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1.
Circ Genom Precis Med ; 11(8): e001977, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30354342

RESUMO

BACKGROUND: Genome-wide association studies have identified multiple loci associated with coronary artery disease and myocardial infarction, but only a few of these loci are current targets for on-market medications. To identify drugs suitable for repurposing and their targets, we created 2 unique pipelines integrating public data on 49 coronary artery disease/myocardial infarction-genome-wide association studies loci, drug-gene interactions, side effects, and chemical interactions. METHODS: We first used publicly available genome-wide association studies results on all phenotypes to predict relevant side effects, identified drug-gene interactions, and prioritized candidates for repurposing among existing drugs. Second, we prioritized gene product targets by calculating a druggability score to estimate how accessible pockets of coronary artery disease/myocardial infarction-associated gene products are, then used again the genome-wide association studies results to predict side effects, excluded loci with widespread cross-tissue expression to avoid housekeeping and genes involved in vital processes and accordingly ranked the remaining gene products. RESULTS: These pipelines ultimately led to 3 suggestions for drug repurposing: pentolinium, adenosine triphosphate, and riociguat (to target CHRNB4, ACSS2, and GUCY1A3, respectively); and 3 proteins for drug development: LMOD1 (leiomodin 1), HIP1 (huntingtin-interacting protein 1), and PPP2R3A (protein phosphatase 2, regulatory subunit b-double prime, α). Most current therapies for coronary artery disease/myocardial infarction treatment were also rediscovered. CONCLUSIONS: Integration of genomic and pharmacological data may prove beneficial for drug repurposing and development, as evidence from our pipelines suggests.


Assuntos
Fármacos Cardiovasculares , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/genética , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos/métodos , Loci Gênicos , Terapia de Alvo Molecular/métodos , Algoritmos , Animais , Fármacos Cardiovasculares/farmacocinética , Fármacos Cardiovasculares/uso terapêutico , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Interação Gene-Ambiente , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Simulação de Acoplamento Molecular , Testes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Fatores de Risco
2.
Sci Rep ; 8(1): 3434, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29467471

RESUMO

Genome-wide association studies (GWAS) have identified over two hundred chromosomal loci that modulate risk of coronary artery disease (CAD). The genes affected by variants at these loci are largely unknown and an untapped resource to improve our understanding of CAD pathophysiology and identify potential therapeutic targets. Here, we prioritized 68 genes as the most likely causal genes at genome-wide significant loci identified by GWAS of CAD and examined their regulatory roles in 286 metabolic and vascular tissue gene-protein sub-networks ("modules"). The modules and genes within were scored for CAD druggability potential. The scoring enriched for targets of cardiometabolic drugs currently in clinical use and in-depth analysis of the top-scoring modules validated established and revealed novel target tissues, biological processes, and druggable targets. This study provides an unprecedented resource of tissue-defined gene-protein interactions directly affected by genetic variance in CAD risk loci.


Assuntos
Doença da Artéria Coronariana/genética , Redes Reguladoras de Genes , Doença da Artéria Coronariana/tratamento farmacológico , Descoberta de Drogas , Redes Reguladoras de Genes/efeitos dos fármacos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Desequilíbrio de Ligação , Terapia de Alvo Molecular , Polimorfismo de Nucleotídeo Único/efeitos dos fármacos , Locos de Características Quantitativas/efeitos dos fármacos
3.
Genome Med ; 9(1): 115, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29268762

