RESUMO
Environmental stimuli commonly act via changes in gene regulation. Human-genome-scale assays to measure such responses are indirect or require knowledge of the transcription factors (TFs) involved. Here, we present the use of human genome-wide high-throughput reporter assays to measure environmentally-responsive regulatory element activity. We focus on responses to glucocorticoids (GCs), an important class of pharmaceuticals and a paradigmatic genomic response model. We assay GC-responsive regulatory activity across >108 unique DNA fragments, covering the human genome at >50×. Those assays directly detected thousands of GC-responsive regulatory elements genome-wide. We then validate those findings with measurements of transcription factor occupancy, histone modifications, chromatin accessibility, and gene expression. We also detect allele-specific environmental responses. Notably, the assays did not require knowledge of GC response mechanisms. Thus, this technology can be used to agnostically quantify genomic responses for which the underlying mechanism remains unknown.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Genoma Humano , Glucocorticoides/farmacologia , Elementos Reguladores de Transcrição/efeitos dos fármacos , Interação Gene-Ambiente , Ensaios de Triagem em Larga Escala , HumanosRESUMO
Cancer cells rely on dysregulated gene expression. This establishes specific transcriptional addictions that may be therapeutically exploited. Yet, the mechanisms that are ultimately responsible for these addictions are poorly understood. Here, we investigated the transcriptional dependencies of transformed cells to the transcription factors YAP and TAZ. YAP/TAZ physically engage the general coactivator bromodomain-containing protein 4 (BRD4), dictating the genome-wide association of BRD4 to chromatin. YAP/TAZ flag a large set of enhancers with super-enhancer-like functional properties. YAP/TAZ-bound enhancers mediate the recruitment of BRD4 and RNA polymerase II at YAP/TAZ-regulated promoters, boosting the expression of a host of growth-regulating genes. Treatment with small-molecule inhibitors of BRD4 blunts YAP/TAZ pro-tumorigenic activity in several cell or tissue contexts, causes the regression of pre-established, YAP/TAZ-addicted neoplastic lesions and reverts drug resistance. This work sheds light on essential mediators, mechanisms and genome-wide regulatory elements that are responsible for transcriptional addiction in cancer and lays the groundwork for a rational use of BET inhibitors according to YAP/TAZ biology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Transcrição Gênica , Neoplasias de Mama Triplo Negativas/genética , Aciltransferases , Carcinogênese/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Células HEK293 , Humanos , Proteínas Nucleares/antagonistas & inibidores , RNA Polimerase II/genética , Elementos Reguladores de Transcrição/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/patologia , Proteínas de Sinalização YAPRESUMO
Although we can increasingly measure transcription, chromatin, methylation, and other aspects of molecular biology at single-cell resolution, most assays survey only one aspect of cellular biology. Here we describe sci-CAR, a combinatorial indexing-based coassay that jointly profiles chromatin accessibility and mRNA (CAR) in each of thousands of single cells. As a proof of concept, we apply sci-CAR to 4825 cells, including a time series of dexamethasone treatment, as well as to 11,296 cells from the adult mouse kidney. With the resulting data, we compare the pseudotemporal dynamics of chromatin accessibility and gene expression, reconstruct the chromatin accessibility profiles of cell types defined by RNA profiles, and link cis-regulatory sites to their target genes on the basis of the covariance of chromatin accessibility and transcription across large numbers of single cells.
Assuntos
Cromatina/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Genômica/métodos , Análise de Célula Única/métodos , Células A549 , Animais , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Elementos Reguladores de Transcrição/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.
