RESUMO
Under the condition of lipopolysaccharide (LPS)/interferon (IFN)-γ activation, macrophage metabolism is converted from oxidative phosphorylation to glycolysis. In the present work, we analysed whether glycolysis could affect interleukin (IL)-1ß expression through altering histone acetylation levels in mouse bone marrow-derived macrophages. Immunocytochemistry and Western blot analysis are used to characterize histone acetylation in macrophages stimulated by LPS/IFN-γ. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay were used to determine IL-1ß production. The metabolism of macrophages was monitored in real-time by the Seahorse test. Our results showed that glycolytic metabolism could enhance histone acetylation and promote IL-1ß production in LPS/IFN-γ-activated macrophages. Moreover, increased production of IL-1ß by glycolysis was mediated through enhanced H3K9 acetylation. Importantly, it was found that a high dose of histone deacetylase inhibitor could also significantly increase the expression of IL-1ß in the absence of glycolytic metabolism. In conclusion, this study demonstrates that glycolytic metabolism could regulate IL-1ß expression by increasing histone acetylation levels in LPS/IFN-γ-stimulated macrophages.
Assuntos
Histonas/metabolismo , Interleucina-1beta/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Acetilação , Animais , Células Cultivadas , Glicólise/imunologia , Interferon gama/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Cultura Primária de Células , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND: p300 (KAT3B) lysine acetyltransferase activity is modulated under different physiological and pathological contexts through the induction of trans-autoacetylation. This phenomenon is mediated by several factors, mechanisms of which are not fully understood. METHODS: Through acetyltransferase assays using full-length, baculovirus-expressed KATs, the specificity of NPM1-mediated enhancement of p300 autoacetylation was tested. Chaperone assays and tryptophan fluorescence studies were performed to evaluate the NPM1-induced protein folding. The NPM1 oligomer-defective mutant characterization was done by glutaraldehyde-crosslinking. The small-molecule inhibitor of NPM1 oligomerization was used to confirm the absolute requirement of multimeric NPM1 in vivo. Immunohistochemistry analysis of oral cancer patient samples was done to uncover the pathophysiological significance of NPM1-induced p300 autoacetylation. RESULTS: We find that the histone chaperone NPM1 is a specific inducer of p300 autoacetylation. Distinct from its histone chaperone activity, NPM1 is a molecular chaperone of p300. The biophysical experiments suggest that there is a reversible binding between NPM1 and p300 which can modulate p300 acetyltransferase activity. Disruption of NPM1 oligomerization suggests that oligomeric NPM1 is essential for the induction of p300 autoacetylation. Significantly, we observe a concomitant hyper-autoacetylation of p300 with overexpression of NPM1 in oral cancer samples. CONCLUSION: NPM1 can specifically modulate p300 acetyltransferase activity through the enhancement of autoacetylation. The molecular chaperone activity and oligomerization of NPM1 play a pivotal role in this phenomenon. GENERAL SIGNIFICANCE: NPM1 is overexpressed in several solid cancers, the significance of which is unknown. Induction of p300 autoacetylation could be the cause of NPM1-mediated tumorigenicity.
Assuntos
Proteína p300 Associada a E1A/química , Proteína p300 Associada a E1A/metabolismo , Histonas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Dobramento de Proteína , Multimerização Proteica , Neoplasias da Língua/metabolismo , Acetilação , Humanos , Nucleofosmina , Ligação Proteica , Conformação Proteica , Neoplasias da Língua/patologia , Células Tumorais CultivadasRESUMO
Fluoxetine (FLX) is an antidepressant drug that belongs to the class of selective serotonin reuptake inhibitors. FLX is known to induce apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of FLX were investigated in SK-N-BE(2)-M17 human neuroblastoma cells. FLX induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid or CHOP siRNA transfection reduced FLX-induced cell death. FLX induced phosphorylation of mitogen-activated protein kinases (MAPKs) family, p38, JNK, and ERK, and an upstream kinase apoptosis signal kinase 1 (ASK1). Inhibition of MAPKs and ASK1 reduced FLX-induced cell death and CHOP expression. We then showed that FLX reduced mitochondrial membrane potential (MMP) and ER stress inhibitors as well as MAPK inhibitors ameliorated FLX-induced loss of MMP. Interestingly, FLX induced hyperacetylation of histone H3 and H4, upregulation of p300 histone acetyltransferase (HAT), and downregulation of histone deacetylases (HDACs). Treatment with a HAT inhibitor anacardic acid or p300 HAT siRNA transfection blocked FLX-induced apoptosis in SK-N-BE(2)-M17 cells. However, FLX did not induce histone acetylation and anacardic acid had no protective effect on FLX-induced cell death and CHOP expression in MYCN non-amplified SH-SY5Y human neuroblastoma and MYCN knockdowned SK-N-BE(2)-M17 cells. These findings suggest that FLX induces apoptosis in neuroblastoma through ER stress and mitochondrial dysfunction via the ASK1 and MAPK pathways and through histone hyperacetylation in a MYCN-dependent manner.
