Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins

Francesca Malvezzi,Thomas A Jowitt,Gianluca Degliesposti,Christopher J Stubbs,Andrew J Bannister,J Mark Skehel,Jon P Degnore,Ian L Dale,Xieyang Guo,Mark S Mcalister

doi:10.1038/s42003-021-02750-6

Abstract

Bromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of more specific therapeutics.

Highlights

Bromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains
Unphosphorylated proteins were produced by expression in E. coli, while phosphorylated samples were generated by expression in insect cells or by in vitro casein kinase 2 (CK2)-mediated phosphorylation
BRD4 has been reported to be an atypical protein kinase with autophosphorylation activity[30], we did not observe any phosphoadducts in the bacterial samples by mass spectrometry and we did not detect any auto-phosphorylation of BRD4 by ADPglo assay in the presence of ATP (Supplementary Fig. 3)

Summary

Introduction

Bromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. The long isoforms of BRD4 and BRDT display an intrinsically disordered region, shown to form in BRD4 phase-separated droplets at the chromatin to compartmentalize transcription[7], and a conserved C-Terminal Motif that, together with BD2, contributes to activate transcription of targeted genes by recruiting the positive transcriptional elongation factor b8,9. The discovery that the transcription of c-MYC and other oncogenic genes is regulated by BRD413 and that selective inhibition of BET bromodomains with small molecules, JQ1 and IBET, is effective against various hematological cancers[14,15,16,17,18], encouraged further development of BET inhibitors towards the clinic. BET inhibitors (biBETs) were developed by three distinct groups, which are able to target two bromodomains (BD1 or BD2) simultaneously and show higher potencies and efficacies compared to monovalent counterparts[22,23,24,25]

Methods

Results

Conclusion