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1.
Elife ; 122023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37470704

RESUMO

The DNA methyltransferase activity of DNMT1 is vital for genomic maintenance of DNA methylation. We report here that DNMT1 function is regulated by O-GlcNAcylation, a protein modification that is sensitive to glucose levels, and that elevated O-GlcNAcylation of DNMT1 from high glucose environment leads to alterations to the epigenome. Using mass spectrometry and complementary alanine mutation experiments, we identified S878 as the major residue that is O-GlcNAcylated on human DNMT1. Functional studies in human and mouse cells further revealed that O-GlcNAcylation of DNMT1-S878 results in an inhibition of methyltransferase activity, resulting in a general loss of DNA methylation that preferentially occurs at partially methylated domains (PMDs). This loss of methylation corresponds with an increase in DNA damage and apoptosis. These results establish O-GlcNAcylation of DNMT1 as a mechanism through which the epigenome is regulated by glucose metabolism and implicates a role for glycosylation of DNMT1 in metabolic diseases characterized by hyperglycemia.


Assuntos
Glucose , Hiperglicemia , Camundongos , Humanos , Animais , Glucose/farmacologia , Epigenoma , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Glicosilação
2.
Genome Res ; 33(8): 1229-1241, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37463750

RESUMO

A primary function of DNA methylation in mammalian genomes is to repress transposable elements (TEs). The widespread methylation loss that is commonly observed in cancer cells results in the loss of epigenetic repression of TEs. The aging process is similarly characterized by changes to the methylome. However, the impact of these epigenomic alterations on TE silencing and the functional consequences of this have remained unclear. To assess the epigenetic regulation of TEs in aging, we profiled DNA methylation in human mammary luminal epithelial cells (LEps)-a key cell lineage implicated in age-related breast cancers-from younger and older women. We report here that several TE subfamilies function as regulatory elements in normal LEps, and a subset of these display consistent methylation changes with age. Methylation changes at these TEs occurred at lineage-specific transcription factor binding sites, consistent with loss of lineage specificity. Whereas TEs mainly showed methylation loss, CpG islands (CGIs) that are targets of the Polycomb repressive complex 2 (PRC2) show a gain of methylation in aging cells. Many TEs with methylation loss in aging LEps have evidence of regulatory activity in breast cancer samples. We furthermore show that methylation changes at TEs impact the regulation of genes associated with luminal breast cancers. These results indicate that aging leads to DNA methylation changes at TEs that undermine the maintenance of lineage specificity, potentially increasing susceptibility to breast cancer.


Assuntos
Neoplasias da Mama , Epigênese Genética , Idoso , Feminino , Humanos , Envelhecimento/genética , Neoplasias da Mama/genética , Metilação de DNA , Elementos de DNA Transponíveis , Retroelementos
3.
Cell Metab ; 35(5): 821-836.e7, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-36948185

RESUMO

The mechanisms that specify and stabilize cell subtypes remain poorly understood. Here, we identify two major subtypes of pancreatic ß cells based on histone mark heterogeneity (ßHI and ßLO). ßHI cells exhibit ∼4-fold higher levels of H3K27me3, distinct chromatin organization and compaction, and a specific transcriptional pattern. ßHI and ßLO cells also differ in size, morphology, cytosolic and nuclear ultrastructure, epigenomes, cell surface marker expression, and function, and can be FACS separated into CD24+ and CD24- fractions. Functionally, ßHI cells have increased mitochondrial mass, activity, and insulin secretion in vivo and ex vivo. Partial loss of function indicates that H3K27me3 dosage regulates ßHI/ßLO ratio in vivo, suggesting that control of ß cell subtype identity and ratio is at least partially uncoupled. Both subtypes are conserved in humans, with ßHI cells enriched in humans with type 2 diabetes. Thus, epigenetic dosage is a novel regulator of cell subtype specification and identifies two functionally distinct ß cell subtypes.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Humanos , Células Secretoras de Insulina/metabolismo , Histonas/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Epigênese Genética , Secreção de Insulina
4.
Subcell Biochem ; 100: 393-426, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36301501

