RESUMO
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , RNA Polimerase II , Transcrição Gênica , Fatores de Elongação da Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Regiões Promotoras Genéticas , Sistemas CRISPR-Cas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Regulação da Expressão Gênica , Fosforilação , Ligação Proteica , Resposta ao Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Nucleossomos/genéticaRESUMO
Heat shock instantly reprograms transcription. Whether gene and enhancer transcription fully recover from stress and whether stress establishes a memory by provoking transcription regulation that persists through mitosis remained unknown. Here, we measured nascent transcription and chromatin accessibility in unconditioned cells and in the daughters of stress-exposed cells. Tracking transcription genome-wide at nucleotide-resolution revealed that cells precisely restored RNA polymerase II (Pol II) distribution at gene bodies and enhancers upon recovery from stress. However, a single heat exposure in embryonic fibroblasts primed a faster gene induction in their daughter cells by increasing promoter-proximal Pol II pausing and by accelerating the pause release. In K562 erythroleukemia cells, repeated stress refined basal and heat-induced transcription over mitotic division and decelerated termination-coupled pre-mRNA processing. The slower termination retained transcripts on the chromatin and reduced recycling of Pol II. These results demonstrate that heat-induced transcriptional memory acts through promoter-proximal pause release and pre-mRNA processing at transcription termination.
Assuntos
Mitose/genética , Regiões Promotoras Genéticas/genética , Estresse Fisiológico/genética , Transcrição Gênica/genética , Linhagem Celular Tumoral , Cromatina/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica/genética , Genoma/genética , Resposta ao Choque Térmico/genética , Humanos , Células K562 , RNA Polimerase II/genética , RNA Mensageiro/genéticaRESUMO
Transcriptionally silent genes must be activated throughout development. This requires nucleosomes be removed from promoters and enhancers to allow transcription factor (TF) binding and recruitment of coactivators and RNA polymerase II (Pol II). Specialized pioneer TFs bind nucleosome-wrapped DNA to perform this chromatin opening by mechanisms that remain incompletely understood. Here, we show that GAGA factor (GAF), a Drosophila pioneer-like factor, functions with both SWI/SNF and ISWI family chromatin remodelers to allow recruitment of Pol II and entry to a promoter-proximal paused state, and also to promote Pol II's transition to productive elongation. We found that GAF interacts with PBAP (SWI/SNF) to open chromatin and allow Pol II to be recruited. Importantly, this activity is not dependent on NURF as previously proposed; however, GAF also synergizes with NURF downstream from this process to ensure efficient Pol II pause release and transition to productive elongation, apparently through its role in precisely positioning the +1 nucleosome. These results demonstrate how a single sequence-specific pioneer TF can synergize with remodelers to activate sets of genes. Furthermore, this behavior of remodelers is consistent with findings in yeast and mice, and likely represents general, conserved mechanisms found throughout eukarya.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina/genética , Ligação Proteica , RNA Polimerase II/metabolismo , Elongação da Transcrição GenéticaRESUMO
Recent advances in sequencing techniques that measure nascent transcripts and that reveal the positioning of RNA polymerase II (Pol II) have shown that the pausing of Pol II in promoter-proximal regions and its release to initiate a phase of productive elongation are key steps in transcription regulation. Moreover, after the release of Pol II from the promoter-proximal region, elongation rates are highly dynamic throughout the transcription of a gene, and vary on a gene-by-gene basis. Interestingly, Pol II elongation rates affect co-transcriptional processes such as splicing, termination and genome stability. Increasing numbers of factors and regulatory mechanisms have been associated with the steps of transcription elongation by Pol II, revealing that elongation is a highly complex process. Elongation is thus now recognized as a key phase in the regulation of transcription by Pol II.
Assuntos
Proteínas de Drosophila/genética , Genoma , Neoplasias/genética , Proteínas Nucleares/genética , Fator B de Elongação Transcricional Positiva/genética , RNA Polimerase II/genética , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Animais , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Instabilidade Genômica , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fator B de Elongação Transcricional Positiva/antagonistas & inibidores , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Our previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Here, through the first genome-wide binding and expression profiles obtained for a FUBP family protein, we demonstrate that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFß signalling). We further demonstrate tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.
