in myeloid leukaemia as well as in several solid tumours, and is associated with a poor patient survival. It was recently shown that EVI-1 associates with two different histone methyltransferases (HMTs), SUV39H1 and G9a. However, the functional roles of these HMTs in EVI-1-mediated leukemogenesis remain unclear. In this study, we showed that EVI-1 physically interacts with SUV39H1 and G9a, but not with Set9. Immunofluorescence analysis revealed that EVI-1 colocalizes with these HMTs in nuclei. We also found that the catalytically inactive form of SUV39H1 abrogates the transcriptional repression mediated by EVI-1, suggesting that SUV39H1 is actively involved in EVI-1-mediated transcriptional repression. Furthermore, RNAi-based knockdown of SUV39H1 or G9a in Evi-1-expressing progenitors significantly reduced their colony-forming activity. In contrast, knockdown of these HMTs did not impair bone marrow immortalization by E2A/HLF. These results indicate that EVI-1 forms higher-order complexes with HMTs, and this association has a role in the transcription repression and bone marrow immortalization. Targeting these HMTs may be of therapeutic benefit in the treatment for EVI-1-related haematological malignancies.