Assuntos
Imidazóis/farmacologia , Indóis/farmacologia , Atrofia Muscular Espinal/tratamento farmacológico , RNA Mensageiro/metabolismo , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Drosophila , Avaliação Pré-Clínica de Medicamentos , Éxons/genética , Células HeLa , Humanos , Imidazóis/química , Imidazóis/uso terapêutico , Indóis/química , Indóis/uso terapêutico , Terapia de Alvo Molecular/métodos , Atrofia Muscular Espinal/genética , Fenótipo , Sítios de Splice de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Elementos Reguladores de Transcrição/efeitos dos fármacos , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Emerging observations link dysregulation of TANK-binding kinase 1 (TBK1) to developmental disorders, inflammatory disease, and cancer. Biochemical mechanisms accounting for direct participation of TBK1 in host defense signaling have been well described. However, the molecular underpinnings of the selective participation of TBK1 in a myriad of additional cell biological systems in normal and pathophysiologic contexts remain poorly understood. To elucidate the context-selective role of TBK1 in cancer cell survival, we employed a combination of broad-scale chemogenomic and interactome discovery strategies to generate data-driven mechanism-of-action hypotheses. This approach uncovered evidence that TBK1 supports AKT/mTORC1 pathway activation and function through direct modulation of multiple pathway components acting both upstream and downstream of the mTOR kinase itself. Furthermore, we identified distinct molecular features in which mesenchymal, Ras-mutant lung cancer is acutely dependent on TBK1-mediated support of AKT/mTORC1 pathway activation for survival. Cancer Res; 77(18); 5077-94. ©2017 AACR.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Mesoderma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesoderma/efeitos dos fármacos , Mesoderma/patologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Elementos Reguladores de Transcrição/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Células Tumorais CultivadasRESUMO
DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation.
Assuntos
Metilação de DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Próstata/metabolismo , Receptores Androgênicos/metabolismo , Elementos Reguladores de Transcrição/genética , Androgênios/farmacologia , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Metilação de DNA/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Masculino , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Receptores Androgênicos/efeitos dos fármacos , Elementos Reguladores de Transcrição/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologiaRESUMO
Nucleotide changes in gene regulatory elements can have a major effect on interindividual differences in drug response. For example, by reviewing all published pharmacogenomic genome-wide association studies, we show here that 96.4% of the associated single nucleotide polymorphisms reside in noncoding regions. We discuss how sequencing technologies are improving our ability to identify drug response-associated regulatory elements genome-wide and to annotate nucleotide variants within them. We highlight specific examples of how nucleotide changes in these elements can affect drug response and illustrate the techniques used to find them and functionally characterize them. Finally, we also discuss challenges in the field of drug-responsive regulatory elements that need to be considered in order to translate these findings into the clinic.
Assuntos
Preparações Farmacêuticas/administração & dosagem , Elementos Reguladores de Transcrição/efeitos dos fármacos , Elementos Reguladores de Transcrição/genética , Animais , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Estudo de Associação Genômica Ampla/tendências , Humanos , Polimorfismo de Nucleotídeo Único/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Sequências Reguladoras de Ácido Nucleico/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Nucleosome positioning influences the access of transcription factors (TFs) to their binding sites and gene expression. Studies in plant, animal, and fungal models demonstrate similar nucleosome positioning patterns along genes and correlations between occupancy and expression. However, the relationships among nucleosome positioning, cis-regulatory element accessibility, and gene expression in plants remain undefined. Here we showed that plant nucleosome depletion occurs on specific 6-mer motifs and this sequence-specific nucleosome depletion is predictive of expression levels. Nucleosome-depleted regions in Arabidopsis thaliana tend to have higher G/C content, unlike yeast, and are centered on specific G/C-rich 6-mers, suggesting that intrinsic sequence properties, such as G/C content, cannot fully explain plant nucleosome positioning. These 6-mer motif sites showed higher DNase I hypersensitivity and are flanked by strongly phased nucleosomes, consistent with known TF binding sites. Intriguingly, this 6-mer-specific nucleosome depletion pattern occurs not only in promoter but also in genic regions and is significantly correlated with higher gene expression level, a phenomenon also found in rice but not in yeast. Among the 6-mer motifs enriched in genes responsive to treatment with the defense hormone jasmonate, there are no significant changes in nucleosome occupancy, suggesting that these sites are potentially preconditioned to enable rapid response without changing chromatin state significantly. Our study provides a global assessment of the joint contribution of nucleosome occupancy and motif sequences that are likely cis-elements to the control of gene expression in plants. Our findings pave the way for further understanding the impact of chromatin state on plant transcriptional regulatory circuits.
