RESUMO
The ex vivo expansion and maintenance of long-term hematopoietic stem cells (LT-HSC) is crucial for stem cell-based gene therapy. A combination of stem cell factor (SCF), thrombopoietin (TPO), FLT3 ligand (FLT3) and interleukin 3 (IL3) cytokines has been commonly used in clinical settings for the expansion of CD34+ from different sources, prior to transplantation. To assess the effect of IL3 on repopulating capacity of cultured CD34+ cells, we employed the commonly used combination of STF, TPO and FILT3 with or without IL3. Expanded cells were transplanted into NSG mice, followed by secondary transplantation. Overall, this study shows that IL3 leads to lower human cell engraftment and repopulating capacity in NSG mice, suggesting a negative effect of IL3 on HSC self-renewal. We, therefore, recommend omitting IL3 from HSC-based gene therapy protocols.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Interleucina-3 , Animais , Humanos , Camundongos , Antígenos CD34 , Células Cultivadas , Citocinas/farmacologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas , Interleucina-3/farmacologia , Fator de Células-Tronco/farmacologia , Trombopoetina/farmacologiaRESUMO
BACKGROUND: Leukocyte infiltration into the central nervous system is an important feature of multiple sclerosis (MS) pathology. Among the infiltrating cells, monocytes comprise the largest population and are considered to play a dual role in the course of the disease. The enzyme tissue transglutaminase (TG2), produced by monocytes, plays a central role in monocyte adhesion/migration in animal models of MS. In the present study, we questioned whether TG2 expression is altered in monocytes from MS patients compared to healthy control (HC) subjects. Moreover, we determined the inflammatory status of these TG2-expressing monocytes, what inflammatory factor regulates TG2 expression, and whether TG2 can functionally contribute to their adhesion/migration processes. METHODS: Primary human monocytes from MS patients and HC subjects were collected, RNA isolated and subjected to qPCR analysis. Human THP-1 monocytes were lentivirally transduced with TG2 siRNA or control and treated with various cytokines. Subsequently, mRNA levels of inflammatory factors, adhesion properties, and activity of RhoA were analyzed in interleukin (IL)-4-treated monocytes. RESULTS: TG2 mRNA levels are significantly increased in monocytes derived from MS patients compared to HC subjects. In addition, correlation analyses indicated that TG2-expressing cells display a more anti-inflammatory, migratory profile in MS patients. Using THP-1 monocytes, we observed that IL-4 is a major trigger of TG2 expression in these cells. Furthermore, knockdown of TG2 expression leads to a pro-inflammatory profile and reduced adhesion/migration properties of IL-4-treated monocytes. CONCLUSIONS: TG2-expressing monocytes in MS patients have a more anti-inflammatory profile. Furthermore, TG2 mediates IL-4-induced anti-inflammatory status in THP-1 monocytes, adhesion, and cytoskeletal rearrangement in vitro. We thus propose that IL-4 upregulates TG2 expression in monocytes of MS patients, driving them into an anti-inflammatory status.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Inflamação/metabolismo , Monócitos/metabolismo , Esclerose Múltipla/metabolismo , Transglutaminases/metabolismo , Adulto , Idoso , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 2 Glutamina gama-Glutamiltransferase , Adulto JovemRESUMO
The Wnt-responsive transcription factor T cell factor 1 (Tcf1) is well known for its role in thymic T cell development and the formation of memory CD8(+) T cells. However, its role in the initial phases of CD8(+) T effector cell formation has remained unexplored. We report that high levels of Wnt signaling and Tcf1 are operational in naive and memory CD8(+) T cells, whereas Wnt signaling and Tcf1 were low in effector CD8(+) T cells. CD8(+) T cells deficient in Tcf1 produce IFN-γ more rapidly, coinciding with increased demethylation of the IFN-γ enhancer and higher expression of the transcription factors Tbet and Blimp1. Moreover, virus-specific Tcf1(-/-) CD8(+) T cells show accelerated expansion in acute infection, which is associated with increased IFN-γ and TNF production and lower viral load. Genetic complementation experiments with various Tcf1 isoforms indicate that Tcf1 dosage and protein stability are critical in suppressing IFN-γ production. Isoforms lacking the ß-catenin binding domain are equally effective in inhibiting CD8(+) effector T cell formation. Thus, Tcf1 functions as a repressor of CD8(+) effector T cell formation in a ß-catenin/Wnt-independent manner.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Interferon gama/metabolismo , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Células Cultivadas , Citotoxicidade Imunológica , Metilação de DNA , Dosagem de Genes , Fator 1-alfa Nuclear de Hepatócito/genética , Memória Imunológica , Interferon gama/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Estabilidade Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Regulação para Cima , Carga Viral , VirosesRESUMO
The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.
