Roles Of N-terminal BRD4 Protein In Liquid Phase Separation And Gene Transcription

Epigenetics is the study of heritable gene expression that are not caused by changes in the actual DNA sequence of genes.This includes chemical modifications of DNA and histone inchromatin,such as DNA methylation,histone acetylation and methylation.They interact to contributes to the stability of intracellular milieu and maintain cell phenotype;The abnormal chemical modification in chromatin can induce occurrence of cancerous tumors and autoimmune diseases.BRD4 is a transcription co-activator that contains two bromodomains and an extra-terminal domain,and is a ‘reader' protein that recognizes epigenetic acetyl-lysine modification.Throughout the cell cycle,BRD4 interacts with acetylated histones through it tandem bromodomains and enrichesin highly acetylated and transcriptionally active chromatin regions(including promoters and enhancers).As a result,BRD4 can recruit different transcription factors such as mediator and P-TEFb to form Nucleation centers.They assemble together as protein complexes to promote RNA polymerase activity and stimulate transcription initiation and extension,and regulate cell growth and differentiation.As the abnormal expression of BRD4 can cause the occurrence and development of various tumors such as acute myeloid leukemia,melanoma,Burkitt's lymphoma,colon cancer,multiple myeloma,NUT midline cancer,and breast cancer,targeted inhibition of BRD4 by small molecules can induce tumor cell apoptosis and slow proliferation,thereby achieving antitumor effects.Therefore,BRD4 is widely recognized as an important target for the development of anticancer drugs.But its specific mechanism is still unclear.There are many organelle-like structures without membranes in the cells.These structures include proteins,nucleic acids and other biological functional molecules.They canform high-density,high-concentration phase-separated droplet-like structures locally,and can exchange substances with the outside environment.Studies have shown that proteins with intrinsically disordered structures are more prone to form phase separation.The N-terminal amino acid sequence of BRD4 contains a large number of intrinsically disordered regions with low complexity,including NPS and CPS domains enriched in threonine and serine residues,and BID domains of lysine.Therefore,we try to understand the specific molecular mechanism of BRD4 protein in regulating gene transcription through BRD4 liquid phase separation,and find a new therapeutic approach for cancer treatment.Firstly,we treated different truncated BRD4 protein fragments with DNA,and found that BRD4 can bind to DNA through ordered structures and disordered regions regardless of the nucleotide sequence of DNA.Notably,the IDRs regions at the N-terminus of BRD4 contribute important associations withDNA.However,BRD4 phosphorylation by CK2 reduces its binding ability to DNA,but treatments of BRD4 with small molecular inhibitors do not affect its binding to DNA,indicating that acetyl-lysine binding pockets ofBRD4 bromodomains are not involved in DNA interaction.Secondly,under a con-focal microscope,we overserved that BRD4 S formed phase separated liquid droplets in vitro,and form nuclear puncta in cells.After treating cells separately with 1,6-hexanediol,high concentration salt or small molecule inhibitor JQ1,BRD4 nuclear puncta reduced in numbers and gradually dispersed into nucleoplasm,indicating their liquid phase separation properties.Finally,we transiently transfected the Flag-tag labeled BRD4 S into C33 A cells,examined mRNA levels of some important genes with different transfection levels of Flag-BRD4 S.We found that the mRNA levels of p21,CDK6,PMSD5 genes enhanced in a BRD4 S transfection-dependent manner,suggesting that BRD4 S promotes gene transcription.These data demonstrate that the BRD4 protein forms bi-valent interaction with acetylated histones and DNA through its disordered domain and ordered bromodomains,synergistically promotes its binding to chromatin.BRD4 phosphorylation changes the electrostatic surface of the protein and reduces the ability to bind DNA.The disordereddomain of IDRs at the N-terminus of BRD4 protein can promote the interaction between BRD4 and DNA,control the formation of liquid phase separation in vitro and in cells,recruit gene transcription machinery protein components to accumulate in chromatin,and then regulate gene transcription.Therefore,our research provides new research mechanism for understanding the gene transcription regulation by BRD4 in tumor cells,and present another approach for searching and optimization of BRD4 innovative small molecular inhibitors.