Assuntos
Arabidopsis/genética , DNA de Plantas/metabolismo , Nucleossomos/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Composição de Bases , Sítios de Ligação/efeitos dos fármacos , Ciclopentanos/farmacologia , DNA de Plantas/química , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Oxilipinas/farmacologia , Elementos Reguladores de Transcrição/efeitos dos fármacosRESUMO
OBJECTIVE: Adrenomedullin (AM), a potent vasodilator peptide, presents in various kinds of tumors and promotes angiogenesis. We have previously reported that AM is expressed in epithelial ovarian carcinoma tissue. Here, we investigated the hypothesis that AM might regulate production of vascular endothelial growth factor (VEGF) in epithelial ovarian carcinoma and further promote angiogenic processes. METHODS: The messenger RNA expression of VEGF in human epithelial ovarian carcinoma cells (HO-8910) was examined by real-time polymerase chain reaction. Transcriptional control was analyzed by transient transfection assay of VEGF promoter-luciferase hybrid genes and chromatin immunoprecipitation assay. Activation of c-Jun N-terminal kinase (JNK) was detected by Western blotting. The formation of capillarylike structures by EA.hy926 cells cocultured with HO-8910 cells on Matrigel was also studied. RESULTS: We found that in HO-8910 cells, AM (10⻹° to 10â»7 mol/L) enhanced VEGF messenger RNA expression in a time- and concentration-dependent manner, as well as promoter activity. Furthermore, JNK was activated by AM stimulation. The AM-induced increase in VEGF expression was significantly attenuated by SP600125, a specific JNK inhibitor. Chromatin immunoprecipitation assay and promoter activity analysis showed that VEGF expression induced by AM required the activator protein 1 motif on the VEGF promoter. In an in vitro angiogenesis system for endothelial cells (EA.hy926) cocultured with HO-8910 cells, we observed that the addition of AM stimulated endothelial cell tube formation, which could be abolished by VEGF neutralizing antibody. CONCLUSIONS: Our findings suggest that the JNK/Activator protein 1 pathway is involved in AM-induced VEGF expression in HO-8910 cells.
Assuntos
Adrenomedulina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Anti-Hipertensivos/farmacologia , Western Blotting , Carcinoma Epitelial do Ovário , Imunoprecipitação da Cromatina , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Neoplasias Epiteliais e Glandulares/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neoplasias Ovarianas/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Elementos Reguladores de Transcrição/efeitos dos fármacos , Elementos Reguladores de Transcrição/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator de Transcrição AP-1/genética , Ativação Transcricional , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development.
Assuntos
Epigênese Genética/efeitos dos fármacos , Proteínas de Homeodomínio/genética , RNA não Traduzido/genética , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Linhagem Celular , Cromatina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Elementos Reguladores de Transcrição/efeitos dos fármacosRESUMO
Methyl CpG Binding Protein 2 (MeCP2) is an important epigenetic factor in the brain. MeCP2 expression is affected by different environmental insults including alcohol exposure. Accumulating evidence supports the role of aberrant MeCP2 expression in ethanol exposure-induced neurological symptoms. However, the underlying molecular mechanisms of ethanol-induced MeCP2 deregulation remain elusive. To study the effect of ethanol on Mecp2/MeCP2 expression during neurodifferentiation, we established an in vitro model of ethanol exposure, using differentiating embryonic brain-derived neural stem cells (NSC). Previously, we demonstrated the impact of DNA methylation at the Mecp2 regulatory elements (REs) on Mecp2/MeCP2 expression in vitro and in vivo. Here, we studied whether altered DNA methylation at these REs is associated with the Mecp2/MeCP2 misexpression induced by ethanol. Binge-like and continuous ethanol exposure upregulated Mecp2/MeCP2, while ethanol withdrawal downregulated its expression. DNA methylation analysis by methylated DNA immunoprecipitation indicated that increased 5-hydroxymethylcytosine (5hmC) and decreased 5-methylcytosine (5mC) enrichment at specific REs were associated with upregulated Mecp2/MeCP2 following continuous ethanol exposure. The reduced Mecp2/MeCP2 expression upon ethanol withdrawal was associated with reduced 5hmC and increased 5mC enrichment at these REs. Moreover, ethanol altered global DNA methylation (5mC and 5hmC). Under the tested conditions, ethanol had minimal effects on NSC cell fate commitment, but caused changes in neuronal morphology and glial cell size. Taken together, our data represent an epigenetic mechanism for ethanol-mediated misexpression of Mecp2/MeCP2 in differentiating embryonic brain cells. We also show the potential role of DNA methylation and MeCP2 in alcohol-related neurological disorders, specifically Fetal Alcohol Spectrum Disorders.