Assuntos
Anticorpos/farmacologia , Antineoplásicos/farmacologia , Apoptose/genética , Cromatina/genética , Neoplasias Colorretais/terapia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Cisplatino/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/radioterapia , Resistencia a Medicamentos Antineoplásicos , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Receptor fas/imunologiaRESUMO
Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining patients carry unknown genetic defects (ICF2) but share with ICF1 patients the same immunological and epigenetic features, including hypomethylation of juxtacentromeric repeat sequences. We performed homozygosity mapping in five unrelated ICF2 patients with consanguineous parents and then performed whole-exome sequencing in one of these patients and Sanger sequencing in all to identify mutations in the zinc-finger- and BTB (bric-a-bric, tramtrack, broad complex)-domain-containing 24 (ZBTB24) gene in four consanguineously descended ICF2 patients. Additionally, we found ZBTB24 mutations in an affected sibling pair and in one patient for whom it was not known whether his parents were consanguineous. ZBTB24 belongs to a large family of transcriptional repressors that include members, such as BCL6 and PATZ1, with prominent regulatory roles in hematopoietic development and malignancy. These data thus indicate that ZBTB24 is involved in DNA methylation of juxtacentromeric DNA and in B cell development and/or B and T cell interactions. Because ZBTB24 is a putative DNA-binding protein highly expressed in the lymphoid lineage, we predict that by studying the molecular function of ZBTB24, we will improve our understanding of the molecular pathophysiology of ICF syndrome and of lymphocyte biology in general.
Assuntos
Centrômero/genética , Metilação de DNA/genética , Proteínas Repressoras/genética , Dedos de Zinco , Adolescente , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA , Epigenômica , Face/anormalidades , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Masculino , Mutação , Linhagem , Doenças da Imunodeficiência PrimáriaRESUMO
Tight regulation of MHC class I gene expression is critical for CD8 T cell activation and host adaptive-immune responses. The promoters of MHC class I genes contain a well-conserved core module, the W/S-X-Y motif, which assembles a nucleoprotein complex termed MHC enhanceosome. A member of the nucleotide-binding domain, leucine-rich repeat (NLR) protein family, NLRC5, is a newly identified transcriptional regulator of MHC class I genes. NLRC5 associates with and transactivates the proximal promoters of MHC class I genes, although the molecular mechanism of transactivation has not been understood. In this article, we show that NLRC5-mediated MHC class I gene induction requires the W/S and X1, X2 cis-regulatory elements. The transcription factors RFX5, RFXAP, and RFXANK/B, which compose the RFX protein complex and associate with the X1 box, cooperate with NLRC5 for MHC class I expression. Coimmunoprecipitation experiments revealed that NLRC5 specifically interacts with the RFX subunit RFXANK/B via its ankyrin repeats. In addition, we show that NLRC5 can cooperate with ATF1 and the transcriptional coactivators CBP/p300 and general control nonderepressible 5, which display histone acetyltransferase activity. Taken together, our data suggest that NLRC5 participates in an MHC class I-specific enhanceosome, which assembles on the conserved W/S-X-Y core module of the MHC class I proximal promoters, including the RFX factor components and CREB/ATF1 family transcription factors, to promote MHC class I gene expression.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Antígenos HLA-B/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fatores de Transcrição/fisiologia , Fator 1 Ativador da Transcrição/genética , Fator 1 Ativador da Transcrição/fisiologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Células HEK293 , Antígenos HLA-B/biossíntese , Humanos , Família Multigênica , Regiões Promotoras Genéticas , Fatores de Transcrição de Fator Regulador X , Sequências Reguladoras de Ácido Nucleico/imunologia , Fatores de Transcrição/genética , Ativação Transcricional/imunologiaRESUMO
Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type-specific expression of CCR5. In this study, we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4(+) T lymphocytes as well as in CD14(+) monocytes. In normal naïve T cells and CD14(+) monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5-expressing CD14(+) monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non-CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14(+) monocytes, this indicates a dominant role for AcH3 in CCR5 transcription. We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukaemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore, RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.
