RESUMO
B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, with long-term overall survival rates of ~85%. However, B-ALL harboring rearrangements of the MLL gene (also known as KMT2A), referred to as MLLr B-ALL, is common in infants and is associated with poor 5-year survival (<30%), frequent relapses, and refractoriness to glucocorticoids (GCs). GCs are an essential part of the treatment backbone for B-ALL and GC resistance is a major clinical predictor of poor outcome. Elucidating the mechanisms of GC resistance in MLLr B-ALL is, therefore, critical to guide therapeutic strategies that deepen the response after induction therapy. Neuron-glial antigen-2 (NG2) expression is a hallmark of MLLr B-ALL and is minimally expressed in healthy hematopoietic cells. We recently reported that NG2 expression is associated with poor prognosis and that anti-NG2 immunotherapy strongly reduces/delays relapse in MLLr B-ALL xenograft models. Despite its contribution to MLLr B-ALL pathogenesis and its diagnostic utility, the role of NG2 in MLLr-mediated leukemogenesis/chemoresistance remains elusive. Here we show that NG2 is an epigenetically regulated direct target gene of the leukemic MLL-AF4 fusion protein. NG2 negatively regulates the expression of the GC receptor NR3C1 and confers GC resistance to MLLr B-ALL cells in vitro and in vivo. Mechanistically, NG2 interacts with FLT3 to render ligand-independent activation of FLT3 signaling (a hallmark of MLLr B-ALL) and downregulation of NR3C1 via AP-1-mediated trans-repression. Collectively, our study elucidates the role of NG2 in GC resistance in MLLr B-ALL through FLT3/AP-1-mediated downregulation of NR3C1, providing novel therapeutic avenues for MLLr B-ALL.
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To make a multicellular organism, genes need to be transcribed at the right developmental stages and in the right tissues. DNA sequences termed 'enhancers' are crucial to achieve this. Despite concerted efforts, the exact mechanisms of enhancer activity remain elusive. Mixed lineage leukemia (MLL or KMT2A) rearrangements (MLLr), commonly observed in cases of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia, produce novel in-frame fusion proteins. Recent work has shown that the MLL-AF4 fusion protein drives aberrant enhancer activity at key oncogenes in ALL, dependent on the continued presence of MLL-AF4 complex components. As well as providing some general insights into enhancer function, these observations may also provide an explanation for transcriptional heterogeneity observed in MLLr patients.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Histona-Lisina N-Metiltransferase , Proteína de Leucina Linfoide-Mieloide , Proteínas de Fusão Oncogênica , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteína de Leucina Linfoide-Mieloide/genética , Humanos , Elementos Facilitadores Genéticos/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Cromatina/genética , Proteínas de Fusão Oncogênica/genética , Rearranjo Gênico/genéticaRESUMO
Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.
Assuntos
Proteínas Nucleares , Fatores de Transcrição , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligantes , Células HEK293 , Fatores de Transcrição/metabolismo , Proteínas Mutantes , Proteínas de Ciclo Celular/genéticaRESUMO
Cellular ontogeny and MLL breakpoint site influence the capacity of MLL-edited CD34+ hematopoietic cells to initiate and recapitulate infant patients' features in pro-B-cell acute lymphoblastic leukemia (B-ALL). We provide key insights into the leukemogenic determinants of MLL-AF4+ infant B-ALL.
Assuntos
Edição de Genes , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Lactente , Humanos , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Células-Tronco Hematopoéticas , Proteínas de Fusão Oncogênica/genéticaRESUMO
In KMT2A-rearranged acute lymphoblastic leukemia (ALL), an aggressive malignancy, oncogenic KMT2A-fusion proteins inappropriately recruit DOT1L to promote leukemogenesis, highlighting DOT1L as an attractive therapeutic target. Unfortunately, treatment with the first-in-class DOT1L inhibitor pinometostat eventually leads to non-responsiveness. To understand this we established acquired pinometostat resistance in pediatric KMT2A::AFF1+ B-ALL cells. Interestingly, these cells became mostly independent of DOT1L-mediated H3K79 methylation, but still relied on the physical presence of DOT1L, HOXA9 and the KMT2A::AFF1 fusion. Moreover, these cells selectively lost the epigenetic regulation and expression of various KMT2A-fusion target genes such as PROM1/CD133, while other KMT2A::AFF1 target genes, including HOXA9 and CDK6 remained unaffected. Concomitantly, these pinometostat-resistant cells showed upregulation of several myeloid-associated genes, including CD33 and LILRB4/CD85k. Taken together, this model comprehensively shows the adaptive potential of KMT2A-rearranged ALL cells upon losing dependency on one of its main oncogenic properties.
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Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT, and the loss of these factors abolishes enhancer-promoter contact. This work not only provides an additional model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation.
