RESUMO
Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well characterized, but little is known about the mechanisms by which ADs effect gene activation. Here, we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation.
Assuntos
Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Receptores de Estrogênio/metabolismo , Ativação Transcricional/fisiologia , Animais , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Fator 3 de Transcrição de Octâmero/genética , Domínios Proteicos , Receptores de Estrogênio/genéticaRESUMO
There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.
Assuntos
Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Fator de Transcrição YY1/metabolismo , Animais , Fator de Ligação a CCCTC/metabolismo , Células-Tronco Embrionárias/metabolismo , Humanos , CamundongosRESUMO
Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer that exhibits extremely high levels of genetic complexity and yet a relatively uniform transcriptional program. We postulate that TNBC might be highly dependent on uninterrupted transcription of a key set of genes within this gene expression program and might therefore be exceptionally sensitive to inhibitors of transcription. Utilizing kinase inhibitors and CRISPR/Cas9-mediated gene editing, we show here that triple-negative but not hormone receptor-positive breast cancer cells are exceptionally dependent on CDK7, a transcriptional cyclin-dependent kinase. TNBC cells are unique in their dependence on this transcriptional CDK and suffer apoptotic cell death upon CDK7 inhibition. An "Achilles cluster" of TNBC-specific genes is especially sensitive to CDK7 inhibition and frequently associated with super-enhancers. We conclude that CDK7 mediates transcriptional addiction to a vital cluster of genes in TNBC and CDK7 inhibition may be a useful therapy for this challenging cancer.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Regulação Neoplásica da Expressão Gênica , Transcrição Gênica , Neoplasias de Mama Triplo Negativas/genética , Animais , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The pluripotent state of embryonic stem cells (ESCs) is produced by active transcription of genes that control cell identity and repression of genes encoding lineage-specifying developmental regulators. Here, we use ESC cohesin ChIA-PET data to identify the local chromosomal structures at both active and repressed genes across the genome. The results produce a map of enhancer-promoter interactions and reveal that super-enhancer-driven genes generally occur within chromosome structures that are formed by the looping of two interacting CTCF sites co-occupied by cohesin. These looped structures form insulated neighborhoods whose integrity is important for proper expression of local genes. We also find that repressed genes encoding lineage-specifying developmental regulators occur within insulated neighborhoods. These results provide insights into the relationship between transcriptional control of cell identity genes and control of local chromosome structure.
Assuntos
Cromossomos de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Animais , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/citologia , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Especificidade de Órgãos , Células-Tronco Pluripotentes/metabolismo , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , CoesinasRESUMO
The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Modelos Animais de Doenças , Neuroblastoma/tratamento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Fenilenodiaminas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/uso terapêutico , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Humanos , Proteína Proto-Oncogênica N-Myc , Transcrição Gênica/efeitos dos fármacos , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Master transcription factors Oct4, Sox2, and Nanog bind enhancer elements and recruit Mediator to activate much of the gene expression program of pluripotent embryonic stem cells (ESCs). We report here that the ESC master transcription factors form unusual enhancer domains at most genes that control the pluripotent state. These domains, which we call super-enhancers, consist of clusters of enhancers that are densely occupied by the master regulators and Mediator. Super-enhancers differ from typical enhancers in size, transcription factor density and content, ability to activate transcription, and sensitivity to perturbation. Reduced levels of Oct4 or Mediator cause preferential loss of expression of super-enhancer-associated genes relative to other genes, suggesting how changes in gene expression programs might be accomplished during development. In other more differentiated cells, super-enhancers containing cell-type-specific master transcription factors are also found at genes that define cell identity. Super-enhancers thus play key roles in the control of mammalian cell identity.