Assuntos
Antagonistas dos Receptores CCR5 , Epigênese Genética/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Linfócitos/metabolismo , Receptores CCR5/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Imunoprecipitação da Cromatina , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Histonas/metabolismo , Humanos , Imunomodulação/genética , Células Jurkat , Linfócitos/efeitos dos fármacos , Modelos Imunológicos , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR5/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This chapter describes recent advances in our understanding how epigenetic events control immune functions with emphasis on transcriptional regulation of major histocompatibility complex ClassI (MIC-I) and Class II (MHC-II) genes. MHC-I and MHC-II molecules play an essential role in the adaptive immune response by virtue of their ability to present peptides, respectively to CD8+ and CD4+ T cells. Central to the onset of an adequate immune response to pathogens is the presentation of pathogen-derived peptides in the context of MHC-II molecules by antigen presenting cells (APCs) to CD4+ T cells of the immune system. In particular dendritic cells are highly specialized APCs that are capable to activate naïve T cells. Given their central role in adaptive immunity, MHC-I and MHC-II genes are regulated in a tight fashion at the transcriptional level to meet with local requirements of an effective antigen-specific immune response. In these regulatory processes the MHC2TA encoded Class II transactivator (CIITA) plays a crucial role. CIITA is essential for transcriptional activation of all MHC-I genes, whereas it plays an ancillary function in the transcriptional control of MHC-I genes. The focus of this chapter therefore will be on the transcription factors that interact with conserved cis-acting promoter elements and epigenetic mechanisms that modulate cell type-specific regulation of MHC-I, MHC-I, and MHC2TA genes. Furthermore, we will also briefly discuss how genetic and epigenetic mechanisms contribute to T helper cell differentiation.
Assuntos
Epigênese Genética , Imunidade , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Animais , Apresentação de Antígeno , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Complexo Principal de Histocompatibilidade , Proteínas Nucleares/genética , Linfócitos T Auxiliares-Indutores/citologia , Transativadores/genéticaRESUMO
Ewing sarcoma (EWS) is a tumour most commonly arising in bone, although on occasion in soft tissue, with a poor prognosis in patients with refractory or relapsed disease, despite multimodal therapy. Immunotherapeutic strategies based on tumour-reactive T and/or natural killer cells may improve the treatment of advanced-stage EWS. Since cellular immune recognition critically depends on human leukocyte antigen (HLA) expression, knowledge about HLA expression in EWS is crucial in the design of cellular immunotherapeutic strategies. Constitutive and IFNgamma-induced HLA class I expression was analysed in EWS cell lines (n = 6) by flow cytometry, using antibodies against both monomorphic and allele-specific antigens. Expression of antigen processing pathway components and beta-2 microglobulin (beta2m) was assessed by western blot. Expression of class II transactivator (CIITA), and its contribution to HLA class II expression, was evaluated by qRT-PCR, transduction assays, and flow cytometry. beta2m/HLA class I and class II expression was validated in EWS tumours (n = 67) by immunofluorescence. Complete or partial absence of HLA class I expression was observed in 79% of EWS tumours. Lung metastases consistently lacked HLA class I and sequential tumours demonstrated a tendency towards decreased expression upon disease progression. Together with absent or low constitutive expression levels of specific HLA class I loci and alleles, and differential induction of identical alleles by IFNgamma in different cell lines, these results may reflect the existence of an immune escape mechanism. Inducible expression of TAP-1/-2, tapasin, LMP-2/-7, and the beta2m/HLA class I complex by IFNgamma suggests that regulatory mechanisms are mainly responsible for heterogeneity in constitutive class I expression. EWSs lack IFNgamma-inducible HLA class II, due to lack of functional CIITA. The majority of EWS tumours, particularly if advanced-stage, exhibit complete or partial absence of both classes of HLA. This knowledge will be instrumental in the design of cellular immunotherapeutic strategies for advanced-stage EWS.
