In Vitro Study Of The BRD4 Short Isoform In Phase Separation And Active Gene Transcription

BRD4 protein,which is a member of BET protein family,plays important roles in gene transcription regulation,DNA replication and repair,and cell cycle,and it has been implicated in numerous forms of human cancers including acute myeloid leukemia,multiple myeloma,Burkitt's lymphoma,NUT midline carcinoma,and colon and breast cancers.As an integral component of transcription regulatory enhancers,BRD4 is required for maintaining higher-order chromatin organization,and mediating muti-factor assembly of gene regulatory enhancers for transcription activation.Due to alternative splicing,there are two isoforms of BRD4,BRD4 long isoform(BRD4L)and BRD4 short isoform(BRD4S).The BRD4S(residues 1-722)contains the same N-terminal segment of BRD4 L,except that BRD4 S has unique three C-terminal residues(GPA)not present in BRD4 L.Many studies involving BRD4 L have been reported,but little was known about the function of BRD4 S.Liquid-Liquid phase separation(LLPS)is a phenomenon that two kinds of liquid are not homogeneously mixed in one phase,but separate into two phases.When solutions of macromolecules such as proteins or nucleic acids undergo LLPS,they condense into a dense phase that often resembles liquid droplets,and this dense phase coexists with a dilute phase.Increasing studies have shown that phase separation is a mechanism by which membraneless compartments form.LLPS is a common phenomenon in organisms,and it plays important roles in chromatin structure,gene transcription regulation,translation and epigenetic regulations.LLPS is involved in normal biological function and development of many kinds of disease.LLPS is driven by multivalent molecules — that is,molecules that harbor multiple elements that govern intra-or inter-molecular interaction.These molecules contain proteins composed of multiple modular interaction domains and proteins containing disordered regions that provide multiple weakly adhesive sequence elements.Liquid-liquid phase separation is driven by 3D folded domain and IDRs,and BRD4 S contains two tandem bromodomains,an ET domain,and is enriched in IDRs.Thus,we speculated that BRD4 S might undergo liquid-liquid phase separation.BRD4 binds to acetylated chromatin with weak affinity,somehow it is stable on chromatin.So we proposed that there are other interactions that facilitate the binding.It is reported that a bromodomain-DNA interaction facilitates acetylation dependent bivalent nucleosome recognition by the BET protein BRDT.Thus,we analysed that BRD4 S might also interact with DNA to enhance its affinity with chromatin.Therefore,we proposed that by bivalent interaction with DNA and acetylated lysine,BRD4 S stably binds to chromatin,and by forming LLPS condensates,BRD4 S recruits transcriptional co-factors and promotes gene transcription.Our study focused on the ability of BRD4 S to form liquid-liquid phase separation in vitro and its role in regulation of gene transcription activity.Here we show the following conclusions:1.BRD4 S undergoes liquid-liquid phase separation in vitro,and BRD4 S droplets are sensitive to salt concentration,and are dynamic and reversible,suggesting the droplets formed by LLPS harbor liquid property.Our LLPS assay of different truncated BRD4 S proteins suggests that the IDRs and bromodomains are both important for LLPS.2.BRD4 S interacts with double-stranded DNA,and longer DNA shows stronger affinity with BRD4 S.Of all the regions in BRD4 S,the lysine-rich BID is crucial for DNA binding.Further more,the binding between BRD4 S and double-stranded DNA enhances LLPS formed by BRD4 S.3.Phosphorylation of BRD4 S induces change of electrostatic charges in the surface of BRD4 S molecue,which leads to the rearrange of BRD4 S structure.As a result,phosphorylation of BRD4 S inhibits its binding affinity to double-stranded DNA and its ability to form LLPS in vitro.4.BRD4 S undergoes LLPS in He La nuclear extracts,and through its bivalent interaction with DNA and acetylated histones,and its ability to form LLPS,BRD4 S facilities the assembly of transcription machinery in chromatin,including RNA Pol ?(S2P,and S5 P Pol ?),BRD4 L,Med1,CDK9 to gene sites,thus promotes the transcriptional activity of DNA templates.In sum,our study provides understanding of a previously unappreciated role of BRD4 S in organizing key chromatin and transcription regulators through dynamic LLPS.And we provide perspectives of how transcriptional co-activators play their roles in gene transcription regulation.