RESUMO

Lysine acetylation is the second most well-studied post-translational modification after phosphorylation. While phosphorylation regulates signaling cascades, one of the most significant roles of acetylation is regulation of chromatin structure. Acetyl-coenzyme A (acetyl-CoA) serves as the acetyl group donor for acetylation reactions mediated by lysine acetyltransferases (KATs). On the other hand, NAD+ serves as the cofactor for lysine deacetylases (KDACs). Both acetyl-CoA and NAD+ are metabolites integral to energy metabolism, and therefore, their metabolic flux can regulate the activity of KATs and KDACs impacting the epigenome. In this chapter, we review our current understanding of how metabolic pathways regulate lysine acetylation in normal and cancer cells.


Assuntos
Lisina , Neoplasias , Humanos , Acetilação , Lisina/metabolismo , Acetilcoenzima A/metabolismo , NAD/metabolismo , Processamento de Proteína Pós-Traducional , Neoplasias/genética
5.
Mol Cell Biol ; 42(2): e0066920, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-34898280

RESUMO

Nucleophosmin (NPM1) is a multifunctional histone chaperone that can activate acetylation-dependent transcription from chromatin templates in vitro. p300-mediated acetylation of NPM1 has been shown to further enhance its transcription activation potential. Acetylated and total NPM1 pools are increased in oral squamous cell carcinoma. However, the role of NPM1 or its acetylated form (AcNPM1) in transcriptional regulation in cells and oral tumorigenesis is not fully elucidated. Using ChIP-seq analyses, we provide the first genome-wide profile of AcNPM1 and show that AcNPM1 is enriched at transcriptional regulatory elements. AcNPM1 co-occupies marks of active transcription at promoters and DNase I hypersensitive sites at enhancers. In addition, using a high-throughput protein interaction profiling approach, we show that NPM1 interacts with RNA Pol II, general transcription factors, mediator subunits, histone acetyltransferase complexes, and chromatin remodelers. NPM1 histone chaperone activity also contributes to its transcription activation potential. Further, NPM1 depletion leads to decreased AcNPM1 occupancy and reduced expression of genes required for proliferative, migratory and invasive potential of oral cancer cells. NPM1 depletion also abrogates the growth of orthotopic tumors in mice. Collectively, these results establish that AcNPM1 functions as a coactivator during during RNA polymerase II-driven transcription and regulates the expression of genes that promote oral tumorigenesis.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Regulação da Expressão Gênica/fisiologia , Chaperonas de Histonas/metabolismo , Neoplasias Bucais/genética , Nucleofosmina/metabolismo , Animais , Carcinogênese/metabolismo , Carcinoma de Células Escamosas/genética , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética
6.
Cancer Prev Res (Phila) ; 14(8): 779-794, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34140348

RESUMO

A robust breast cancer prevention strategy requires risk assessment biomarkers for early detection. We show that expression of ELF5, a transcription factor critical for normal mammary development, is downregulated in mammary luminal epithelia with age. DNA methylation of the ELF5 promoter is negatively correlated with expression in an age-dependent manner. Both ELF5 methylation and gene expression were used to build biological clocks to estimate chronological ages of mammary epithelia. ELF5 clock-based estimates of biological age in luminal epithelia from average-risk women were within three years of chronological age. Biological ages of breast epithelia from BRCA1 or BRCA2 mutation carriers, who were high risk for developing breast cancer, suggested they were accelerated by two decades relative to chronological age. The ELF5 DNA methylation clock had better performance at predicting biological age in luminal epithelial cells as compared with two other epigenetic clocks based on whole tissues. We propose that the changes in ELF5 expression or ELF5-proximal DNA methylation in luminal epithelia are emergent properties of at-risk breast tissue and constitute breast-specific biological clocks. PREVENTION RELEVANCE: ELF5 expression or DNA methylation level at the ELF5 promoter region can be used as breast-specific biological clocks to identify women at higher than average risk of breast cancer.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Mama/metabolismo , Relógios Circadianos/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Adulto , Biomarcadores Tumorais/genética , Mama/patologia , Neoplasias da Mama/patologia , Transformação Celular Neoplásica , Células Cultivadas , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Detecção Precoce de Câncer/métodos , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença/genética , Testes Genéticos/métodos , Humanos , Pessoa de Meia-Idade , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
7.
Nat Commun ; 11(1): 3326, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620791