Assuntos
Proteínas de Drosophila , Drosophila , Proteínas de Ligação a RNA , Animais , Divisão Celular , Proliferação de Células , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ativação TranscricionalRESUMO
We report the immediate effects of estrogen signaling on the transcriptome of breast cancer cells using global run-on and sequencing (GRO-seq). The data were analyzed using a new bioinformatic approach that allowed us to identify transcripts directly from the GRO-seq data. We found that estrogen signaling directly regulates a strikingly large fraction of the transcriptome in a rapid, robust, and unexpectedly transient manner. In addition to protein-coding genes, estrogen regulates the distribution and activity of all three RNA polymerases and virtually every class of noncoding RNA that has been described to date. We also identified a large number of previously undetected estrogen-regulated intergenic transcripts, many of which are found proximal to estrogen receptor binding sites. Collectively, our results provide the most comprehensive measurement of the primary and immediate estrogen effects to date and a resource for understanding rapid signal-dependent transcription in other systems.
Assuntos
Neoplasias da Mama/genética , Biologia Computacional/métodos , Estrogênios/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Técnicas Genéticas , Humanos , RNA não Traduzido/genética , Análise de Sequência de DNA , Transdução de SinaisRESUMO
Following the discovery of widespread enhancer transcription, enhancers and promoters have been found to be far more similar than previously thought. In this issue of Genes & Development, two studies (Henriques and colleagues [pp. 26-41] and Mikhaylichenko and colleagues [pp. 42-57]) shine new light on the transcriptional nature of promoters and enhancers in Drosophila Together, these studies support recent work in mammalian cells that indicates that most active enhancers drive local transcription using factors and mechanisms similar to those of promoters. Intriguingly, enhancer transcription is shown to be coordinated by SPT5- and P-TEFb-mediated pause-release, but the pause half-life is shorter, and termination is more rapid at enhancers than at promoters. Moreover, bidirectional transcription from promoters is associated with enhancer activity, lending further credence to models in which regulatory elements exist along a spectrum of promoter-ness and enhancer-ness. We propose a general unified model to explain possible functions of transcription at enhancers.
Assuntos
Drosophila/genética , Elementos Facilitadores Genéticos , Animais , Regiões Promotoras GenéticasRESUMO
The programmes that direct an organism's development and maintenance are encoded in its genome. Decoding of this information begins with regulated transcription of genomic DNA into RNA. Although transcription and its control can be tracked indirectly by measuring stable RNAs, it is only by directly measuring nascent RNAs that the immediate regulatory changes in response to developmental, environmental, disease and metabolic signals are revealed. Multiple complementary methods have been developed to quantitatively track nascent transcription genome-wide at nucleotide resolution, all of which have contributed novel insights into the mechanisms of gene regulation and transcription-coupled RNA processing. Here we critically evaluate the array of strategies used for investigating nascent transcription and discuss the recent conceptual advances they have provided.
Assuntos
Regulação da Expressão Gênica/fisiologia , Interação Gene-Ambiente , Genoma Humano/fisiologia , RNA Mensageiro/biossíntese , Transcrição Gênica/fisiologia , Animais , Humanos , RNA Mensageiro/genéticaRESUMO
Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. Here, we identify a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. We find that Drosophila CBP inhibition results in "dribbling" of Pol II from the pause site to positions further downstream but impedes transcription through the +1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Nucleossomos/enzimologia , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Nucleossomos/genética , Ligação Proteica , RNA Polimerase II/genética , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Transcrição de p300-CBP/genéticaRESUMO
Proteotoxic stress, that is, stress caused by protein misfolding and aggregation, triggers the rapid and global reprogramming of transcription at genes and enhancers. Genome-wide assays that track transcriptionally engaged RNA polymerase II (Pol II) at nucleotide resolution have provided key insights into the underlying molecular mechanisms that regulate transcriptional responses to stress. In addition, recent kinetic analyses of transcriptional control under heat stress have shown how cells 'prewire' and rapidly execute genome-wide changes in transcription while concurrently becoming poised for recovery. The regulation of Pol II at genes and enhancers in response to heat stress is coupled to chromatin modification and compartmentalization, as well as to co-transcriptional RNA processing. These mechanistic features seem to apply broadly to other coordinated genome-regulatory responses.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Resposta ao Choque Térmico , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Cromatina/genética , Elementos Facilitadores Genéticos , Humanos , Regiões Promotoras Genéticas , RNA Polimerase II/efeitos dos fármacosRESUMO
The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity 1 . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3'-end formation and termination 2 , but how this sequence is initiated remains unclear. In a chemical-genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 (CycT1) complex 3 . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 4 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat 5 . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing 6 ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling-a switch that is analogous to a Cdk1-PP1 circuit that controls mitotic progression 4 .