Assuntos
Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fluoxetina/farmacologia , Histonas/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Neuroblastoma/patologia , Animais , Antineoplásicos/farmacologia , Caspases Iniciadoras/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , MAP Quinase Quinase Quinase 5/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genéticaRESUMO
In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Here we report that, in mouse primary or embryonic stem cell-derived cardiomyocytes, increased calcium levels induce robust and reversible skipping of several alternative exons from endogenously expressed genes. Interestingly, we demonstrate a calcium-mediated splicing regulatory mechanism that depends on changes of histone modifications. Specifically, the regulation occurs through changes in calcium-responsive kinase activities that lead to alterations in histone modifications and subsequent changes in the transcriptional elongation rate and exon skipping. We demonstrate that increased intracellular calcium levels lead to histone hyperacetylation along the body of the genes containing calcium-responsive alternative exons by disrupting the histone deacetylase-to-histone acetyltransferase balance in the nucleus. Consequently, the RNA polymerase II elongation rate increases significantly on those genes, resulting in skipping of the alternative exons. These studies reveal a mechanism by which calcium-level changes in cardiomyocytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns.
Assuntos
Processamento Alternativo , Sinalização do Cálcio/fisiologia , Histona Desacetilases/fisiologia , Histonas/metabolismo , Miócitos Cardíacos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Acetilação , Processamento Alternativo/efeitos dos fármacos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Éxons , Regulação da Expressão Gênica/fisiologia , Genes da Neurofibromatose 1 , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Neurofibromina 1/biossíntese , Neurofibromina 1/genética , Cloreto de Potássio/farmacologia , Inibidores de Proteínas Quinases/farmacologia , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Canais de Cátion TRPP/fisiologia , Elongação da Transcrição GenéticaRESUMO
The aim of the study was to investigate the in vivo epigenetic influences of dietary butyrate supplementation on the acetylation state of core histones and the activity of drug-metabolising microsomal cytochrome P450 (CYP) enzymes in the liver of broiler chickens in the starter period. One-day-old Ross 308 broilers were fed a starter diet without or with sodium butyrate (1.5 g/kg feed) for 21 days. After slaughtering, nucleus and microsome fractions were isolated from the exsanguinated liver by multi-step differential centrifugation. Histone acetylation level was detected from hepatocyte nuclei by Western blotting, while microsomal CYP activity was examined by specific enzyme assays. Hyperacetylation of hepatic histone H2A at lysine 5 was observed after butyrate supplementation, providing modifications in the epigenetic regulation of cell function. No significant changes could be found in the acetylation state of the other core histones at the acetylation sites examined. Furthermore, butyrate did not cause any changes in the drugmetabolising activity of hepatic microsomal CYP2H and CYP3A37 enzymes, which are mainly involved in the biotransformation of most xenobiotics in chicken. These data indicate that supplementation of the diet with butyrate probably does not have any pharmacokinetic interactions with simultaneously applied xenobiotics.