RESUMO

Tumour cells adapt to nutrient deprivation in vivo, yet strategies targeting the nutrient poor microenvironment remain unexplored. In melanoma, tumour cells often experience low glutamine levels, which promote cell dedifferentiation. Here, we show that dietary glutamine supplementation significantly inhibits melanoma tumour growth, prolongs survival in a transgenic melanoma mouse model, and increases sensitivity to a BRAF inhibitor. Metabolomic analysis reveals that dietary uptake of glutamine effectively increases the concentration of glutamine in tumours and its downstream metabolite, αKG, without increasing biosynthetic intermediates necessary for cell proliferation. Mechanistically, we find that glutamine supplementation uniformly alters the transcriptome in tumours. Our data further demonstrate that increase in intra-tumoural αKG concentration drives hypomethylation of H3K4me3, thereby suppressing epigenetically-activated oncogenic pathways in melanoma. Therefore, our findings provide evidence that glutamine supplementation can serve as a potential dietary intervention to block melanoma tumour growth and sensitize tumours to targeted therapy via epigenetic reprogramming.


Assuntos
Proliferação de Células/efeitos dos fármacos , Suplementos Nutricionais , Epigênese Genética/efeitos dos fármacos , Glutamina/farmacologia , Melanoma/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Epigênese Genética/genética , Glutamina/administração & dosagem , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Melanoma/genética , Melanoma/patologia , Metilação/efeitos dos fármacos , Camundongos Nus , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
8.
Epigenetics Chromatin ; 12(1): 44, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315653

RESUMO

BACKGROUND: Hyperinsulinemia, the presence of excess insulin relative to glucose in the blood, is considered to be a poor prognostic indicator for patients with triple-negative breast cancer (TNBC). mTOR, a downstream effector of insulin, enhances mitochondrial biogenesis and activity, thereby increasing acetyl-CoA precursors. Increased acetyl-CoA can, in turn, be utilized by nuclear acetyltransferases for histone acetylation, a critical feature of genome regulation. While signaling pathways downstream of insulin have been established for sometime, the effect of insulin on chromatin remains unclear. We hypothesized that hyperinsulinemia-induced metabolic changes lead to genome-wide changes in histone acetylation in TNBC. RESULTS: MDA-MB-231 cells were xenografted into hyperinsulinemic and wild-type mice. Tumors in the hyperinsulinemic mice displayed elevated levels of histone acetylation compared to tumors in normal insulin conditions. We show that insulin treatment in vitro leads to global increase in chromatin-associated histone acetylation, in particular at H3K9, through the PI3K/AKT/mTOR pathway. Genome-wide analyses revealed that most promoter regions have an increase in histone acetylation upon insulin treatment. In addition, insulin induces higher levels of reactive oxygen species and DNA damage foci in cells. CONCLUSIONS: These results demonstrate the impact of hyperinsulinemia on altered gene regulation through chromatin and the importance of targeting hyperinsulinemia-induced processes that lead to chromatin dysfunction in TNBC.


