RESUMO
Growing evidence suggests prevalence of transcriptional condensates on chromatin, yet their mechanisms of formation and functional significance remain largely unclear. In human cancer, a series of mutations in the histone acetylation reader ENL create gain-of-function mutants with increased transcriptional activation ability. Here, we show that these mutations, clustered in ENL's structured acetyl-reading YEATS domain, trigger aberrant condensates at native genomic targets through multivalent homotypic and heterotypic interactions. Mechanistically, mutation-induced structural changes in the YEATS domain, ENL's two disordered regions of opposing charges, and the incorporation of extrinsic elongation factors are all required for ENL condensate formation. Extensive mutagenesis establishes condensate formation as a driver of oncogenic gene activation. Furthermore, expression of ENL mutants beyond the endogenous level leads to non-functional condensates. Our findings provide new mechanistic and functional insights into cancer-associated condensates and support condensate dysregulation as an oncogenic mechanism.
Assuntos
Neoplasias , Corpos Nucleares , Humanos , Domínios Proteicos , Cromatina/genética , Mutação , Neoplasias/genéticaRESUMO
Genome-wide transcriptional activity involves the binding of many transcription factors (TFs) to thousands of sites in the genome. Pioneer TFs are a class of TFs that maintain open chromatin and allow non-pioneer TFs access to their target sites. Determining which TF binding sites directly drive transcription remains a challenge. Here, we use acute protein depletion of the pioneer TF SOX2 to establish its functionality in maintaining chromatin accessibility. We show that thousands of accessible sites are lost within an hour of protein depletion, indicating rapid turnover of these sites in the absence of the pioneer factor. To understand the relationship with transcription, we performed nascent transcription analysis and found that open chromatin sites that are maintained by SOX2 are highly predictive of gene expression, in contrast to all other SOX2 binding sites. We use CRISPR-Cas9 genome editing in the Klf2 locus to functionally validate a predicted regulatory element. We conclude that the regulatory activity of SOX2 is exerted mainly at sites where it maintains accessibility and that other binding sites are largely dispensable for gene regulation.
Assuntos
Cromatina , Fatores de Transcrição SOXB1 , Fatores de Transcrição , Sítios de Ligação , Cromatina/genética , Regulação da Expressão Gênica , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , CamundongosRESUMO
Micro Capture-C (MCC) is a chromatin conformation capture (3C) method for visualizing reproducible three-dimensional contacts of specified regions of the genome at base pair resolution. These methods are an established family of techniques that use proximity ligation to assay the topology of chromatin. MCC can generate data at substantially higher resolution than previous techniques through multiple refinements of the 3C method. Using a sequence agnostic nuclease, the maintenance of cellular integrity and full sequencing of the ligation junctions, MCC achieves subnucleosomal levels of resolution, which can be used to reveal transcription factor binding sites analogous to DNAse I footprinting. Gene dense regions, close-range enhancer-promoter contacts, individual enhancers within super-enhancers and multiple other types of loci or regulatory regions that were previously challenging to assay with conventional 3C techniques, are readily observed using MCC. MCC requires training in common molecular biology techniques and bioinformatics to perform the experiment and analyze the data. The protocol can be expected to be completed in a 3 week timeframe for experienced molecular biologists.
Assuntos
Cromatina , Cromossomos , Cromatina/genética , Genoma , Biologia Computacional/métodos , Sequências Reguladoras de Ácido NucleicoRESUMO
The chromatin reader eleven-nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9-mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line- and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach. SIGNIFICANCE: AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483.
Assuntos
Leucemia Mieloide Aguda , Lisina , Humanos , Leucemia Mieloide Aguda/genética , Histonas/metabolismo , Cromatina , Proteína de Leucina Linfoide-Mieloide/metabolismoRESUMO
Extracellular vesicles (EVs) have been highlighted as novel drug carriers due to their unique structural properties and intrinsic features, including high stability, biocompatibility, and cell-targeting properties. Although many efforts have been made to harness these features to develop a clinically effective EV-based therapeutic system, the clinical translation of EV-based nano-drugs is hindered by their low yield and loading capacity. Herein, we present an engineering strategy that enables upscaled EV production with increased loading capacity through the secretion of EVs from cells via cytochalasin-B (CB) treatment and reduction of EV intravesicular contents through hypo-osmotic stimulation. CB (10 µg/mL) promotes cells to extrude EVs, producing ~three-fold more particles than through natural EV secretion. When CB is induced in hypotonic conditions (223 mOsm/kg), the produced EVs (hypo-CIMVs) exhibit ~68% less intravesicular protein, giving 3.4-fold enhanced drug loading capacity compared to naturally secreted EVs. By loading doxorubicin (DOX) into hypo-CIMVs, we found that hypo-CIMVs efficiently deliver their drug cargos to their target and induce up to ~1.5-fold more cell death than the free DOX. Thus, our EV engineering offers the potential for leveraging EVs as an effective drug delivery vehicle for cancer treatment.