Regulation Mechanism Of BRD4-NUT In Gene Transcription And Epithelial-Mesenchymal Transition

Background:NUT carcinoma(NC)is a rare,aggressive cancer,the cell of origin is not known.Typically,NC arises from midline anatomical sites of adolescent body,therefore,it is always called NUT midline carcinoma(NMC).In NMC cells,the coding sequence of NUT on chromosome 15q14 is fused with BRD4,creating a fusion gene that encodes BRD4-NUT fusion protein.This fusion protein binds lysine-acetylated histones tails through its tandem bromodomains(Br Ds)in the BRD4 moiety,while the NUT moiety recruits and activates the histone acetyltransferase p300/CBP,leading to hyperacetylated chromatin regions(mega-domains)and aberrant gene transcriptions.NUT midline carcinoma(NMC)is one of the most therapy-resistant tumors.It is often widely metastatic at the time of diagnosis and rapidly lethal.The median survival time is about 6.7months.There is currently no effective treatment of NUT carcinoma.The prognosis of traditional surgery or radiotherapy and chemotherapy is poor,targeted therapy has become a new therapeutical approach.BET inhibitors(BETi)could block the interaction between bromodomains and acetyl-histone tails and have been in clinical trials for the treatment of NMCs.However,the response rate in NMC to BETis was only 20% and patients eventually developed resistance.Another concern issue is the toxicity of pan-BETi,leading to some side-effects in some patients during treatment.Therefore,alternative regiments or therapies need to be developed.The transcription co-activator,p300 is a large protein that contains folded or disordered domains.The interaction between NUT and p300 could recruits and activates p300 to hyperacetylate histones in chromatin.Knockdown of BRD4-NUT protein in NMC cells could reduce the catalytic activity of p300 and inhibit tumor cell proliferation.Thus,inhibition of p300 binding to BRD4-NUT may reduce p300-HAT activity,which can be a putative way for NMC treatment.At present,p300 inhibitors,which have been used in clinical trials,are designed against the catalytic domain(HAT).p300 HAT inhibitors have been applied to NMC treatments and show selective potency against NMC cell lines.However,the HAT domain of p300 is the key acetyl-transferase for histone lysine acetylation and plays an important role in sustaining the growth and development of normal cells.p300 HAT inhibitor may have potential toxicity towards normal tissues.prompting the search for new drug target.So,exploring the structural mechanism of BRD4-NUT association with p300 could provide theoretical basis for the development of novel p300 inhibitors.Liquid-Liquid phase separation(LLPS)is a physical process that occurs when a supersaturated solution of components spontaneously separates into two phases,a dense phase and a dilute phase,that then stably coexist.Biological macromolecules such as proteins or nucleic acids in cell can concentrated locally by multivalent interactions.Molecules will be miscible in solution until they reach their solubility limit,the threshold concentration at which they phase separate.Phase separation play key roles in many biological processes such as stress signal transduction,DNA damage response and gene transcriptional regulation.The study on liquid-liquid phase separation(LLPS)of biomacromolecules have been increased rapidly in the past decade.The most important step toward phase separation is the establishment of a dense network of multivalent interactions between macromolecules.Extensive work on the molecular features that promote phase separation has delineated two archetypes of phase separating proteins:(1)proteins with multiple folded domains or modules and(2)intrinsically disordered regions(IDRs).Bioinformatic prediction of ordered/disordered protein sequence revealed that NUT consists of extended intrinsically disordered regions(IDRs).We speculated that the NUT moiety of BRD4-NUT could mediate LLPS to recruit p300 and other transcriptional factors in chromatin,activate gene expression.Epithelial-mesenchymal transition(EMT)is a reversible cellular program that transiently places epithelial cells into mesenchymal cell states.EMT is known to be crucial for embryogenesis,wound healing and malignant progression.EMT pathway is precisely regulated by EMT transcription factors(EMT-TF)such as SNAIL and TWIST.However,the expression level or activity of EMT-TF is always abnormal in carcinoma cells,promoting the metastasis ability of cancer cells.ALX1(Aristaless-like Homeobox protein 1),a member of ALX protein family could mediate the survival and development of vertebrate mesenchymal cell.Its mutation will prevent the fusion of frontal nose and maxilla.ALX1/Snail is a novel epithelial-to-mesenchymal transition(EMT)regulating axis associated with tumor metastasis.ALX1 gene is highly expressed in NMCs,but its function remains elusive.We speculate that ALX1 could be directly regulated by BRD4-NUT.In NMCs,BRD4-NUT could activate EMT by up-regulate ALX1 gene expression,promote cellular migration and invasion.Objective:To investigate the structural mechanism between BRD4-NUT and p300.The effect of BRD4-NUT mediated LLPS on gene transcription and promoting cell metastasis are also explored.Methods:Structurally,the key sequence of NUT binding to p300-TAZ2 was confirmed by GST pulldown and NMR HSQC assays.High-resolution three-dimensional structures of NUT/p300-TAZ2 complexes were determined by triple-resonance NMR spectroscopy methods.Mechanistically,EGFP-tagged NUT fragment and Cherry-labeled p300-TAZ2 domain were purified for the investigation of the liquid-liquid phase separation capability of NUT in vitro.In cell,the liquid properties of EGFP-BRD4-NUT protein was validated by FRAP and other experiments.BRD4-NUT condensates components were analyzed by immunofluorescence assay.BRD4-NUT-F1 c deletion construct(deletion of TAZ2 binding sequence)was also subjected for the study of the effect of NUT/p300 interaction on p300 activity and gene transcription.At last,we sought to explore the possible epigenetic regulatory role of BRD4-NUT in EMT with the help of experiments including quantitative PCR,immunoblotting and chromatin immunoprecipitation.Conclusions:(1)NUT constitutes two binding sequences for p300 TAZ2 domain;(2)x Px xx is one of the binding motif that recognized by p300-TAZ2,it can folded into ? helix(where P represents proline,is a hydrophobic amino acid,and xany amino acid);(3)NUT could phase separate in vitro,p300-TAZ2 domain promotes NUT phase sepatation;(4)BRD4-NUT constitutes discrete condensates through LLPS to drive gene transcription in cells;(5)BRD4-NUT promotes the activation of ALX1 for EMT.