Assuntos
Histonas/sangue , Hiperinsulinismo/sangue , Neoplasias de Mama Triplo Negativas/sangue , Acetilação , Animais , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Drosophila , Feminino , Estudo de Associação Genômica Ampla , Xenoenxertos , Histona Acetiltransferases/sangue , Histona Acetiltransferases/genética , Histonas/metabolismo , Humanos , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patologia , Insulina/sangue , Insulina/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
9.
Circ Res ; 123(12): 1298-1312, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30566058

RESUMO

RATIONALE: AngII (angiotensin II)-mediated vascular smooth muscle cell (VSMC) dysfunction plays a major role in hypertension. Long noncoding RNAs have elicited much interest, but their molecular roles in AngII actions and hypertension are unclear. OBJECTIVE: To investigate the regulation and functions of a novel long noncoding RNA growth factor- and proinflammatory cytokine-induced vascular cell-expressed RNA ( Giver), in AngII-mediated VSMC dysfunction. METHODS AND RESULTS: RNA-sequencing and real-time quantitative polymerase chain reactions revealed that treatment of rat VSMC with AngII increased the expression of Giver and Nr4a3, an adjacent gene encoding a nuclear receptor. Similar changes were observed in rat and mouse aortas treated ex vivo with AngII. RNA-FISH (fluorescence in situ hybridization) and subcellular fractionation showed predominantly nuclear localization of Giver. AngII increased Giver expression via recruitment of Nr4a3 to Giver promoter. Microarray profiling and real-time quantitative polymerase chain reaction validation in VSMC showed that Giver knockdown attenuated the expression of genes involved in oxidative stress ( Nox1) and inflammation ( Il6, Ccl2, Tnf) but increased Nr4a3. Conversely, endogenous Giver overexpression showed opposite effects supporting its role in oxidative stress and inflammation. Chromatin immunoprecipitation assays showed Giver overexpression also increased Pol II (RNA polymerase II) enrichment and decreased repressive histone modification histone H3 trimethylation on lysine 27 at Nox1 and inflammatory gene promoters. Accordingly, Giver knockdown inhibited AngII-induced oxidative stress and proliferation in rat VSMC. RNA-pulldown combined with mass spectrometry showed Giver interacts with nuclear and chromatin remodeling proteins and corepressors, including NONO (non-pou domain-containing octamer-binding protein). Moreover, NONO knockdown elicited similar effects as Giver knockdown on the expression of key Giver-regulated genes. Notably, GIVER and NR4A3 were increased in AngII-treated human VSMC and in arteries from hypertensive patients but attenuated in hypertensive patients treated with ACE (angiotensin-converting enzyme) inhibitors or angiotensin receptor blockers. Furthermore, human GIVER also exhibits partial functional conservation with rat Giver. CONCLUSIONS: Giver and its regulator Nr4a3 are important players in AngII-mediated VSMC dysfunction and could be novel targets for antihypertensive therapy.


Assuntos
Proliferação de Células , Citocinas/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Estresse Oxidativo , RNA Longo não Codificante/genética , Animais , Células Cultivadas , Humanos , Hipertensão/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , NADPH Oxidase 1/genética , NADPH Oxidase 1/metabolismo , RNA Longo não Codificante/metabolismo , Ratos , Ratos Sprague-Dawley
10.
FEBS J ; 285(18): 3503-3524, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30085406

RESUMO

Nucleophosmin (NPM1) is a nucleolar protein that is frequently overexpressed in various types of solid tumors. NPM1 is involved in several cellular processes that might contribute significantly to the increased proliferation potential of cancers. Previous reports suggest that NPM1 expression is highly increased in response to mitogenic and oncogenic signals, the mechanisms of which have not been elucidated extensively. Using constructs incorporating different fragments of the NPM1 promoter upstream to a Luciferase reporter gene, we have identified the minimal promoter of NPM1 and candidate transcription factors regulating NPM1 promoter activity by luciferase reporter assays. We have validated the roles of a few candidate factors at the transcriptional and protein level by quantitative reverse transcriptase PCR, immunoblotting and immunohistochemistry, and explored the mechanism of regulation of NPM1 expression using immunoprecipitation and chromatin immunoprecipitation assays. We show here that the expression of NPM1 is regulated by transcription factor c-fos, a protein that is strongly activated by growth factor signals. In addition, mutant p53 (R175H) overexpression also enhances NPM1 expression possibly through c-myc and c-fos. Moreover, both c-fos and mutant p53 are overexpressed in oral tumor tissues that showed NPM1 overexpression. Collectively, our results suggest that c-fos and mutant p53 R175H positively regulate NPM1 expression, possibly in synergism, that might lead to oncogenic manifestation.


