RESUMO
In Drosophila, ubiquitous expression of a short Cyclin G isoform generates extreme developmental noise estimated by fluctuating asymmetry (FA), providing a model to tackle developmental stability. This transcriptional cyclin interacts with chromatin regulators of the Enhancer of Trithorax and Polycomb (ETP) and Polycomb families. This led us to investigate the importance of these interactions in developmental stability. Deregulation of Cyclin G highlights an organ intrinsic control of developmental noise, linked to the ETP-interacting domain, and enhanced by mutations in genes encoding members of the Polycomb Repressive complexes PRC1 and PR-DUB. Deep-sequencing of wing imaginal discs deregulating CycG reveals that high developmental noise correlates with up-regulation of genes involved in translation and down-regulation of genes involved in energy production. Most Cyclin G direct transcriptional targets are also direct targets of PRC1 and RNAPolII in the developing wing. Altogether, our results suggest that Cyclin G, PRC1 and PR-DUB cooperate for developmental stability.
Assuntos
Ciclina G/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Complexo Repressor Polycomb 1/metabolismo , Animais , Animais Geneticamente Modificados , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Ciclina G/genética , Regulação para Baixo , Proteínas de Drosophila/genética , Feminino , Redes Reguladoras de Genes/fisiologia , Masculino , Complexo Repressor Polycomb 1/genética , Ligação Proteica/genética , Regulação para Cima , Asas de Animais/embriologiaRESUMO
Chromodomains are found in many regulators of chromatin structure, and most of them recognize methylated lysines on histones. Here, we investigate the role of the Drosophila melanogaster protein Corto's chromodomain. The Enhancer of Trithorax and Polycomb Corto is involved in both silencing and activation of gene expression. Over-expression of the Corto chromodomain (CortoCD) in transgenic flies shows that it is a chromatin-targeting module, critical for Corto function. Unexpectedly, mass spectrometry analysis reveals that polypeptides pulled down by CortoCD from nuclear extracts correspond to ribosomal proteins. Furthermore, real-time interaction analyses demonstrate that CortoCD binds with high affinity RPL12 tri-methylated on lysine 3. Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes in open chromatin. RNA-seq based transcriptomes of wing imaginal discs over-expressing either CortoCD or RPL12 reveal that both factors deregulate large sets of common genes, which are enriched in heat-response and ribosomal protein genes, suggesting that they could be implicated in dynamic coordination of ribosome biogenesis. Chromatin immunoprecipitation experiments show that Corto and RPL12 bind hsp70 and are similarly recruited on gene body after heat shock. Hence, Corto and RPL12 could be involved together in regulation of gene transcription. We discuss whether pseudo-ribosomal complexes composed of various ribosomal proteins might participate in regulation of gene expression in connection with chromatin regulators.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Complexo Repressor Polycomb 1/metabolismo , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Expressão Gênica , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Proteínas de Choque Térmico HSP70/genética , Lisina/metabolismo , Metilação , Dados de Sequência Molecular , Fenótipo , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Alinhamento de Sequência , Transcrição Gênica , TranscriptomaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0273198.].
RESUMO
The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs.
Assuntos
Proteínas de Drosophila , Lisina , Proteínas Ribossômicas , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Lisina/genética , Lisina/metabolismo , Mutação , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ativação Transcricional/genéticaRESUMO
Immune complexes can trigger a SHIP-1-independent proapoptotic signal in mouse class-switched IgG(+) B cells and plasma cells by binding to Fc gammaRIIB, in the absence of concomitant coaggregation with BCR, hence regulating plasma cell survival and participating in the selection of B cells producing high affinity Abs during secondary Ab responses. By contrast, we demonstrate in the present study that the unique aggregation of Fc gammaRIIB on human peripheral IgM(+) B cells does not induce apoptosis but transiently inhibits B cell proliferation and calcium influx triggered by BCR cross-linking. Using human peripheral B cells and IIA1.6 lymphoma B cells expressing wild-type human Fc gammaRIIB (IIA1.6-Fc gammaRIIB), we also show that the unique aggregation of human Fc gammaRIIB induces ITIM phosphorylation. This aggregation provokes the recruitment of phosphorylated SHIP-1 by Fc gammaRIIB and inhibits the constitutive phosphorylation of Akt in human IIA1.6-Fc gammaRIIB cells. This inhibitory signaling pathway is abrogated in IIA1.6 cells expressing ITIM-mutated Fc gammaRIIB (Fc gammaRIIB(Y292G)), suggesting that ITIM phosphorylation is necessary for Fc gammaRIIB-induced B cell blockade. Overall, we demonstrate that the unique aggregation of Fc gammaRIIB on human peripheral IgM(+) B cells is sufficient to transiently down-regulate their activation without inducing apoptosis. Our results suggest that Fc gammaRIIB could negatively regulate IgM(+) B cells before class-switch occurrence and that its unique engagement by immune complexes represents a reversible checkpoint for peripheral IgM(+) B cells.