Assuntos
Leucemia , Proteínas Nucleares , Humanos , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Sequências Reguladoras de Ácido Nucleico , Leucemia/genética , Regiões Promotoras Genéticas/genética , Proteínas de Ciclo Celular , Proteínas de Fusão Oncogênica/genética , Proteína de Leucina Linfoide-Mieloide/genéticaRESUMO
Acute myeloid leukaemia (AML) is an aggressive type of leukaemia with low rates of long-term survival. While the current standard of care is based on cytotoxic chemotherapy, a promising emerging approach is differentiation therapy. However, most current differentiating agents target specific mutations and are effective only in certain patient subtypes. To identify agents which may be effective in wider population cohorts, we performed a phenotypic screen with the myeloid marker CD11b and identified a compound series that was able to differentiate AML cell lines in vitro regardless of their mutation status. Structure-activity relationship studies revealed that replacing the formamide and catechol methyl ether groups with sulfonamide and indazole respectively improved the in vitro metabolic profile of the series while maintaining the differentiation profile in multiple cell lines. This optimisation exercise enabled progression of a lead compound to in vivo efficacy testing. Our work supports the promise of phenotypic screening to identify novel small molecules that induce differentiation in a wide range of AML subtypes.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular , Diferenciação Celular , Piridinas/farmacologiaRESUMO
Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with ß-alanine buildup. Raising ß-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.
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Despite much progress in developing better drugs, many patients with acute myeloid leukemia (AML) still die within a year of diagnosis. This is partly because it is difficult to identify therapeutic targets that are effective across multiple AML subtypes. One common factor across AML subtypes is the presence of a block in differentiation. Overcoming this block should allow for the identification of therapies that are not dependent on a specific mutation for their efficacy. Here, we used a phenotypic screen to identify compounds that stimulate differentiation in genetically diverse AML cell lines. Lead compounds were shown to decrease tumor burden and to increase survival in vivo. Using multiple complementary target deconvolution approaches, these compounds were revealed to be anti-mitotic tubulin disruptors that cause differentiation by inducing a G2-M mitotic arrest. Together, these results reveal a function for tubulin disruptors in causing differentiation of AML cells.
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Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated "striated muscle contraction" as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.
Assuntos
Núcleo Celular , Miocárdio , Animais , Expressão Gênica , Mamíferos , Contração Miocárdica , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Chromosome conformation capture (3C) methods measure the spatial proximity between DNA elements in the cell nucleus. Many methods have been developed to sample 3C material, including the Capture-C family of protocols. Capture-C methods use oligonucleotides to enrich for interactions of interest from sequencing-ready 3C libraries. This approach is modular and has been adapted and optimized to work for sampling of disperse DNA elements (NuTi Capture-C), including from low cell inputs (LI Capture-C), as well as to generate Hi-C like maps for specific regions of interest (Tiled-C) and to interrogate multiway interactions (Tri-C). We present the design, experimental protocol and analysis pipeline for NuTi Capture-C in addition to the variations for generation of LI Capture-C, Tiled-C and Tri-C data. The entire procedure can be performed in 3 weeks and requires standard molecular biology skills and equipment, access to a next-generation sequencing platform, and basic bioinformatic skills. Implemented with other sequencing technologies, these methods can be used to identify regulatory interactions and to compare the structural organization of the genome in different cell types and genetic models.
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CromossomosRESUMO
The prognosis for B-cell precursor acute lymphoblastic leukemia patients with Mixed-Lineage Leukemia (MLL) gene rearrangements (MLLr BCP-ALL) is still extremely poor. Inhibition of anti-apoptotic protein BCL-2 with venetoclax emerged as a promising strategy for this subtype of BCP-ALL, however, lack of sufficient responses in preclinical models and the possibility of developing resistance exclude using venetoclax as monotherapy. Herein, we aimed to uncover potential mechanisms responsible for limited venetoclax activity in MLLr BCP-ALL and to identify drugs that could be used in combination therapy. Using RNA-seq, we observed that long-term exposure to venetoclax in vivo in a patient-derived xenograft model leads to downregulation of several tumor protein 53 (TP53)-related genes. Interestingly, auranofin, a thioredoxin reductase inhibitor, sensitized MLLr BCP-ALL to venetoclax in various in vitro and in vivo models, independently of the p53 pathway functionality. Synergistic activity of these drugs resulted from auranofin-mediated upregulation of NOXA pro-apoptotic protein and potent induction of apoptotic cell death. More specifically, we observed that auranofin orchestrates upregulation of the NOXA-encoding gene Phorbol-12-Myristate-13-Acetate-Induced Protein 1 (PMAIP1) associated with chromatin remodeling and increased transcriptional accessibility. Altogether, these results present an efficacious drug combination that could be considered for the treatment of MLLr BCP-ALL patients, including those with TP53 mutations.