Assuntos
Carcinoma de Células Escamosas/patologia , Regulação Neoplásica da Expressão Gênica , Genes fos , Neoplasias Bucais/patologia , Mutação , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células , Humanos , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Proteínas Nucleares/metabolismo , Nucleofosmina , Prognóstico , Regiões Promotoras Genéticas , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética
11.
Arterioscler Thromb Vasc Biol ; 38(8): 1806-1820, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29930005

RESUMO

Objective- Macrophages play key roles in inflammation and diabetic vascular complications. Emerging evidence implicates long noncoding RNAs in inflammation, but their role in macrophage dysfunction associated with inflammatory diabetic complications is unclear and was therefore investigated in this study. Approach and Results- RNA-sequencing and real-time quantitative PCR demonstrated that a long noncoding RNA Dnm3os (dynamin 3 opposite strand) is upregulated in bone marrow-derived macrophages from type 2 diabetic db/db mice, diet-induced insulin-resistant mice, and diabetic ApoE-/- mice, as well as in monocytes from type 2 diabetic patients relative to controls. Diabetic conditions (high glucose and palmitic acid) induced Dnm3os in mouse and human macrophages. Promoter reporter analysis and chromatin immunoprecipitation assays demonstrated that diabetic conditions induce Dnm3os via NF-κB activation. RNA fluorescence in situ hybridization and real-time quantitative PCRs of subcellular fractions demonstrated nuclear localization and chromatin enrichment of Dnm3os in macrophages. Stable overexpression of Dnm3os in macrophages altered global histone modifications and upregulated inflammation and immune response genes and phagocytosis. Conversely, RNAi-mediated knockdown of Dnm3os attenuated these responses. RNA pull-down assays with macrophage nuclear lysates identified nucleolin and ILF-2 (interleukin enhancer-binding factor 2) as protein binding partners of Dnm3os, which was further confirmed by RNA fluorescence in situ hybridization immunofluorescence. Furthermore, nucleolin levels were decreased in diabetic conditions, and its knockdown enhanced Dnm3os-induced inflammatory gene expression and histone H3K9-acetylation at their promoters. Conclusions- These results demonstrate novel mechanisms involving upregulation of long noncoding RNA Dnm3os, disruption of its interaction with nucleolin, and epigenetic modifications at target genes that promote macrophage inflammatory phenotype in diabetes mellitus. The data could lead to long noncoding RNA-based therapies for inflammatory diabetes mellitus complications.


Assuntos
Núcleo Celular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Estudos de Casos e Controles , Núcleo Celular/genética , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Epigênese Genética , Feminino , Humanos , Inflamação/genética , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Fagocitose , Fenótipo , Fosfoproteínas/metabolismo , Ligação Proteica , Células RAW 264.7 , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Estreptozocina , Regulação para Cima , Nucleolina
12.
Biochim Biophys Acta Gen Subj ; 1862(8): 1729-1741, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29746960

RESUMO

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 Cultivadas
13.
Nat Commun ; 8(1): 1467, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29133788

RESUMO

Angiotensin II (AngII) promotes hypertension and atherosclerosis by activating growth-promoting and pro-inflammatory gene expression in vascular smooth muscle cells (VSMCs). Enhancers and super-enhancers (SEs) play critical roles in driving disease-associated gene expression. However, enhancers/SEs mediating VSMC dysfunction remain uncharacterized. Here, we show that AngII alters vascular enhancer and SE repertoires in cultured VSMCs in vitro, ex vivo, and in AngII-infused mice aortas in vivo. AngII-induced enhancers/SEs are enriched in binding sites for signal-dependent transcription factors and dependent on key signaling kinases. Moreover, CRISPR-Cas9-mediated deletion of candidate enhancers/SEs, targeting SEs with the bromodomain and extra-terminal domain inhibitor JQ1, or knockdown of overlapping long noncoding RNAs (lncRNAs) blocks AngII-induced genes associated with growth-factor signaling and atherosclerosis. Furthermore, JQ1 ameliorates AngII-induced hypertension, medial hypertrophy and inflammation in vivo in mice. These results demonstrate AngII-induced signals integrate enhancers/SEs and lncRNAs to increase expression of genes involved in VSMC dysfunction, and could uncover novel therapies.