RESUMO

BACKGROUND: Monitoring treatment and early detection of fatal breast cancer (BC) remains a major unmet need. Aberrant circulating DNA methylation (DNAme) patterns are likely to provide a highly specific cancer signal. We hypothesized that cell-free DNAme markers could indicate disseminated breast cancer, even in the presence of substantial quantities of background DNA. METHODS: We used reduced representation bisulfite sequencing (RRBS) of 31 tissues and established serum assays based on ultra-high coverage bisulfite sequencing in two independent prospective serum sets (n = 110). The clinical use of one specific region, EFC#93, was validated in 419 patients (in both pre- and post-adjuvant chemotherapy samples) from SUCCESS (Simultaneous Study of Gemcitabine-Docetaxel Combination adjuvant treatment, as well as Extended Bisphosphonate and Surveillance-Trial) and 925 women (pre-diagnosis) from the UKCTOCS (UK Collaborative Trial of Ovarian Cancer Screening) population cohort, with overall survival and occurrence of incident breast cancer (which will or will not lead to death), respectively, as primary endpoints. RESULTS: A total of 18 BC specific DNAme patterns were discovered in tissue, of which the top six were further tested in serum. The best candidate, EFC#93, was validated for clinical use. EFC#93 was an independent poor prognostic marker in pre-chemotherapy samples (hazard ratio [HR] for death = 7.689) and superior to circulating tumor cells (CTCs) (HR for death = 5.681). More than 70% of patients with both CTCs and EFC#93 serum DNAme positivity in their pre-chemotherapy samples relapsed within five years. EFC#93-positive disseminated disease in post-chemotherapy samples seems to respond to anti-hormonal treatment. The presence of EFC#93 serum DNAme identified 42.9% and 25% of women who were diagnosed with a fatal BC within 3-6 and 6-12 months of sample donation, respectively, with a specificity of 88%. The sensitivity with respect to detecting fatal BC was ~ 4-fold higher compared to non-fatal BC. CONCLUSIONS: Detection of EFC#93 serum DNAme patterns offers a new tool for early diagnosis and management of disseminated breast cancers. Clinical trials are required to assess whether EFC#93-positive women in the absence of radiological detectable breast cancers will benefit from anti-hormonal treatment before the breast lesions become clinically apparent.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Ácidos Nucleicos Livres/genética , Metilação de DNA , Adulto , Idoso , Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia
4.
Genome Med ; 9(1): 116, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29268796

RESUMO

BACKGROUND: Despite a myriad of attempts in the last three decades to diagnose ovarian cancer (OC) earlier, this clinical aim still remains a significant challenge. Aberrant methylation patterns of linked CpGs analyzed in DNA fragments shed by cancers into the bloodstream (i.e. cell-free DNA) can provide highly specific signals indicating cancer presence. METHODS: We analyzed 699 cancerous and non-cancerous tissues using a methylation array or reduced representation bisulfite sequencing to discover the most specific OC methylation patterns. A three-DNA-methylation-serum-marker panel was developed using targeted ultra-high coverage bisulfite sequencing in 151 women and validated in 250 women with various conditions, particularly in those associated with high CA125 levels (endometriosis and other benign pelvic masses), serial samples from 25 patients undergoing neoadjuvant chemotherapy, and a nested case control study of 172 UKCTOCS control arm participants which included serum samples up to two years before OC diagnosis. RESULTS: The cell-free DNA amount and average fragment size in the serum samples was up to ten times higher than average published values (based on samples that were immediately processed) due to leakage of DNA from white blood cells owing to delayed time to serum separation. Despite this, the marker panel discriminated high grade serous OC patients from healthy women or patients with a benign pelvic mass with specificity/sensitivity of 90.7% (95% confidence interval [CI] = 84.3-94.8%) and 41.4% (95% CI = 24.1-60.9%), respectively. Levels of all three markers plummeted after exposure to chemotherapy and correctly identified 78% and 86% responders and non-responders (Fisher's exact test, p = 0.04), respectively, which was superior to a CA125 cut-off of 35 IU/mL (20% and 75%). 57.9% (95% CI 34.0-78.9%) of women who developed OC within two years of sample collection were identified with a specificity of 88.1% (95% CI = 77.3-94.3%). Sensitivity and specificity improved further when specifically analyzing CA125 negative samples only (63.6% and 87.5%, respectively). CONCLUSIONS: Our data suggest that DNA methylation patterns in cell-free DNA have the potential to detect a proportion of OCs up to two years in advance of diagnosis and may potentially guide personalized treatment. The prospective use of novel collection vials, which stabilize blood cells and reduce background DNA contamination in serum/plasma samples, will facilitate clinical implementation of liquid biopsy analyses.


Assuntos
Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/genética , Metilação de DNA , Neoplasias Ovarianas/genética , Adulto , Idoso , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/normas , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Distribuição Aleatória , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas
5.
Cancer Res ; 76(10): 3097-108, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27197233