Assuntos
Apoptose/imunologia , Imunoglobulina M , Ativação Linfocitária/imunologia , Plasmócitos/imunologia , Proteínas Proto-Oncogênicas c-bcr/imunologia , Receptores de IgG/imunologia , Substituição de Aminoácidos/imunologia , Animais , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Apoptose/genética , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Humanos , Inositol Polifosfato 5-Fosfatases , Ativação Linfocitária/genética , Camundongos , Mutação de Sentido Incorreto/imunologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Plasmócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcr/genética , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Receptores de IgG/genéticaRESUMO
BACKGROUND: The role of tumor-infiltrating immune cells in the early metastatic invasion of colorectal cancer is unknown. METHODS: We studied pathological signs of early metastatic invasion (venous emboli and lymphatic and perineural invasion) in 959 specimens of resected colorectal cancer. The local immune response within the tumor was studied by flow cytometry (39 tumors), low-density-array real-time polymerase-chain-reaction assay (75 tumors), and tissue microarrays (415 tumors). RESULTS: Univariate analysis showed significant differences in disease-free and overall survival according to the presence or absence of histologic signs of early metastatic invasion (P<0.001). Multivariate Cox analysis showed that an early conventional pathological tumor-node-metastasis stage (P<0.001) and the absence of early metastatic invasion (P=0.04) were independently associated with increased survival. As compared with tumors with signs of early metastatic invasion, tumors without such signs had increased infiltrates of immune cells and increased levels of messenger RNA (mRNA) for products of type 1 helper effector T cells (CD8, T-BET [T-box transcription factor 21], interferon regulatory factor 1, interferon-gamma, granulysin, and granzyme B) but not increased levels of inflammatory mediators or immunosuppressive molecules. The two types of tumors had significant differences in the levels of expression of 65 combinations of T-cell markers, and hierarchical clustering showed that markers of T-cell migration, activation, and differentiation were increased in tumors without signs of early metastatic invasion. The latter type of tumors also had increased numbers of CD8+ T cells, ranging from early memory (CD45RO+CCR7-CD28+CD27+) to effector memory (CD45RO+CCR7-CD28-CD27-) T cells. The presence of high levels of infiltrating memory CD45RO+ cells, evaluated immunohistochemically, correlated with the absence of signs of early metastatic invasion, a less advanced pathological stage, and increased survival. CONCLUSIONS: Signs of an immune response within colorectal cancers are associated with the absence of pathological evidence of early metastatic invasion and with prolonged survival.
Assuntos
Neoplasias Colorretais/imunologia , Metástase Neoplásica/imunologia , Linfócitos T/fisiologia , Análise de Variância , Linfócitos T CD8-Positivos/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/secundário , Embolia/etiologia , Citometria de Fluxo , Expressão Gênica , Humanos , Antígenos Comuns de Leucócito , Metástase Linfática/imunologia , Linfócitos do Interstício Tumoral/fisiologia , Análise em Microsséries , Invasividade Neoplásica/imunologia , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Modelos de Riscos Proporcionais , Proteína Tirosina Fosfatase não Receptora Tipo 1 , RNA Mensageiro/biossíntese , Análise de Sobrevida , Linfócitos T/imunologiaRESUMO
A role for the immune system in controlling the progression of solid tumors has been established in several mouse models. However, the effect of immune responses and tumor escape on patient prognosis in the context of human cancer is poorly understood. Here, we investigate the cellular and molecular parameters that could describe in situ immune responses in human colorectal cancer according to clinical parameters of metastatic lymph node or distant organ invasion (META- or META+ patients). Primary tumor samples of colorectal carcinoma were analyzed by integrating large-scale phenotypic (flow cytometry, 39 patients) and gene expression (real time reverse transcription-PCR, 103 patients) data sets related to immune and protumoral processes. In META- colorectal cancer primary tumors with high densities of T cells, we observed significant positive correlations between markers of innate immune cells [tumor-associated macrophages, dendritic cells, natural killer (NK) cells, and NKT cells] and markers of early-activated T cells. Significant correlations were also observed between markers of cytotoxic and effector memory T-cell subpopulations. These correlation profiles were absent in tumors with low T-cell infiltrates and were altered in META+ tumors with high T-cell infiltrates. We show that the coexpression of genes mediating cytotoxicity (GNLY) and Th1 adaptive immune responses (IRF1) accurately predicted patient survival independently of the metastatic status. High intratumoral mRNA expression of the proangiogenic mediator vascular endothelial growth factor was associated with significantly reduced survival rates in patients expressing high mRNA levels of GNLY. Investigation of the colorectal cancer primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response.
Assuntos
Neoplasias Colorretais/imunologia , Recidiva Local de Neoplasia/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/patologia , Humanos , Memória Imunológica , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Prognóstico , Linfócitos T/imunologia , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The role of the adaptive immune response in controlling the growth and recurrence of human tumors has been controversial. We characterized the tumor-infiltrating immune cells in large cohorts of human colorectal cancers by gene expression profiling and in situ immunohistochemical staining. Collectively, the immunological data (the type, density, and location of immune cells within the tumor samples) were found to be a better predictor of patient survival than the histopathological methods currently used to stage colorectal cancer. The results were validated in two additional patient populations. These data support the hypothesis that the adaptive immune response influences the behavior of human tumors. In situ analysis of tumor-infiltrating immune cells may therefore be a valuable prognostic tool in the treatment of colorectal cancer and possibly other malignancies.