Assuntos
Angiotensina II/metabolismo , Aterosclerose/genética , Hipertensão/genética , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , RNA Longo não Codificante/genética , Animais , Aorta/citologia , Aorta/patologia , Aterosclerose/tratamento farmacológico , Azepinas/farmacologia , Células Cultivadas , Regulação da Expressão Gênica , Histonas/metabolismo , Hipertensão/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Triazóis/farmacologia
14.
Sci Rep ; 6: 26580, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27253419

RESUMO

More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering.


Assuntos
Proteínas 14-3-3/metabolismo , Centrossomo/metabolismo , Instabilidade Cromossômica , Neoplasias/patologia , Proteínas 14-3-3/genética , Aneuploidia , Animais , Ciclo Celular , Linhagem Celular Tumoral , Centrossomo/patologia , Deleção de Genes , Células HCT116 , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosforilação , Treonina/química , Fosfatases cdc25/metabolismo
15.
Methods Mol Biol ; 1288: 375-94, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25827892

RESUMO

Histone chaperones are histone interacting proteins that are involved in various stages of histone metabolism in the cell such as histone storage, transport, nucleosome assembly and disassembly. Histone assembly and disassembly are essential processes in certain DNA-templated phenomena such as replication, repair and transcription in eukaryotes. Since the first histone chaperone Nucleoplasmin was discovered in Xenopus, a plethora of histone chaperones have been identified, characterized and their functional significance elucidated in the last 35 years or so. Some of the histone chaperone containing complexes such as FACT have been described to play a significant role in nucleosome disassembly during transcription elongation. We have reported earlier that human Nucleophosmin (NPM1), a histone chaperone belonging to the Nucleoplasmin family, is a co-activator of transcription. In this chapter, we describe several methods that are used to study the histone chaperone activity of proteins and their role in transcription.


Assuntos
Cromatina/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Nucleossomos/genética , Transcrição Gênica , Animais , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Humanos , Técnicas In Vitro , Camundongos , Nucleofosmina , Nucleossomos/metabolismo
16.
FEBS Lett ; 588(14): 2198-205, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24857377

RESUMO

The functional association of NPM1 with Aurora kinases is well documented. Surprisingly, although NPM1 is a well characterized phosphoprotein, it is unknown whether it is a substrate of Aurora kinases. We have found that Aurora kinases A and B can phosphorylate NPM1 at a single serine residue, Ser125, in vitro and in vivo. Phosphorylated-S125-NPM1 (pS125-NPM1) localizes to the midbody region during late cytokinesis where it colocalizes with Aurora B. The overexpression of mutant (S125A) NPM1 resulted in the deregulation of centrosome duplication and mitotic defects possibly due to cytokinesis failure. These data suggest that Aurora kinase B-mediated phosphorylation of NPM1 plays a critical role during mitosis, which could have wider implications in oncogenesis.


Assuntos
Aurora Quinase B/fisiologia , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Aurora Quinase A/química , Aurora Quinase B/química , Carcinoma de Células Escamosas/enzimologia , Transformação Celular Neoplásica/metabolismo , Centrossomo/metabolismo , Células HEK293 , Humanos , Camundongos , Neoplasias Bucais/enzimologia , Células NIH 3T3 , Proteínas Nucleares/química , Nucleofosmina , Fosforilação , Transporte Proteico , Telófase
17.
J Biochem ; 156(4): 221-7, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24839294

RESUMO

Mammalian centromeric histone H3 variant, CENP-A, is involved in maintaining the functional integrity and epigenetic inheritance of the centromere. CENP-A causes transcriptional repression of centromeric chromatin through an unknown mechanism. Here, we report that reconstituted CENP-A nucleosomes are amenable to ATP-dependent SWI/SNF-mediated remodelling but are less permissive to acetylation and acetylation-dependent in vitro chromatin transcription. Remarkably, the transcriptional repression of the CENP-A chromatinized template could be relieved by the ectopic addition of histone chaperone, nucleophosmin.