Assuntos
5-Metilcitosina/metabolismo , Diferenciação Celular/fisiologia , Citosina/análogos & derivados , Etanol/toxicidade , Proteína 2 de Ligação a Metil-CpG/biossíntese , Células-Tronco Neurais/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citosina/metabolismo , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Elementos Reguladores de Transcrição/efeitos dos fármacos , Elementos Reguladores de Transcrição/fisiologiaRESUMO
Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , RNA Mensageiro/metabolismo , Elementos Reguladores de Transcrição/efeitos dos fármacos , Plântula/efeitos dos fármacos , Plântula/metabolismoRESUMO
Melanoma causes more deaths than any other skin cancer, and its incidence in the US continues to rise. Current medical therapies are insufficient to control this deadly neoplasm, necessitating the development of new target-based approaches. The objective of this study was to determine the role and functional significance of the class III histone deacetylase SIRT1 in melanoma. We have found that SIRT1 is overexpressed in clinical human melanoma tissues and human melanoma cell lines (Sk-Mel-2, WM35, G361, A375, and Hs294T) compared to normal skin and normal melanocytes, respectively. In addition, treatment of melanoma cell lines A375, Hs294T, and G361 with Tenovin-1, a small molecule SIRT1 inhibitor, resulted in a significant decrease in cell growth and cell viability. Further, Tenovin-1 treatment also resulted in a marked decrease in the clonogenic survival of melanoma cells. Further experiments showed that the anti-proliferative response of Tenovin-1 was accompanied by an increase in the protein as well as activity of the tumor suppressor p53. This increase in p53 activity was substantiated by an increase in the protein level of its downstream target p21. Overall, these data suggest that small molecule inhibition of SIRT1 causes anti-proliferative effects in melanoma cells. SIRT1 appears to be acting through the activity of the tumor suppressor p53, which is not mutated in the majority of melanomas. However, future detailed studies are needed to further explore the role and mechanism of SIRT1 in melanoma development and progression and its usefulness in melanoma treatment.
Assuntos
Acetanilidas/farmacologia , Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Tioureia/análogos & derivados , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanoma/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Elementos Reguladores de Transcrição/efeitos dos fármacos , Sirtuína 1/genética , Neoplasias Cutâneas/patologia , Tioureia/farmacologia , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Two cytidine analogues, gemcitabine and cytosine arabinoside (AraC), are widely used in the treatment of a variety of cancers with a large individual variation in response. To identify potential genetic biomarkers associated with response to these two drugs, we used a human lymphoblastoid cell line (LCL) model system with extensive genomic data, including 1.3 million SNPs and 54,000 basal expression probesets to perform genome-wide association studies (GWAS) with gemcitabine and AraC IC50 values. RESULTS: We identified 11 and 27 SNP loci significantly associated with gemcitabine and AraC IC50 values, respectively. Eleven candidate genes were functionally validated using siRNA knockdown approach in multiple cancer cell lines. We also characterized the potential mechanisms of genes by determining their influence on the activity of 10 cancer-related signaling pathways using reporter gene assays. Most SNPs regulated gene expression in a trans manner, except 7 SNPs in the PIGB gene that were significantly associated with both the expression of PIGB and gemcitabine cytotoxicity. CONCLUSION: These results suggest that genetic variation might contribute to drug response via either cis- or trans- regulation of gene expression. GWAS analysis followed by functional pharmacogenomics studies might help identify novel biomarkers contributing to variation in response to these two drugs and enhance our understanding of underlying mechanisms of drug action.