Assuntos
Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Auranofina/farmacologia , Auranofina/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Humanos , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Sulfonamidas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Although 90% of children with acute lymphoblastic leukemia (ALL) are now cured, the prognosis for infant-ALL remains dismal. Infant-ALL is usually caused by a single genetic hit that arises in utero: an MLL/KMT2A gene rearrangement (MLL-r). This is sufficient to induce a uniquely aggressive and treatment-refractory leukemia compared to older children. The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. Using the most common MLL-r in infant-ALL, MLL-AF4, as a disease model, we show that fetal-specific gene expression programs are maintained in MLL-AF4 infant-ALL but not in MLL-AF4 childhood-ALL. We use CRISPR-Cas9 gene editing of primary human fetal liver hematopoietic cells to produce a t(4;11)/MLL-AF4 translocation, which replicates the clinical features of infant-ALL and drives infant-ALL-specific and fetal-specific gene expression programs. These data support the hypothesis that fetal-specific gene expression programs cooperate with MLL-AF4 to initiate and maintain the distinct biology of infant-ALL.
Assuntos
Feto , Regulação Neoplásica da Expressão Gênica , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Animais , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA , Feminino , Edição de Genes , Histona-Lisina N-Metiltransferase , Humanos , Fígado , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fatores de Elongação da TranscriçãoRESUMO
Acute myeloid leukemia (AML) is the most aggressive type of blood cancer, and there is a continued need for new treatments that are well tolerated and improve long-term survival rates in patients. Induction of differentiation has emerged as a promising alternative to conventional cytotoxic chemotherapy, but known agents lack efficacy in genetically distinct patient populations. Previously, we established a phenotypic screen to identify small molecules that could stimulate differentiation in a range of AML cell lines. Utilising this strategy, a 1,5-dihydrobenzo[e][1,4]oxazepin-2(3H)-one hit compound was identified. Herein, we report the hit validation in vitro, structure-activity relationship (SAR) studies and the pharmacokinetic profiles for selected compounds.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Química Sintética , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mieloide Aguda , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Induction of differentiation is a promising therapeutic strategy against acute myeloid leukemia. However, current differentiation therapies are effective only to specific patient populations. To identify novel differentiation agents with wider efficacy, we developed a phenotypic high-throughput screen with a range of genetically diverse cell lines. From the resulting hits, one chemical scaffold was optimized in terms of activity and physicochemical properties to yield OXS007417, a proof-of-concept tool compound, which was also able to decrease tumor volume in a murine in vivo xenograft model.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Fenótipo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the lysine methyltransferase 2A (KMT2A) gene produce KMT2A fusion proteins such as KMT2A-AFF1 (previously MLL-AF4), causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs). KMT2A-AFF1 drives leukemogenesis through direct binding and inducing the aberrant overexpression of key genes, such as the anti-apoptotic factor BCL2 and the proto-oncogene MYC However, studying direct binding alone does not incorporate possible network-generated regulatory outputs, including the indirect induction of gene repression. To better understand the KMT2A-AFF1-driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq, and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors such as RUNX1 that regulate target genes downstream of KMT2A-AFF1 using feed-forward loop (FFL) and cascade motifs. A core set of nodes are present in both ALL and AML, and CRISPR screening revealed several factors that help mediate response to the drug venetoclax. Using our GRN, we then identified a KMT2A-AFF1:RUNX1 cascade that represses CASP9, as well as KMT2A-AFF1-driven FFLs that regulate BCL2 and MYC through combinatorial TF activity. This illustrates how our GRN can be used to better connect KMT2A-AFF1 behavior to downstream pathways that contribute to leukemogenesis, and potentially predict shifts in gene expression that mediate drug response.
RESUMO
In higher eukaryotes, many genes are regulated by enhancers that are 104-106 base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around 7-22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion1 is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding.
Assuntos
Pareamento de Bases/genética , Genoma/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Elementos Facilitadores Genéticos/genética , Células Eritroides/citologia , Células Eritroides/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , alfa-Globinas/genética , CoesinasRESUMO
Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPß binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPß, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPß binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.
Assuntos
Arginina/metabolismo , Cromatina/metabolismo , Evasão da Resposta Imune/genética , Neoplasias/genética , Linfócitos T/metabolismo , Animais , HumanosRESUMO
Enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes in higher eukaryotes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. Bromodomain and Extra-Terminal domain (BET) and Mediator proteins have been shown capable of forming phase condensates and are thought to be essential for super-enhancer function. Here, we show that targeting of cells with inhibitors of BET proteins or pharmacological degradation of BET protein Bromodomain-containing protein 4 (BRD4) has a strong impact on transcription but very little impact on enhancer-promoter interactions. Dissolving phase condensates reduces BRD4 and Mediator binding at enhancers and can also strongly affect gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancer-promoter interactions are separable events. Our findings further indicate that enhancer-promoter interactions are not dependent on high levels of BRD4 and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and, at some sites, CTCF and cohesin.