Assuntos
Autoantígenos/metabolismo , Centrômero/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Animais , Autoantígenos/genética , Proteína Centromérica A , Proteínas Cromossômicas não Histona/genética , Histonas/genética , Proteínas Nucleares/genética , Nucleofosmina , Xenopus laevis
18.
Mol Cancer ; 13: 66, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24655440

RESUMO

BACKGROUND: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been linked with proliferation, survival, invasion and angiogenesis of a variety of human cancer cells, including hepatocellular carcinoma (HCC). Thus, novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. Here we report, garcinol, a polyisoprenylated benzophenone, could suppress STAT3 activation in HCC cell lines and in xenografted tumor of HCC in nude mice model. EXPERIMENTAL DESIGN: Different HCC cell lines have been treated with garcinol and the inhibition of STAT3 activation, dimerization and acetylation have been checked by immunoblotting, immuno-fluorescence, and DNA binding assays. Xenografted tumor model has been generated in nude mice using HCC cell line and effect of garcinol in the inhibition of tumor growth has been investigated. RESULTS: Garcinol could inhibit both constitutive and interleukin (IL-6) inducible STAT3 activation in HCC cells. Computational modeling showed that garcinol could bind to the SH2 domain of STAT3 and suppress its dimerization in vitro. Being an acetyltransferase inhibitor, garcinol also inhibits STAT3 acetylation and thus impairs its DNA binding ability. The inhibition of STAT3 activation by garcinol led to the suppression of expression of various genes involved in proliferation, survival, and angiogenesis. It also suppressed proliferation and induced substantial apoptosis in HCC cells. Remarkably, garcinol inhibited the growth of human HCC xenograft tumors in athymic nu/nu mice, through the inhibition of STAT3 activation. CONCLUSION: Overall, our results suggest that garcinol exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Fator de Transcrição STAT3/biossíntese , Terpenos/administração & dosagem , Acetilação/efeitos dos fármacos , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dimerização , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos , Fosforilação , Fator de Transcrição STAT3/antagonistas & inibidores
19.
PLoS One ; 8(2): e57693, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23460895

RESUMO

The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as "chromatin remodeling". In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance.


Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/metabolismo , DNA/química , Distamicinas/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Animais , Cromatina/química , Dicroísmo Circular , DNA/metabolismo , Distamicinas/metabolismo , Histonas/metabolismo , Masculino , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
20.
FEBS Lett ; 587(5): 417-24, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23353999

RESUMO

Nucleolin is a multifunctional protein that carries several post-translational modifications. We characterized nucleolin acetylation and developed antibodies specific to nucleolin K88 acetylation. Using this antibody we show that nucleolin is acetylated in vivo and is not localized in the nucleoli, but instead is distributed throughout the nucleoplasm. Immunofluorescence studies indicate that acetylated nucleolin is co-localized with the splicing factor SC35 and partially with Y12. Acetylated nucleolin is expressed in all tested proliferating cell types. Our findings show that acetylation defines a new pool of nucleolin which support a role for nucleolin in the regulation of mRNA maturation and transcription by RNA polymerase II.


Assuntos
Nucléolo Celular/metabolismo , Lisina/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Western Blotting , Núcleo Celular/metabolismo , Células HeLa , Humanos , Soros Imunes/química , Leucócitos Mononucleares/metabolismo , Lisina/química , Lisina/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fosfoproteínas/química , Fosfoproteínas/imunologia , Transporte Proteico , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/imunologia , Coelhos , Nucleolina
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