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Elongação da Transcrição Genética , Terminação da Transcrição Genética , Sequência de Aminoácidos , Quinase 9 Dependente de Ciclina/química , Humanos , Mitose , Fosfoproteínas Fosfatases/química , Fosforilação , RNA Polimerase II/química , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/química , Transdução de Sinais , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismoRESUMO
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
The coordinated regulation of gene expression at the transcriptional level is fundamental to development and homeostasis. Inducible systems are invaluable when studying transcription because the regulatory process can be triggered instantaneously, allowing the tracking of ordered mechanistic events. Here, we use precision run-on sequencing (PRO-seq) to examine the genome-wide heat shock (HS) response in Drosophila and the function of two key transcription factors on the immediate transcription activation or repression of all genes regulated by HS. We identify the primary HS response genes and the rate-limiting steps in the transcription cycle that GAGA-associated factor (GAF) and HS factor (HSF) regulate. We demonstrate that GAF acts upstream of promoter-proximally paused RNA polymerase II (Pol II) formation (likely at the step of chromatin opening) and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing but is critical for the release of paused Pol II into the gene body at a subset of highly activated genes. Additionally, HSF has no detectable role in the rapid HS repression of thousands of genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulação da Expressão Gênica/genética , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , DNA Polimerase II/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Fatores de Transcrição de Choque Térmico , Regiões Promotoras Genéticas/genética , Interferência de RNA , Fatores de Transcrição/genéticaRESUMO
To efficiently transcribe genes, RNA Polymerase II (Pol II) must overcome barriers imposed by nucleosomes and higher-order chromatin structure. Many genes, including Drosophila melanogaster Hsp70, undergo changes in chromatin structure upon activation. To characterize these changes, we mapped the nucleosome landscape of Hsp70 after an instantaneous heat shock at high spatial and temporal resolution. Surprisingly, we find an initial disruption of nucleosomes across the entire gene within 30 s after activation, faster than the rate of Pol II transcription, followed by a second further disruption within 2 min. This initial change occurs independently of Pol II transcription. Furthermore, the rapid loss of nucleosomes extends beyond Hsp70 and halts at the scs and scs' insulating elements. An RNAi screen of 28 transcription and chromatin-related factors reveals that depletion of heat shock factor, GAGA Factor, or Poly(ADP)-Ribose Polymerase or its activity abolishes the loss of nucleosomes upon Hsp70 activation.
Assuntos
Cromatina/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , Animais , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Drosophila melanogaster/metabolismo , Temperatura Alta , Poli(ADP-Ribose) Polimerases/metabolismo , Transcrição GênicaRESUMO
This corrects the article DOI: 10.1038/nature23884.
RESUMO
The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.
