The Interaction Of BRD4-NUT With P300 And Its Regulation Roles In Gene Transcription

Gene fusions,resulting from chromosomal rearrangements,are strong driver mutations present in a wide variety of cancers.They provide the first-line indicators for diagnosis,prognosis,and cancer subtype biomarkers and serve as specific targets for drug development due to advance progresses in molecular cytogenetics.Studies of most recurrent gene fusions have elucidated a variety of biological functions: 1.The dysregulation of parent gene expression of as the expression control of a seemingly normal gene in the fusion gene are removed;2.The fusions to involve in kinases,which make kinase activity abnormally activated after the fusion;3.The function loss of tumor suppressor genes as a result of chromosomal fusion rearrangement;4.Fusion genes/proteins are capable of regulating expression of tumor-related genes through recruiting transcription factors and epigenetic modulations.These four mechanisms can be divided into two ways: affecting the function of fusion genes themselves or the expression of other tumor-related genes.NUT carcinoma is a rare and highly lethal squamous cell carcinoma.Compared with other squamous cell carcinoma,the NUT carcinoma has stronger invasion ability,which has a wide range of spread during the process of diagnosis,and the survival of patients is generally less than one year.Because of no standard and effective treatment for the NUT carcinoma currently,patients with NUT cancer respond poorly to conventional treatments such as chemotherapy and radiation,and soon relapse,deteriorate and die.Although the targeted inhibitors for BET and HDAC have initial efficacy on treatment,patients will develop drug resistance after long-term treating,so patients still remain uncured.Therefore,the development of new therapeutic regiments or the discovery of new potentially targeted therapeutic molecules are both critical to save the lives of patients with NUT cancer.The NUT carcinoma has a unique molecular genetics characteristics.Typically,chromosome rearrangement occurs on chromosome 19 and chromosome 15,resulting in the fusion of NUT exon 2 and BRD4 exon 11 and the formation of the BRD4-NUT fusion gene.BRD4-NUT recognizes acetylated histones that locating on the promoters or enhancers of the target genes via the tandem bromine domain and is recruited to the transcriptional regulatory elements of the target genes.BRD4-NUT recruits and activates acetyltransferase p300 through the F1 c domain,which makes histones highly acetylated and activates gene transcription.At present,the p300 inhibitors are mainly designed to target the HAT domain which has the acetylation activity.However,as the p300-HAT domain is important to maintain the physiological function,and fully suppressing the p300-HAT activity may seriously affect the growth and development of normal cells and cause serious side effects.Therefore,blocking of the specific interaction between p300 and BRD4-NUT provides a rationality for the potential development of inhibitors targeting NUT cancer.Objective:In this study,we study the interaction between BRD4-NUT and p300-TAZ2,and explore its influence on gene transcription.Methods:Firstly,we construct plasmids,which expressing different lengths of NUT-F1 C proteins with GST-labeled.We perform GST pull-down assays,and find the key protein fragments essential in the interaction between p300-TAZ2 and NUT-F1 c.Then using ITC assays verify the results of GST pull-down assays and calculate the affinity coefficient.NUT proteins lack 3D structure and harbor intrinsically disordered regions,which may promote NUT to form condensation through liquid-liquid phase separation(LLPS).Therefore we perform LLPS experiments,the liquid droplets of NUT-F1 c are dependent on the protein concentration and the ionic strength(salt concentration).And whether it is affected by the interaction between p300-TAZ2 protein and NUT-F1 c protein.In cells,BRD4-NUT accumulates the p300 in the transcriptional regulatory elements and abnormally activates gene transcription.Furthermore,we transfect BRD4-NUT wild-type plasmids or BRD4-NUT mutant plasmids(lacks F1 c domain)into HEK393 cells to study the regulation effect of F1 c domain on gene transcription.Results:1.Fragments 377-418 and 419-470 of NUT-F1 C can directly interact with p300-TAZ2;2.The NUT-F1 C domain can form droplets by liquid-liquid separation in vitro;3.The interaction between the NUT-F1 C domain and the P300-TAZ2 domain promotes the liquid-liquid separation process;4.BRD4-NUT promotes the expression of SOX2,LIN28,IDH3 B and other genes through its F1 C domain,and promotes the self-acetylation of p300.Conclusion:In conclusion,the BRD4-NUT fusion protein binds to p300-TAZ2 protein through sequences 377-418 and 419-470 in the F1 c domain,which affects the transcription of related genes.The explicit binding sequence provides a potential target for the design of specific targeting inhibitors of NUT cancer.