Assuntos
Butiratos , Galinhas , Acetilação , Animais , Biotransformação , Galinhas/metabolismo , Epigênese Genética , HistonasRESUMO
In maturing sperm cells, a major genome re-organization takes place, which includes a global increase in the acetylation of histones prior to their replacement by protamines, the latter being responsible for the tight packaging of the male genome. Understanding the function of the oncogenic BRD4-NUT fusion protein in NUT carcinoma (NC) cells has proven to be essential in uncovering the mechanisms underlying histone hyperacetylation in spermatogenic cells. Indeed, these studies have revealed the mechanism by which a cooperation between BRD4, a bromodomain factor of the BET family, NUT, a normally testis-specific factor, and the histone acetyltransferase p300, induces the generation of hyperacetylated chromatin domains which are present in NC cells. The generation of Nut ko mice enabled us to demonstrate a genetic interaction between Nut and Brdt, encoding BRDT, a testis-specific BRD4-like factor. Indeed, in spermatogenic cells, NUT and p300 interact, which results in an increased acetylation of histone H4 at both positions K5 and K8. These two positions, when both acetylated, are specifically recognized by the first bromodomain of BRDT, which then mediates the removal of histone and their replacement by protamines. Taken together, these investigations show that the fusion of NUT to BRD4 in NUT Carcinoma cells reconstitutes, in somatic cells, a functional loop, which normally drives histone hyperacetylation and chromatin binding by a BET factor in spermatogenic cells.
RESUMO
Plants possess the structural maintenance of chromosome (SMC) protein complexes cohesin, condensin, and SMC5/6, which function in fundamental biological processes such as sister chromatid cohesion, chromosome condensation and segregation, and damaged DNA repair. Recently, increasing evidence in several organisms has suggested that condensin is involved in chromatin organizations during interphase. In Arabidopsis thaliana, condensin II is localized in the nucleus throughout interphase and is suggested to be required for keeping centromeres apart and the assembly of euchromatic chromosome arms. However, it remains unclear how condensin II organizes chromatin associations. Here, we first showed the high possibility that the function of condensin II as a complex is required for the disassociation of centromeres. Analysis of the rDNA array distribution revealed that condensin II is also indispensable for the association of centromeres with rDNA arrays. Reduced axial compaction of chromosomes and impaired genome integrity in condensin II mutants are not related to the disruption of chromatin organization. In contrast, the axial compaction of chromosomes by condensin II produces the force leading to the disassociation of heterologous centromeres in Drosophila melanogaster. Taken together, our data imply that the condensin II function in chromatin organization differs among eukaryotes.
Assuntos
Adenosina Trifosfatases/metabolismo , Arabidopsis/enzimologia , Centrômero/metabolismo , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Complexos Multiproteicos/genética , MutaçãoRESUMO
We have shown that melatonin induces histone hyperacetylation in vitro and in vivo. To clarify the mechanisms involved, we have now investigated its effects on histone acetylation and signaling pathways in human SH-SY5Y neuroblastoma cells, which express melatonin MT1 receptors. Melatonin caused significant concentration-dependent increases in both histone H3 and H4 acetylation. Blockade of melatonin receptors with luzindole abolished the histone hyperacetylating effect of melatonin, whereas inhibition of MAPK-ERK by PD98059 attenuated but did not block this effect. Melatonin treatment for 24-h decreased the levels of phospho-ERK1/2, but significantly increased Akt phosphorylation and protein expression of the histone acetyltransferase, p300. These findings suggest that the epigenetic effects of melatonin in SH-SY5Y cells are mediated by G protein-coupled MT1 melatonin receptors. Furthermore, upregulation of the histone acetyltransferase/transcriptional co-activator p300, along with phosphorylation of Akt, which is essential for p300 activation, appear to be key mechanisms underlying the epigenetic effects of melatonin.
Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Melatonina/fisiologia , Acetilação , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Humanos , Neuroblastoma , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor MT1 de Melatonina/antagonistas & inibidores , Receptor MT1 de Melatonina/metabolismo , Transdução de Sinais , Triptaminas/farmacologia , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
In contrast to cell type-specific pre-mRNA alternative splicing, mechanisms controlling activity-dependent alternative splicing is under-studied and not well understood. In a recent study, we conducted a comprehensive analysis of calcium-mediated mechanism that regulates alternative exon skipping in mouse cardiomyocytes. Our results reveal a strong link between histone hyperacetylation and skipping of cassette exons, and provide support to the kinetic coupling model of the epigenetic regulation of alternative splicing at the chromatin level.