Assuntos
Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Complexo Mediador/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Transcrição GênicaRESUMO
Super-enhancers are large clusters of transcriptional enhancers that drive expression of genes that define cell identity. Improved understanding of the roles that super-enhancers play in biology would be afforded by knowing the constellation of factors that constitute these domains and by identifying super-enhancers across the spectrum of human cell types. We describe here the population of transcription factors, cofactors, chromatin regulators, and transcription apparatus occupying super-enhancers in embryonic stem cells and evidence that super-enhancers are highly transcribed. We produce a catalog of super-enhancers in a broad range of human cell types and find that super-enhancers associate with genes that control and define the biology of these cells. Interestingly, disease-associated variation is especially enriched in the super-enhancers of disease-relevant cell types. Furthermore, we find that cancer cells generate super-enhancers at oncogenes and other genes important in tumor pathogenesis. Thus, super-enhancers play key roles in human cell identity in health and in disease.
Assuntos
Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Neoplasias/genética , Animais , Cromatina/metabolismo , Humanos , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-ß, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator ß-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity.
Assuntos
Elementos Facilitadores Genéticos/genética , Complexo Mediador/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Complexo Mediador/fisiologia , Fatores de Transcrição STAT/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad3/metabolismo , Proteínas da Superfamília de TGF-beta/metabolismo , Transcrição Gênica , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex1-4. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus5,6. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain7-12. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers7,8, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites9-12. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference.
Assuntos
Complexo Mediador/química , Complexo Mediador/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Splicing de RNA , Transcrição Gênica , Animais , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Humanos , Complexo Mediador/genética , Camundongos , Fosforilação , Domínios Proteicos , RNA Polimerase II/genética , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
Super-enhancers and stretch enhancers (SEs) drive expression of genes that play prominent roles in normal and disease cells, but the functional importance of these clustered enhancer elements is poorly understood, so it is not clear why genes key to cell identity have evolved regulation by such elements. Here, we show that SEs consist of functional constituent units that concentrate multiple developmental signaling pathways at key pluripotency genes in embryonic stem cells and confer enhanced responsiveness to signaling of their associated genes. Cancer cells frequently acquire SEs at genes that promote tumorigenesis, and we show that these genes are especially sensitive to perturbation of oncogenic signaling pathways. Super-enhancers thus provide a platform for signaling pathways to regulate genes that control cell identity during development and tumorigenesis.
Assuntos
Elementos Facilitadores Genéticos , Neoplasias/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , CamundongosRESUMO
The bromodomain and extraterminal motif (BET) proteins are critical drug targets for diseases. The precise functions and relationship of BRD2 with other BET proteins remain elusive mechanistically. Here, we used acute protein degradation and quantitative genomic and proteomic approaches to investigate the primary functions of BRD2 in transcription. We report that BRD2 is required for TAF3-mediated Pol II initiation at promoters with low levels of H3K4me3 and for R-loop suppression during Pol II elongation. Single and double depletion revealed that BRD2 and BRD3 function additively, independently, or perhaps antagonistically in Pol II transcription at different promoters. Furthermore, we found that BRD2 regulates the expression of different genes during embryonic body differentiation processes by promoter priming in embryonic stem cells. Therefore, our results suggest complex interconnections between BRD2 and BRD3 at promoters to fine-tune Pol II initiation and elongation for control of cell state.
Assuntos
Proteômica , Fatores de Transcrição , Diferenciação Celular , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
LIN28B is highly expressed in neuroblastoma and promotes tumorigenesis, at least, in part, through inhibition of let-7 microRNA biogenesis. Here, we report that overexpression of either wild-type (WT) LIN28B or a LIN28B mutant that is unable to inhibit let-7 processing increases the penetrance of MYCN-induced neuroblastoma, potentiates the invasion and migration of transformed sympathetic neuroblasts, and drives distant metastases in vivo. Genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and coimmunoprecipitation experiments show that LIN28B binds active gene promoters in neuroblastoma cells through protein-protein interaction with the sequence-specific zinc-finger transcription factor ZNF143 and activates the expression of downstream targets, including transcription factors forming the adrenergic core regulatory circuitry that controls the malignant cell state in neuroblastoma as well as GSK3B and L1CAM that are involved in neuronal cell adhesion and migration. These findings reveal an unexpected let-7-independent function of LIN28B in transcriptional regulation during neuroblastoma pathogenesis.