RESUMO

Aberrant hypermethylation of CpG islands (CGI) in human tumors occurs predominantly at repressed genes in the host tissue, but the preceding events driving this phenomenon are poorly understood. In this study, we temporally tracked epigenetic and transcriptomic perturbations that occur in a mouse model of liver carcinogenesis. Hypermethylated CGI events in the model were predicted by enrichment of the DNA modification 5-hydroxymethylcytosine (5hmC) and the histone H3 modification H3K27me3 at silenced promoters in the host tissue. During cancer progression, selected CGIs underwent hypo-hydroxymethylation prior to hypermethylation, while retaining H3K27me3. In livers from mice deficient in Tet1, a tumor suppressor involved in cytosine demethylation, we observed a similar loss of promoter core 5hmC, suggesting that reduced Tet1 activity at CGI may contribute to epigenetic dysregulation during hepatocarcinogenesis. Consistent with this possibility, mouse liver tumors exhibited reduced Tet1 protein levels. Similar to humans, DNA methylation changes at CGI in mice did not appear to be direct drivers of hepatocellular carcinoma progression, rather, dynamic changes in H3K27me3 promoter deposition correlated strongly with tumor-specific activation and repression of transcription. Overall, our results suggest that loss of promoter-associated 5hmC in liver tumors licenses reprograming of DNA methylation at silent CGI during progression. Cancer Res; 76(10); 3097-108. ©2016 AACR.


Assuntos
5-Metilcitosina/análogos & derivados , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Neoplasias Hepáticas Experimentais/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/toxicidade , Animais , Carcinoma Hepatocelular , Diferenciação Celular , Histonas/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
6.
Epigenomics ; 6(3): 311-27, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25111485

RESUMO

Breast, ovarian and endometrial cancers cause significant morbidity and mortality. Despite the presence of existing screening, diagnostic and treatment modalities, they continue to pose considerable unsolved challenges. Overdiagnosis is a growing problem in breast cancer screening and neither screening nor early diagnosis of ovarian or endometrial cancer is currently possible. Moreover, treatment of the diversity of these cancers presenting in the clinic is not sufficiently personalized at present. Recent technological advances, including reduced representation bisulfite sequencing, methylation arrays, digital PCR, next-generation sequencing and advanced statistical data analysis, enable the analysis of methylation patterns in cell-free tumor DNA in serum/plasma. Ongoing work is bringing these methods together for the analysis of samples from large clinical trials, which have been collected well in advance of cancer diagnosis. These efforts pave the way for the development of a noninvasive method that would enable us to overcome existing challenges to personalized medicine.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Metilação de DNA , Detecção Precoce de Câncer , Neoplasias dos Genitais Femininos/diagnóstico , Detecção Precoce de Câncer/métodos , Feminino , Genômica , Humanos , Medicina de Precisão
7.
Toxicol Sci ; 139(2): 501-11, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24690595

RESUMO

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.


Assuntos
Ciclo Celular/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fenobarbital/toxicidade , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Transcriptoma/efeitos dos fármacos , Animais , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Receptor Constitutivo de Androstano , Perfilação da Expressão Gênica , Humanos , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenobarbital/farmacocinética , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Especificidade da Espécie , Xenobióticos/farmacocinética , Xenobióticos/toxicidade
8.
Int J Cancer ; 135(7): 1574-85, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24535843

RESUMO

The process of hepatocarcinogenesis in the diethylnitrosamine (DEN) initiation/phenobarbital (PB) promotion mouse model involves the selective clonal outgrowth of cells harboring oncogene mutations in Ctnnb1, while spontaneous or DEN-only-induced tumors are often Ha-ras- or B-raf-mutated. The molecular mechanisms and pathways underlying these different tumor sub-types are not well characterized. Their identification may help identify markers for xenobiotic promoted versus spontaneously occurring liver tumors. Here, we have characterized mouse liver tumors harboring either Ctnnb1 or Ha-ras mutations via integrated molecular profiling at the transcriptional, translational and post-translational levels. In addition, metabolites of the intermediary metabolism were quantified by high resolution (1)H magic angle nuclear magnetic resonance. We have identified tumor genotype-specific differences in mRNA and miRNA expression, protein levels, post-translational modifications, and metabolite levels that facilitate the molecular and biochemical stratification of tumor phenotypes. Bioinformatic integration of these data at the pathway level led to novel insights into tumor genotype-specific aberrant cell signaling and in particular to a better understanding of alterations in pathways of the cell intermediary metabolism, which are driven by the constitutive activation of the ß-Catenin and Ha-ras oncoproteins in tumors of the two genotypes.


Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Genes ras/genética , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Metabolômica , Mutação/genética , beta Catenina/genética , Animais , Biomarcadores Tumorais/metabolismo , Western Blotting , Redes e Vias Metabólicas , Camundongos , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Nucleic Acids Res ; 41(11): 5639-54, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23598998

RESUMO

Aberrant DNA methylation is a common feature of neoplastic lesions, and early detection of such changes may provide powerful mechanistic insights and biomarkers for carcinogenesis. Here, we investigate dynamic changes in the mouse liver DNA methylome associated with short (1 day) and prolonged (7, 28 and 91 days) exposure to the rodent liver non-genotoxic carcinogen, phenobarbital (PB). We find that the distribution of 5mC/5hmC is highly consistent between untreated individuals of a similar age; yet, changes during liver maturation in a transcriptionally dependent manner. Following drug treatment, we identify and validate a series of differentially methylated or hydroxymethylated regions: exposure results in staged transcriptional responses with distinct kinetic profiles that strongly correlate with promoter proximal region 5hmC levels. Furthermore, reciprocal changes for both 5mC and 5hmC in response to PB suggest that active demethylation may be taking place at each set of these loci via a 5hmC intermediate. Finally, we identify potential early biomarkers for non-genotoxic carcinogenesis, including several genes aberrantly expressed in liver cancer. Our work suggests that 5hmC profiling can be used as an indicator of cell states during organ maturation and drug-induced responses and provides novel epigenetic signatures for non-genotoxic carcinogen exposure.


Assuntos
Citosina/análogos & derivados , Metilação de DNA , Epigênese Genética , Fígado/metabolismo , 5-Metilcitosina/metabolismo , Animais , Carcinógenos/toxicidade , Transformação Celular Neoplásica , Sistema Enzimático do Citocromo P-450/genética , Citosina/metabolismo , Epigênese Genética/efeitos dos fármacos , Marcadores Genéticos , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fenobarbital/toxicidade , Regiões Promotoras Genéticas , Transcriptoma/efeitos dos fármacos
10.
Toxicol Sci ; 131(2): 375-86, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23091169

RESUMO

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for ß-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and ß-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.


Assuntos
Biomarcadores Tumorais/genética , Impressão Genômica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Iodeto Peroxidase/genética , Neoplasias Hepáticas Experimentais/genética , Família Multigênica , RNA não Traduzido/genética , Animais , Proteínas de Ligação ao Cálcio , Receptor Constitutivo de Androstano , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Reação em Cadeia da Polimerase , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Transcriptoma , beta Catenina/metabolismo
11.
Genome Biol ; 13(10): R93, 2012 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-23034186

RESUMO

BACKGROUND: Induction and promotion of liver cancer by exposure to non-genotoxic carcinogens coincides with epigenetic perturbations, including specific changes in DNA methylation. Here we investigate the genome-wide dynamics of 5-hydroxymethylcytosine (5hmC) as a likely intermediate of 5-methylcytosine (5mC) demethylation in a DNA methylation reprogramming pathway. We use a rodent model of non-genotoxic carcinogen exposure using the drug phenobarbital. RESULTS: Exposure to phenobarbital results in dynamic and reciprocal changes to the 5mC/5hmC patterns over the promoter regions of a cohort of genes that are transcriptionally upregulated. This reprogramming of 5mC/5hmC coincides with characteristic changes in the histone marks H3K4me2, H3K27me3 and H3K36me3. Quantitative analysis of phenobarbital-induced genes that are involved in xenobiotic metabolism reveals that both DNA modifications are lost at the transcription start site, while there is a reciprocal relationship between increasing levels of 5hmC and loss of 5mC at regions immediately adjacent to core promoters. CONCLUSIONS: Collectively, these experiments support the hypothesis that 5hmC is a potential intermediate in a demethylation pathway and reveal precise perturbations of the mouse liver DNA methylome and hydroxymethylome upon exposure to a rodent hepatocarcinogen.


Assuntos
Citosina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Fígado/metabolismo , Fenobarbital/farmacologia , 5-Metilcitosina/análogos & derivados , Animais , Citosina/metabolismo , Epigenômica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/efeitos dos fármacos
12.
PLoS One ; 6(3): e18216, 2011 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-21455306

RESUMO

Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Epigenômica/métodos , Fenobarbital/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Esteroide Hidroxilases/genética , Animais , Imunoprecipitação da Cromatina , Receptor Constitutivo de Androstano , Ilhas de CpG/efeitos dos fármacos , Ilhas de CpG/genética , Família 2 do Citocromo P450 , Metilação de DNA/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
13.
Curr Opin Cell Biol ; 21(6): 855-63, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19796927