Assuntos
Neoplasias Ósseas/imunologia , Antígenos de Histocompatibilidade Classe I/análise , Sarcoma de Ewing/imunologia , Adolescente , Apresentação de Antígeno , Biomarcadores/análise , Western Blotting , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Citometria de Fluxo , Antígenos HLA-DR/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Imunização , Imuno-Histoquímica , Interferon gama/farmacologia , Estimativa de Kaplan-Meier , Proteínas Nucleares/genética , Sarcoma de Ewing/mortalidade , Sarcoma de Ewing/secundário , Estatísticas não Paramétricas , Transativadores/genética , Células Tumorais Cultivadas , Evasão Tumoral , Microglobulina beta-2/análiseRESUMO
Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen's flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.
Assuntos
Ensaio de Amplificação de Sinal de DNA Ramificado , DNA/biossíntese , Citometria de Fluxo , Regulação da Expressão Gênica , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Análise de Célula Única , Animais , Células Cultivadas , Vetores Genéticos , Humanos , Camundongos , Transdução Genética , TransgenesRESUMO
MHC-II deficiency is recognized by defects in components of the RFX complex or CIITA. In this study, we have characterized at the molecular level the putative defect in MHC-II regulatory factors of a recently identified MHC-II deficiency patient (FGK). We found that this patient lacked detectable levels of mRNA and protein of the RFX complex subunit RFXAP. It was subsequently established that the RFXAP gene in FGK differed from wild type RFXAP by a homozygous 75bp insertion in the 5'-UTR, which impaired the activity of the FGK RFXAP promoter. The transcriptional silent state of RFXAP correlated with reduced recruitment of RNA polymerase II to FGK RFXAP chromatin. Together, this insertion in the promoter region represents a novel type of MHC-II gene silencing in MHC-II deficiency patients.
Assuntos
Inativação Gênica , Imunodeficiência Combinada Severa/genética , Fatores de Transcrição/genética , Transcrição Gênica , Regiões 5' não Traduzidas/química , Regiões 5' não Traduzidas/genética , Pareamento de Bases , Sequência de Bases , Southern Blotting , Pré-Escolar , Cromatina/metabolismo , Feminino , Células HeLa , Antígenos de Histocompatibilidade Classe I/imunologia , Homozigoto , Humanos , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Mutagênese Insercional , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
Cell lines established from tumor tissue of cutaneous melanoma biopsies often display constitutive and IFNgamma-inducible expression of MHC class II molecules. The expression of MHC class II molecules in melanoma is associated with an overall poor prognosis and unfavorable clinical outcome. We have analyzed the DNA elements and interacting transcription factors that control the constitutive and IFNgamma-inducible expression of the class II transactivator (CIITA), a co-activator essential for transcription of all MHC class II genes. Our studies reveal the activation of multiple CIITA promoter regions (CIITA-PII, -PIII and -PIV) in melanoma cell lines for both the constitutive and IFNgamma-inducible expression of MHC class II molecules. Furthermore, we show that constitutive and IFNgamma-inducible expression of the CIITA-PIII isoform is governed by separate regulatory elements within the PIII upstream regulatory region (PURR). Similarly constitutive activation in melanoma of CIITA-PII, CIITA-PIII, and CIITA-PIV does not require components of the IFNgamma signaling pathway. However, these components are readily recruited to the PURR and CIITA-PIV after exposure of cells to IFNgamma and account for the IFNgamma-induced expression of CIITA. Together, our data reveal the contribution of distinct elements and factors in the constitutive and IFNgamma-inducible expression of CIITA in melanoma cell lines of the skin.