Assuntos
Citarabina/toxicidade , Desoxicitidina/análogos & derivados , Marcadores Genéticos/genética , Elementos Reguladores de Transcrição/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/toxicidade , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Desequilíbrio de Ligação , Manosiltransferases/antagonistas & inibidores , Manosiltransferases/genética , Polimorfismo de Nucleotídeo Único , Interferência de RNA , RNA Interferente Pequeno/metabolismo , GencitabinaRESUMO
We applied a metagenomics approach to screen for transcriptional regulators that sense aromatic compounds. The library was constructed by cloning environmental DNA fragments into a promoter-less vector containing green fluorescence protein. Fluorescence-based screening was then performed in the presence of various aromatic compounds. A total of 12 clones were isolated that fluoresced in response to salicylate, 3-methyl catechol, 4-chlorocatechol and chlorohydroquinone. Sequence analysis revealed at least 1 putative transcriptional regulator, excluding 1 clone (CHLO8F). Deletion analysis identified compound-specific transcriptional regulators; namely, 8 LysR-types, 2 two-component-types and 1 AraC-type. Of these, 9 representative clones were selected and their reaction specificities to 18 aromatic compounds were investigated. Overall, our transcriptional regulators were functionally diverse in terms of both specificity and induction rates. LysR- and AraC- type regulators had relatively narrow specificities with high induction rates (5-50 fold), whereas two-component-types had wide specificities with low induction rates (3 fold). Numerous transcriptional regulators have been deposited in sequence databases, but their functions remain largely unknown. Thus, our results add valuable information regarding the sequence-function relationship of transcriptional regulators.
Assuntos
Bactérias/genética , Hidrocarbonetos Aromáticos/toxicidade , Metagenoma/genética , Elementos Reguladores de Transcrição/efeitos dos fármacos , Elementos Reguladores de Transcrição/genética , Sequência de Bases , Catecóis , Clonagem Molecular , Escherichia coli , Biblioteca Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Hidroquinonas , Metagenômica , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNAAssuntos
Doença de Alzheimer , Ácidos Cumáricos/farmacologia , Heme Oxigenase-1/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Elementos Reguladores de Transcrição/efeitos dos fármacos , Tacrina/análogos & derivados , Doença de Alzheimer/metabolismo , Doença de Alzheimer/prevenção & controle , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Transformada , Ácidos Cumáricos/química , Ácidos Cumáricos/uso terapêutico , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/fisiologia , Elementos Reguladores de Transcrição/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tacrina/química , Tacrina/farmacologia , Tacrina/uso terapêuticoRESUMO
Our previous study shows that inhibiting activator protein one (AP1) transcription factor function in murine epidermis, using dominant-negative c-jun (TAM67), increases cell proliferation and delays differentiation. To understand the mechanism of action, we compare TAM67 impact in mouse epidermis and in cultured normal human keratinocytes. We show that TAM67 localizes in the nucleus where it forms TAM67 homodimers that competitively interact with AP1 transcription factor DNA binding sites to reduce endogenous jun and fos factor binding. Involucrin is a marker of keratinocyte differentiation that is expressed in the suprabasal epidermis and this expression requires AP1 factor interaction at the AP1-5 site in the promoter. TAM67 interacts competitively at this site to reduce involucrin expression. TAM67 also reduces endogenous c-jun, junB and junD mRNA and protein level. Studies with c-jun promoter suggest that this is due to reduced transcription of the c-jun gene. We propose that TAM67 suppresses keratinocyte differentiation by interfering with endogenous AP1 factor binding to regulator elements in differentiation-associated target genes, and by reducing endogenous c-jun factor expression.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Queratinócitos/fisiologia , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Ligação Competitiva , Diferenciação Celular/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Queratinócitos/metabolismo , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Precursores de Proteínas , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Elementos Reguladores de Transcrição/efeitos dos fármacos , Elementos Reguladores de Transcrição/genéticaRESUMO
Estrogen Receptor (ER) is a nuclear receptor that mediates the actions of estrogen and tamoxifen. ER is expressed in a major fraction of human breast cancers. Recently, genomic maps for estrogen- and tamoxifen-ER have been published. Interestingly, estrogen and tamoxifen induce similar genomic interactions and both ligands have been shown to use co-operating factors. The interactions of these co-operating factors within ER regions have impact both on ER-DNA interactions and gene expression regulated by estrogen and tamoxifen. Moreover, the study of chromatin changes induced by these factors has also provided significant insight into our understanding of ER transcriptional regulation. This methods review describes some functional genomic methods to study the influence of both ER ligands and ER co-operating factors. The analysis of protein-DNA interactions and chromatin changes can be explored by using classical and novel methods such as Chromatin Immunoprecipitation (ChIP) or Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE). This review also explores the properties of each of these methods and the advantages of combining them with high throughput sequencing.