RESUMO

Ribosomes provide the basis for protein production and this drives cell growth. Recent studies, both in yeast and in higher eukaryotes, are beginning to reveal new mechanisms underlying the elaborate control of ribosome biogenesis, which requires coordinate regulation of all three RNA polymerases. Transcription of ribosomal RNA is finely tuned to cellular energy status and linked to the production of ribosomal proteins. Several autoregulatory mechanisms controlling various aspects of ribosome biogenesis have been uncovered and reveal new connections to cell-cycle and cell-size control. Ribosome biogenesis has now been clearly linked to disease, particularly to cancer and anemia, and also to aging. A challenge for future studies will be to elucidate further the molecular mechanisms underlying these connections.


Assuntos
Ribossomos/metabolismo , Animais , Ciclo Celular , DNA Ribossômico/metabolismo , Eucariotos/metabolismo , Crescimento/fisiologia , Humanos , Modelos Biológicos , Biogênese de Organelas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transcrição Gênica
14.
EMBO J ; 28(20): 3067-73, 2009 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-19779456

RESUMO

Phosphatidylinositol-3 kinase-related kinases (PIKKs) comprise a family of protein kinases that respond to various stresses, including DNA damage, blocks in DNA replication, availability of nutrients and errors in mRNA splicing. PIKKs are characterized by the presence of a conserved kinase domain (KD), whose activity is regulated by two C-terminal regions, referred to as PIKK-regulatory domain (PRD) and FRAP-ATM-TRRAP-C-terminal (FATC), respectively. Here, we review functional and structural data that implicate the PRD and FATC domains in regulation of PIKK activity, drawing parallels to phosphatidylinositol-3 kinases (PI3K), lipid kinases that have sequence similarity to PIKKs. The PI3K C-terminus, which we propose to be equivalent to the PRD and FATC domains of PIKKs, is in close proximity to the activation loop of the KD, suggesting that in PIKKs, the PRD and FATC domains may regulate kinase activity by targeting the activation loop.


Assuntos
Regulação Enzimológica da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Humanos , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Estrutura Terciária de Proteína , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologia
15.
Mol Cell ; 33(6): 704-16, 2009 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-19328065

RESUMO

Ribosome biogenesis drives cell growth, and the large transcriptional output underlying this process is tightly regulated. The Target of Rapamycin (TOR) kinase is part of a highly conserved signaling pathway linking nutritional and stress signals to regulation of ribosomal protein (RP) and ribosome biogenesis (Ribi) gene transcription. In Saccharomyces cerevisiae, one of the downstream effectors of TOR is Sfp1, a transcriptional activator that regulates both RP and Ribi genes. Here, we report that Sfp1 interacts directly with TOR complex 1 (TORC1) in a rapamycin-regulated manner, and that phosphorylation of Sfp1 by this kinase complex regulates its function. Sfp1, in turn, negatively regulates TORC1 phosphorylation of Sch9, another key TORC1 target that acts in parallel with Sfp1, revealing a feedback mechanism controlling the activity of these proteins. Finally, we show that the Sfp1-interacting protein Mrs6, a Rab escort protein involved in membrane trafficking, regulates both Sfp1 nuclear localization and TORC1 signaling.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Imunoprecipitação , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia
16.
Mol Biol Cell ; 20(3): 1048-57, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19073887

RESUMO

The conserved Target Of Rapamycin (TOR) growth control signaling pathway is a major regulator of genes required for protein synthesis. The ubiquitous toxic metalloid arsenic, as well as mercury and nickel, are shown here to efficiently inhibit the rapamycin-sensitive TORC1 (TOR complex 1) protein kinase. This rapid inhibition of the TORC1 kinase is demonstrated in vivo by the dephosphorylation and inactivation of its downstream effector, the yeast S6 kinase homolog Sch9. Arsenic, mercury, and nickel cause reduction of transcription of ribosome biogenesis genes, which are under the control of Sfp1, a TORC1-regulated transcriptional activator. We report that arsenic stress deactivates Sfp1 as it becomes dephosphorylated, dissociates from chromatin, and exits the nucleus. Curiously, whereas loss of SFP1 function leads to increased arsenic resistance, absence of TOR1 or SCH9 has the opposite effect suggesting that TORC1 has a role beyond down-regulation of Sfp1. Indeed, we show that arsenic activates the transcription factors Msn2 and Msn4 both of which are targets of TORC1 and protein kinase A (PKA). In contrast to TORC1, PKA activity is not repressed during acute arsenic stress. A normal level of PKA activity might serve to dampen the stress response since hyperactive Msn2 will decrease arsenic tolerance. Thus arsenic toxicity in yeast might be determined by the balance between chronic activation of general stress factors in combination with lowered TORC1 kinase activity.