Assuntos
Antivirais/farmacologia , Regulação Neoplásica da Expressão Gênica , Interferon gama/farmacologia , Melanoma/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares/biossíntese , Elementos de Resposta , Transativadores/biossíntese , Antivirais/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Genes MHC da Classe II/imunologia , Células HeLa , Humanos , Interferon gama/imunologia , Células Jurkat , Melanoma/genética , Melanoma/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Elementos de Resposta/imunologia , Transativadores/genética , Transativadores/imunologiaRESUMO
Lack of expression of major histocompatibility complex (MHC) molecules of both classes is frequently noted on tumour cells . It is thought that in this way tumour cells escape immunosurveillance. The genes encoding both classes of MHC molecules are localized on the distal part of chromosome 6 (6p21.3). The class II transactivator (CIITA), encoded by the MHC2TA gene, is essential for transcriptional activation of all MHC-II genes, while it has a helper function in the transcriptional regulation of MHC-I genes (with the exception of human leukocyte antigen (HLA)-G) and of the gene encoding beta2-microglobulin (beta2m) . Here we discuss our current knowledge on the expression characteristics of MHC2TA and argue for an important role of epigenetic factors and mechanisms in the transcriptional silencing of MHC2TA in cancer cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Genes MHC da Classe II , Neoplasias/genética , Proteínas Nucleares/genética , Transativadores/genética , Transcrição Gênica , Animais , HumanosRESUMO
Overexpression of LMO2 is known to be one of the causes of T-cell acute lymphoblastic leukemia (T-ALL) development; however, the mechanisms behind its oncogenic activity are incompletely understood. LMO2-overexpressing transgenic mouse models suggest an accumulation of immature T-cell progenitors in the thymus as the main preleukemic event. The effects of LMO2 overexpression on human T-cell development in vivo are unknown. Here, we report studies of a humanized mouse model transplanted with LMO2-transduced human hematopoietic stem/progenitor cells. The effects of LMO2 overexpression were confined to the T-cell lineage; however, initially, multipotent cells were transduced. Three effects of LMO2 on human T-cell development were observed: (1) a block at the double-negative/immature single-positive stage, (2) an accumulation of CD4(+)CD8(+) double-positive CD3(-) cells, and (3) an altered CD8/CD4 ratio with enhanced peripheral T lymphocytes. Microarray analysis of sorted double-positive cells overexpressing LMO2 led to the identification of an LMO2 gene set that clustered with human T-ALL patient samples of the described "proliferative" cluster. In this article, we demonstrate previously unrecognized mechanisms by which LMO2 alters human T-cell development in vivo; these mechanisms correlate with human T-ALL leukemogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Expressão Gênica , Proteínas com Domínio LIM/genética , Proteínas Proto-Oncogênicas/genética , Linfócitos T/metabolismo , Animais , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Perfilação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismo , Camundongos , Linfócitos T/patologia , Transdução GenéticaRESUMO
PURPOSE: Monosomy 3 (M3) or the presence of a specific RNA expression profile, known as class 2, is strongly associated with death from uveal melanoma (UM). Given the important role of epigenetic processes in cancer development and progression, we compared the transcriptional profiles of a selection of epigenetic regulators between primary UM with a good and a bad prognosis. METHODS: Transcriptional levels of 59 epigenetic regulator genes were measured by quantitative PCR (qPCR) in 20 UM, 12 with monosomy of chromosome 3 (M3) and 8 with disomy of chromosome 3 (D3). Validation was performed in an independent cohort. Expression levels were compared to clinicopathological characteristics, including class type. Bisulfite sequencing was used to evaluate the role of DNA methylation in gene silencing. RESULTS: In the first set of tumors, general downregulation of transcription of the genes encoding epigenetic regulatory enzymes was seen in association with M3. The 10 genes with the highest differential expression between M3 and D3 were selected and were analyzed in a second set of tumors. In the validation set, significantly lower levels of KAT2B (P = 0.008), HDAC11 (P = 0.009), KMT1C (P = 0.05), KDM4B (P = 0.003), KDM6B (P = 0.04), and BMI-1 (P = 0.001) transcripts were found in tumors with M3/class 2. Methylation of C-phosphate-G (CpG) residues was not observed on the putative regulatory regions of KAT2B, KDM4B, or KDM6B. CONCLUSIONS: Expression levels of a number of histone-modifying genes and polycomb family members are significantly lower in uveal melanoma with monosomy 3/class 2, supporting a general dysregulation of epigenetic modifiers in UM with a bad prognosis.