Assuntos
Neoplasias da Mama/metabolismo , Genômica/métodos , Glândulas Mamárias Humanas/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Antineoplásicos Hormonais/metabolismo , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Reagentes de Ligações Cruzadas/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Glândulas Mamárias Humanas/efeitos dos fármacos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Elementos Reguladores de Transcrição/efeitos dos fármacos , Elementos de Resposta/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/metabolismo , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: Successful drug development has been hampered by a limited understanding of how to translate laboratory-based biological discoveries into safe and effective medicines. We have developed a generic method for predicting the effects of drugs on biological processes. Information derived from the chemical structure and experimental omics data from short-term efficacy studies are combined to predict the possible protein targets and cellular pathways affected by drugs. RESULTS: Validation of the method with anti-atherosclerotic compounds (fenofibrate, rosuvastatin, LXR activator T0901317) demonstrated a great conformity between the computationally predicted effects and the wet-lab biochemical effects. Comparative genome-wide pathway mapping revealed that the biological drug effects were realized largely via different pathways and mechanisms. In line with the predictions, the drugs showed differential effects on inflammatory pathways (downstream of PDGF, VEGF, IFNγ, TGFß, IL1ß, TNFα, LPS), transcriptional regulators (NFκB, C/EBP, STAT3, AP-1) and enzymes (PKCδ, AKT, PLA2), and they quenched different aspects of the inflammatory signaling cascade. Fenofibrate, the compound predicted to be most efficacious in inhibiting early processes of atherosclerosis, had the strongest effect on early lesion development. CONCLUSION: Our approach provides mechanistic rationales for the differential and common effects of drugs and may help to better understand the origins of drug actions and the design of combination therapies.
Assuntos
Anti-Inflamatórios/farmacologia , Aterosclerose/tratamento farmacológico , Fármacos Cardiovasculares/farmacologia , Desenho de Fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Biologia de Sistemas/métodos , Animais , Anti-Inflamatórios/uso terapêutico , Fármacos Cardiovasculares/uso terapêutico , Fenofibrato , Fluorbenzenos , Humanos , Hidrocarbonetos Fluorados , Camundongos , Análise em Microsséries , Pirimidinas , Elementos Reguladores de Transcrição/efeitos dos fármacos , Rosuvastatina Cálcica , Relação Estrutura-Atividade , SulfonamidasRESUMO
Histone deacetylase inhibitors (HDACIs) are promising anti-tumor agents that selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Fundamentally, HDACIs are proposed to function by activating the transcription of genes, including the potent cyclin dependent kinase inhibitor p21(WAF1). However, HDACIs primarily increase p21(WAF1) expression at the post-transcriptional level in HepG2 cells, implying that these anti-tumor agents regulate genes at multiple levels. Here, two novel cis-acting elements in the 3' untranslated region (UTR) of p21(WAF1) are identified that control the ability of HDACIs to induce p21(WAF1) mRNA stabilization. Collectively, these studies highlight the complexity of HDACIs in gene regulation.