Assuntos
Arsênio/toxicidade , Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Estresse Fisiológico/efeitos dos fármacos , Sequência de Bases , Cromatina/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/efeitos dos fármacos
17.
Mol Endocrinol ; 19(9): 2258-72, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15905360

RESUMO

Ligand-dependent signal transduction by nuclear receptors (NRs) includes dynamic exchanges of coactivator (CoA) and corepressor (CoR) proteins. Here we focused on the structural determinants of the antagonist- and inverse agonist-enhanced interaction of the endocrine NR vitamin D receptor (VDR) and the adopted orphan NR constitutive androstane receptor (CAR) from two species with the CoR NR corepressor. We found that the pure VDR antagonist ZK168281 and the human CAR inverse agonist clotrimazole are both effective inhibitors of the CoA interaction of their respective receptors, whereas ZK168281 resembled more the mouse CAR inverse agonist androstanol in its ability to recruit CoR proteins. Molecular dynamics simulations resulted in comparable models for the CoR receptor interaction domain peptide bound to VDR/antagonist or CAR/inverse agonist complexes. A salt bridge between the CoR and a conserved lysine in helix 4 of the NR is central to this interaction, but also helix 12 was stabilized by direct contacts with residues of the CoR. Fixation of helix 12 in the antagonistic/inverse agonistic conformation prevents an energetically unfavorable free floatation of the C terminus. The comparable molecular mechanisms that explain the similar functional profile of antagonist and inverse agonists are likely to be extended from VDR and CAR to other members of the NR superfamily and may lead to the design of even more effective ligands.


Assuntos
Proteínas Nucleares/metabolismo , Receptores de Calcitriol/antagonistas & inibidores , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/agonistas , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Androstanóis/química , Androstanóis/farmacologia , Animais , Calcitriol/análogos & derivados , Calcitriol/química , Calcitriol/farmacologia , Células Cultivadas , Clotrimazol/química , Clotrimazol/farmacologia , Simulação por Computador , Receptor Constitutivo de Androstano , Dimerização , Regulação da Expressão Gênica , Humanos , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Correpressor 1 de Receptor Nuclear , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Receptores de Calcitriol/química , Receptores Citoplasmáticos e Nucleares/química , Receptores X de Retinoides/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/química
18.
J Biol Chem ; 279(32): 33558-66, 2004 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-15151997

RESUMO

The constitutive androstane receptor (CAR) is an interesting member of the nuclear receptor superfamily because of its exceptionally high constitutive activity due to ligand-independent interaction of the ligand-binding domain with co-activator proteins. This study compares the agonist-dependent and agonist-independent activities of human CAR with those of mouse CAR and the vitamin D receptor and demonstrates that the constitutive activity of CAR is mediated by at least three contacts between the amino acids of helix 12, partner amino acids in helices 4 and 11, and a charge clamp between helices 12 and 3. The stabilization of helix 12 by a contact between its C terminus and the lysine of helix 4 has the same impact in human and mouse CARs. In addition, the charge clamp between the glutamate in helix 12 and the lysine in helix 3 is also important for the constitutive activity of both receptor orthologs but less critical for the agonist-dependent stabilization of their respective helices 12. Interestingly, Cys-357 in mouse CAR has significantly more impact on the stabilization of helix 12 than does the orthologous position Cys-347 in human CAR. This deficit appears to be compensated by a more dominant role of Ile-330 in human CAR over Leu-340 in mouse CAR because it is more efficient than Cys-347 in controlling the flexibility of helix 12 in the presence of an agonist. The constitutive activity of other members of the nuclear receptor superfamily could be explained by a homologous hydrophobic interaction between large, non-polar amino acids of helices 11 and 12.


Assuntos
Sequência de Aminoácidos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/agonistas , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Sítios de Ligação , Neoplasias da Mama , Receptor Constitutivo de Androstano , Cisteína , Dimerização , Estabilidade de Medicamentos , Ácido Glutâmico , Humanos , Isoleucina , Leucina , Lisina , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Transcrição/química , Transfecção , Células Tumorais Cultivadas
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