Assuntos
Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Melanoma/genética , Neoplasias Uveais/genética , Adulto , Idoso , Metilação de DNA/genética , Progressão da Doença , Regulação para Baixo/genética , Feminino , Inativação Gênica , Genes Reguladores/genética , Humanos , Masculino , Melanoma/mortalidade , Melanoma/patologia , Pessoa de Meia-Idade , Prognóstico , Transcrição Gênica/genética , Úvea/patologia , Neoplasias Uveais/mortalidade , Neoplasias Uveais/patologiaRESUMO
AIMS: Alterations in epigenetic processes are frequently noted in human disease. These epigenetic processes involve methylation of DNA and post-translational modifications of histones. It is well established that in particular histone methylation plays a key role in gene transcription. In this study, we have investigated the relationship between triple methylation of lysine 27 in histone H3 (H3K27Me3) modifications and atherosclerotic plaque stage. MATERIALS AND METHODS: 28 peri-renal aortic tissue patches covering the entire spectrum of atherosclerotic plaque development were evaluated by immunohistochemistry for the levels of H3K27Me3, EZH2, JMJD3 and BMI1. KEY FINDINGS: The results of our studies are in support of a reduction in global levels of the H3K27Me3 modification in vessels with advanced atherosclerotic plaques. This reduction in H3K27Me3 levels is not accompanied by alterations in global levels of the corresponding histone methyltransferase EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2). Likewise no alterations in global levels of BMI1, a component of the PRC1 complex, which binds to H3K27Me3-modified histones or the global expression levels of the histone demethylase JMJD3, which removes the methyl marks on H3K27, were observed. SIGNIFICANCE: Together, our data show that in atherosclerosis development alterations in global levels of H3K27Me3 occur. The reduction in the number of nuclei in the tunica media that display the repressive H3K27Me3 mark in vessels with advanced atherosclerosis plaques therefore could be a reflection of the dynamic pattern of smooth muscle cell differentiation and proliferation associated with atherosclerotic disease.
Assuntos
Aorta/patologia , Metilação de DNA/fisiologia , Histonas/metabolismo , Placa Aterosclerótica/patologia , Análise de Variância , Aorta/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Placa Aterosclerótica/metabolismo , Complexo Repressor Polycomb 2/metabolismoRESUMO
Monocytes play a key role in immune system function. Chromatin remodeling is crucial for various differentiation and gene regulation processes and is rather well studied in T cells. However, for monocytes not much is known regarding how the epigenetic machinery influences the differentiation into various effector cell types. In the work presented here, we explore the epigenetic underpinnings of monocyte differentiation. By transcriptional profiling we show that transcription of lysine methyltransferases (KMTs) and in particular KMT1c is markedly up regulated after differentiation of monocytes into immature dendritic cells (iDCs). Specifically inhibiting KMT1c function, using the small-molecule inhibitor BIX-01294, changes the transcription levels of the DC marker DC-SIGN, but does not affect surface protein expression. Blocking global KMT activity, using DZNep, does influence monocyte differentiation into iDCs, indicated by a loss of DC-SIGN surface expression. When BIX-01294 and DZNep treatment was combined DC-SIGN expression was almost lost completely. This work shows that the activities of KMTs are required for successful differentiation of monocyte-derived dendritic cells. Furthermore it shows the importance of KMT inhibitors in the field of epigenetic immune therapy, which is still much focused around HDAC inhibitors.