Assuntos
Núcleo Celular/genética , Núcleo Celular/fisiologia , Genoma , Modelos Moleculares , Imagem Molecular/métodos , Análise Espaço-Temporal , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromossomos/química , Cromossomos/genética , Cromossomos/metabolismo , Genômica/métodos , Genômica/organização & administração , Objetivos , Humanos , Disseminação de Informação , Camundongos , Modelos Biológicos , Reprodutibilidade dos Testes , Análise de Célula ÚnicaRESUMO
To initiate X-Chromosome inactivation (XCI), the long noncoding RNA Xist mediates chromosome-wide gene silencing of one X Chromosome in female mammals to equalize gene dosage between the sexes. The efficiency of gene silencing is highly variable across genes, with some genes even escaping XCI in somatic cells. A gene's susceptibility to Xist-mediated silencing appears to be determined by a complex interplay of epigenetic and genomic features; however, the underlying rules remain poorly understood. We have quantified chromosome-wide gene silencing kinetics at the level of the nascent transcriptome using allele-specific Precision nuclear Run-On sequencing (PRO-seq). We have developed a Random Forest machine-learning model that can predict the measured silencing dynamics based on a large set of epigenetic and genomic features and tested its predictive power experimentally. The genomic distance to the Xist locus, followed by gene density and distance to LINE elements, are the prime determinants of the speed of gene silencing. Moreover, we find two distinct gene classes associated with different silencing pathways: a class that requires Xist-repeat A for silencing, which is known to activate the SPEN pathway, and a second class in which genes are premarked by Polycomb complexes and tend to rely on the B repeat in Xist for silencing, known to recruit Polycomb complexes during XCI. Moreover, a series of features associated with active transcriptional elongation and chromatin 3D structure are enriched at rapidly silenced genes. Our machine-learning approach can thus uncover the complex combinatorial rules underlying gene silencing during X inactivation.
Assuntos
Epigênese Genética , Inativação Gênica , Aprendizado de Máquina , RNA Longo não Codificante/fisiologia , Inativação do Cromossomo X/genética , Animais , Linhagem Celular , Células-Tronco Embrionárias , Feminino , Genes Ligados ao Cromossomo X , Genoma , Cinética , Camundongos , Modelos GenéticosRESUMO
The aberrant activities of transcription factors such as the androgen receptor (AR) underpin prostate cancer development. While the AR cis-regulation has been extensively studied in prostate cancer, information pertaining to the spatial architecture of the AR transcriptional circuitry remains limited. In this paper, we propose a novel framework to profile long-range chromatin interactions associated with AR and its collaborative transcription factor, erythroblast transformation-specific related gene (ERG), using chromatin interaction analysis by paired-end tag (ChIA-PET). We identified ERG-associated long-range chromatin interactions as a cooperative component in the AR-associated chromatin interactome, acting in concert to achieve coordinated regulation of a subset of AR target genes. Through multifaceted functional data analysis, we found that AR-ERG interaction hub regions are characterized by distinct functional signatures, including bidirectional transcription and cotranscription factor binding. In addition, cancer-associated long noncoding RNAs were found to be connected near protein-coding genes through AR-ERG looping. Finally, we found strong enrichment of prostate cancer genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) at AR-ERG co-binding sites participating in chromatin interactions and gene regulation, suggesting GWAS target genes identified from chromatin looping data provide more biologically relevant findings than using the nearest gene approach. Taken together, our results revealed an AR-ERG-centric higher-order chromatin structure that drives coordinated gene expression in prostate cancer progression and the identification of potential target genes for therapeutic intervention.
Assuntos
Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Receptores Androgênicos/metabolismo , Transcrição Gênica , Linhagem Celular Tumoral , Cromatina/química , Redes Reguladoras de Genes , Genoma Humano , Humanos , Masculino , Proteínas de Fusão Oncogênica/análise , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/metabolismo , RNA Longo não Codificante/metabolismo , Regulador Transcricional ERG/metabolismo , Regulador Transcricional ERG/fisiologiaRESUMO
Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.
Assuntos
Aptâmeros de Nucleotídeos/química , Fatores de Transcrição de Choque Térmico/química , Substâncias Macromoleculares/isolamento & purificação , Espectrometria de Massas , Aptâmeros de Nucleotídeos/genética , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/genética , Histonas/química , Humanos , Imunoprecipitação , Substâncias Macromoleculares/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Peptídeos/química , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes in transcription that are accompanied by chromatin decondensation and nucleosome loss at HS loci. Here we have employed in situ Hi-C to determine how heat stress affects long-range chromatin conformation in human and Drosophila cells. We found that compartments and topologically associating domains (TADs) remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. Although most interactions between HSF1 binding sites and target promoters were established in the nonheat shock (NHS) condition, a subset increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the HSF1 bound sites (putative enhancers), and contact frequency with a target promoter accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is preestablished in NHS cells of diverse metazoan species.