Assuntos
Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Animais Geneticamente Modificados , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/fisiopatologia , Ligação Proteica , Proteínas de Ligação a RNA/genética , Transativadores/genética , Peixe-ZebraRESUMO
Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA , Hematopoese , Animais , Diferenciação Celular , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Células-Tronco Hematopoéticas , Humanos , Masculino , Camundongos , Baço/citologia , Peixe-ZebraRESUMO
BACKGROUND: Genome-wide protein-DNA binding is popularly assessed using specific antibody pulldown in Chromatin Immunoprecipitation Sequencing (ChIP-Seq) or Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing experiments. These technologies generate high-throughput sequencing data that necessitate the use of multiple sophisticated, computationally intensive genomic tools to make discoveries, but these genomic tools often have a high barrier to use because of computational resource constraints. RESULTS: We present a comprehensive, infrastructure-independent, computational pipeline called SEAseq, which leverages field-standard, open-source tools for processing and analyzing ChIP-Seq/CUT&RUN data. SEAseq performs extensive analyses from the raw output of the experiment, including alignment, peak calling, motif analysis, promoters and metagene coverage profiling, peak annotation distribution, clustered/stitched peaks (e.g. super-enhancer) identification, and multiple relevant quality assessment metrics, as well as automatic interfacing with data in GEO/SRA. SEAseq enables rapid and cost-effective resource for analysis of both new and publicly available datasets as demonstrated in our comparative case studies. CONCLUSIONS: The easy-to-use and versatile design of SEAseq makes it a reliable and efficient resource for ensuring high quality analysis. Its cloud implementation enables a broad suite of analyses in environments with constrained computational resources. SEAseq is platform-independent and is aimed to be usable by everyone with or without programming skills. It is available on the cloud at https://platform.stjude.cloud/workflows/seaseq and can be locally installed from the repository at https://github.com/stjude/seaseq .
Assuntos
Cromatina , Software , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Computação em Nuvem , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Genome-wide association studies (GWAS) have identified numerous genetic variants associated with complex diseases, but mechanistic insights are impeded by a lack of understanding of how specific risk variants functionally contribute to the underlying pathogenesis. It has been proposed that cis-acting effects of non-coding risk variants on gene expression are a major factor for phenotypic variation of complex traits and disease susceptibility. Recent genome-scale epigenetic studies have highlighted the enrichment of GWAS-identified variants in regulatory DNA elements of disease-relevant cell types. Furthermore, single nucleotide polymorphism (SNP)-specific changes in transcription factor binding are correlated with heritable alterations in chromatin state and considered a major mediator of sequence-dependent regulation of gene expression. Here we describe a novel strategy to functionally dissect the cis-acting effect of genetic risk variants in regulatory elements on gene expression by combining genome-wide epigenetic information with clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in human pluripotent stem cells. By generating a genetically precisely controlled experimental system, we identify a common Parkinson's disease associated risk variant in a non-coding distal enhancer element that regulates the expression of α-synuclein (SNCA), a key gene implicated in the pathogenesis of Parkinson's disease. Our data suggest that the transcriptional deregulation of SNCA is associated with sequence-dependent binding of the brain-specific transcription factors EMX2 and NKX6-1. This work establishes an experimental paradigm to functionally connect genetic variation with disease-relevant phenotypes.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença/genética , Doença de Parkinson/genética , alfa-Sinucleína/genética , Alelos , Encéfalo/metabolismo , Sistemas CRISPR-Cas/genética , Epigênese Genética/genética , Engenharia Genética , Genoma Humano/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Genéticos , Células-Tronco Pluripotentes/metabolismo , Risco , Fatores de Transcrição/metabolismoRESUMO
Neuroblastoma is a paediatric malignancy that typically arises in early childhood, and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumours with excellent outcomes to widely metastatic disease in which long-term survival is approximately 40% despite intensive therapy. A previous genome-wide association study identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumour cells. Here we investigate the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation and transcription factor binding sites. We show that SNP rs2168101 G>T is the most highly associated variant (combined P = 7.47 × 10(-29), odds ratio 0.65, 95% confidence interval 0.60-0.70), and resides in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G allele that is associated with tumour formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P = 0.028) in neuroblastoma primary tumours, and ablates GATA3 binding (P < 0.0001). We demonstrate allelic imbalance favouring the G-containing strand in tumours heterozygous for this SNP, as demonstrated both by RNA sequencing (P < 0.0001) and reporter assays (P = 0.002). These findings indicate that a recently evolved polymorphism within a super-enhancer element in the first intron of LMO1 influences neuroblastoma susceptibility through differential GATA transcription factor binding and direct modulation of LMO1 expression in cis, and this leads to an oncogenic dependency in tumour cells.
Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença/genética , Proteínas com Domínio LIM/genética , Neuroblastoma/genética , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética , Acetilação , Alelos , Desequilíbrio Alélico , Sítios de Ligação , Epigenômica , Fator de Transcrição GATA3/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Estudo de Associação Genômica Ampla , Genótipo , Histonas/química , Histonas/metabolismo , Humanos , Íntrons/genética , Lisina/metabolismo , Especificidade de Órgãos , Reprodutibilidade dos TestesRESUMO
The transcription factor GATA3 plays an essential role during T cell development and T helper 2 (Th2) cell differentiation. To understand GATA3-mediated gene regulation, we identified genome-wide GATA3 binding sites in ten well-defined developmental and effector T lymphocyte lineages. In the thymus, GATA3 directly regulated many critical factors, including Th-POK, Notch1, and T cell receptor subunits. In the periphery, GATA3 induced a large number of Th2 cell-specific as well as Th2 cell-nonspecific genes, including several transcription factors. Our data also indicate that GATA3 regulates both active and repressive histone modifications of many target genes at their regulatory elements near GATA3 binding sites. Overall, although GATA3 binding exhibited both shared and cell-specific patterns among various T cell lineages, many genes were either positively or negatively regulated by GATA3 in a cell type-specific manner, suggesting that GATA3-mediated gene regulation depends strongly on cofactors existing in different T cells.
Assuntos
Fator de Transcrição GATA3/metabolismo , Proteínas Mutantes/metabolismo , Subpopulações de Linfócitos T/metabolismo , Células Th2/metabolismo , Animais , Linhagem da Célula/genética , Metilação de DNA , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica , Genoma/imunologia , Estudo de Associação Genômica Ampla , Histonas/genética , Histonas/metabolismo , Linfopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Ligação Proteica , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Células Th2/imunologia , Células Th2/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.
Assuntos
Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologia , Pirimidinas/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Cisteína/metabolismo , Humanos , Células Jurkat , Fosforilação/efeitos dos fármacosRESUMO
A small set of core transcription factors (TFs) dominates control of the gene expression program in embryonic stem cells and other well-studied cellular models. These core TFs collectively regulate their own gene expression, thus forming an interconnected auto-regulatory loop that can be considered the core transcriptional regulatory circuitry (CRC) for that cell type. There is limited knowledge of core TFs, and thus models of core regulatory circuitry, for most cell types. We recently discovered that genes encoding known core TFs forming CRCs are driven by super-enhancers, which provides an opportunity to systematically predict CRCs in poorly studied cell types through super-enhancer mapping. Here, we use super-enhancer maps to generate CRC models for 75 human cell and tissue types. These core circuitry models should prove valuable for further investigating cell-type-specific transcriptional regulation in healthy and diseased cells.
Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sítios de Ligação , Linhagem Celular , Células-Tronco Embrionárias Humanas , Humanos , Especificidade de Órgãos , Ligação ProteicaRESUMO
Somatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5'-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy-induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.