Assuntos
Células Dendríticas/metabolismo , Epigênese Genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Monócitos/metabolismo , Acetilação , Adenosina/análogos & derivados , Adenosina/farmacologia , Azepinas/farmacologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Metilação , Monócitos/citologia , Monócitos/efeitos dos fármacos , Cultura Primária de Células , Quinazolinas/farmacologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Statins exert anti-inflammatory characteristics, besides their lipid lowering properties, and may display beneficial effects for the treatment of inflammatory diseases. One possible explanation is that statins interfere in the deregulated gene transcription patterns associated with immune-mediated diseases, although the precise mechanism is not fully understood. Besides gene regulatory proteins, epigenetic mechanisms play an important role in the orchestration of gene expression. Disturbances in the tightly controlled epigenetic mechanisms influence the cellular portrait of expressed genes resulting in the protein dysfunctions found in many inflammatory diseases. In this study, we found that simvastatin reduces secretion and gene expression of CCL2 in monocyte-derived immature dendritic cells and in type 1 macrophages, which is accompanied by increased levels of the 3meK27H3 and 3meK9H3 repressive histone marks and decreased levels of the permissive histone marks AcH3 and 3meK4H3 in CCL2 promoter chromatin. The repressive chromatin status of the CCL2 promoter region affected recruitment of the NF-κB p65 subunit, which controls CCL2 transcription. The down-regulation of CCL2 in these immune cells may therefore impact their chemotactic activity and reduce their recruitment to sites of tissue injury.
Assuntos
Quimiocina CCL2/genética , Cromatina/efeitos dos fármacos , Cromatina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Sinvastatina/farmacologia , Células Cultivadas , Quimiocina CCL2/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/citologia , Regiões Promotoras GenéticasRESUMO
Multiple Sclerosis (MS) is a demyelinating disease characterized by chronic inflammation of the central nervous system (CNS) gray and white matter. Although the cause of MS is unknown, it is widely appreciated that innate and adaptive immune processes contribute to its pathogenesis. These include microglia/macrophage activation, pro-inflammatory T-cell (Th1) responses and humoral responses. Additionally, there is evidence indicating that MS has a neurodegenerative component since neuronal and axonal loss occurs even in the absence of overt inflammation. These aspects also form the rationale for clinical management of the disease. However, the currently available therapies to control the disease are only partially effective at best indicating that more effective therapeutic solutions are urgently needed. It is appreciated that in the immune-driven and neurodegenerative processes MS-specific deregulation of gene expressions and resulting protein dysfunction are thought to play a central role. These deviations in gene expression patterns contribute to the inflammatory response in the CNS, and to neuronal or axonal loss. Epigenetic mechanisms control transcription of most, if not all genes, in nucleated cells including cells of the CNS and in haematopoietic cells. MS-specific alterations in epigenetic regulation of gene expression may therefore lie at the heart of the deregulation of gene expression in MS. As such, epigenetic mechanisms most likely play an important role in disease pathogenesis. In this review we discuss a role for MS-specific deregulation of epigenetic features that control gene expression in the CNS and in the periphery. Furthermore, we discuss the application of small molecule inhibitors that target the epigenetic machinery to ameliorate disease in experimental animal models, indicating that such approaches may be applicable to MS patients.
RESUMO
Immunodeficiency with centromeric instability and facial anomalies (ICF) syndrome is a primary immunodeficiency, predominantly characterized by agammaglobulinemia or hypoimmunoglobulinemia, centromere instability and facial anomalies. Mutations in two genes have been discovered to cause ICF syndrome: DNMT3B and ZBTB24. To characterize the clinical features of this syndrome, as well as genotype-phenotype correlations, we compared clinical and genetic data of 44 ICF patients. Of them, 23 had mutations in DNMT3B (ICF1), 13 patients had mutations in ZBTB24 (ICF2), whereas for 8 patients, the gene defect has not yet been identified (ICFX). While at first sight these patients share the same immunological, morphological and epigenetic hallmarks of the disease, systematic evaluation of all reported informative cases shows that: (1) the humoral immunodeficiency is generally more pronounced in ICF1 patients, (2) B- and T-cell compartments are both involved in ICF1 and ICF2, (3) ICF2 patients have a significantly higher incidence of intellectual disability and (4) congenital malformations can be observed in some ICF1 and ICF2 cases. It is expected that these observations on prevalence and clinical presentation will facilitate mutation-screening strategies and help in diagnostic counseling.