RESUMO
The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.
Assuntos
Neoplasias Encefálicas/genética , Mapeamento Cromossômico/métodos , Glioblastoma/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Cromatina/genética , Cromatina/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano , Glioblastoma/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Células Jurkat , Desequilíbrio de Ligação , Camundongos , Camundongos Nus , Elongação da Transcrição GenéticaRESUMO
The heat shock response (HSR) is critical for survival of all organisms. However, its scope, extent, and the molecular mechanism of regulation are poorly understood. Here we show that the genome-wide transcriptional response to heat shock in mammals is rapid and dynamic and results in induction of several hundred and repression of several thousand genes. Heat shock factor 1 (HSF1), the "master regulator" of the HSR, controls only a fraction of heat shock-induced genes and does so by increasing RNA polymerase II release from promoter-proximal pause. Notably, HSF2 does not compensate for the lack of HSF1. However, serum response factor appears to transiently induce cytoskeletal genes independently of HSF1. The pervasive repression of transcription is predominantly HSF1-independent and is mediated through reduction of RNA polymerase II pause release. Overall, mammalian cells orchestrate rapid, dynamic, and extensive changes in transcription upon heat shock that are largely modulated at pause release, and HSF1 plays a limited and specialized role.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico , Mamíferos/genética , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Linhagem Celular , Fibroblastos/citologia , Regulação da Expressão Gênica , Fatores de Transcrição de Choque Térmico , Mamíferos/metabolismo , Camundongos , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fator de Resposta Sérica/genéticaRESUMO
Heat shock factor 1 (HSF1) is a master regulator that coordinates chaperone protein expression to enhance cellular survival in the face of heat stress. In cancer cells, HSF1 drives a transcriptional program distinct from heat shock to promote metastasis and cell survival. Its strong association with the malignant phenotype implies that HSF1 antagonists may have general and effective utilities in cancer therapy. For this purpose, we had identified an avid RNA aptamer for HSF1 that is portable among different model organisms. Extending our previous work in yeast and Drosophila, here we report the activity of this aptamer in human cancer cell lines. When delivered into cells using a synthetic gene and strong promoter, this aptamer was able to prevent HSF1 from binding to its DNA regulation elements. At the cellular level, expression of this aptamer induced apoptosis and abolished the colony-forming capability of cancer cells. At the molecular level, it reduced chaperones and attenuated the activation of the MAPK signaling pathway. Collectively, these data demonstrate the advantage of aptamers in drug target validation and support the hypothesis that HSF1 DNA binding activity is a potential target for controlling oncogenic transformation and neoplastic growth.
Assuntos
Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , RNA/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Fatores de Transcrição de Choque Térmico , Resposta ao Choque Térmico/genética , Temperatura Alta , Humanos , Células MCF-7 , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
Heat shock transcription factor (HSF1) is a conserved master regulator that orchestrates the protection of normal cells from stress. However, HSF1 also protects abnormal cells and is required for carcinogenesis. Here, we generate an highly specific RNA aptamer (iaRNA(HSF1)) that binds Drosophila HSF1 and inhibits HSF1 binding to DNA. In Drosophila animals, iaRNA(HSF1) reduces normal Hsp83 levels and promotes developmental abnormalities, mimicking the spectrum of phenotypes that occur when Hsp83 activity is reduced. The HSF1 aptamer also effectively suppresses the abnormal growth phenotypes induced by constitutively active forms of the EGF receptor and Raf oncoproteins. Our results indicate that HSF1 contributes toward the morphological development of animal traits by controlling the expression of molecular chaperones under normal growth conditions. Additionally, our study demonstrates the utility of the RNA aptamer technology as a promising chemical genetic approach to investigate biological mechanisms, including cancer and for identifying effective drug targets in vivo.
