Targeted protein degradation via intramolecular bivalent glues

Chemical synthesis

Additional details are provided in the Supplementary Methods.

Plasmids and oligonucleotides

The design and construction of the human CRL-focused sgRNA library used for BRD4 stability screens, lentiviral sgRNA expression vectors used for single gene knockouts, as well as viral vectors used for the engineering of inducible Cas9 cell lines have been described previously48,49. For the engineering of the fluorescent protein stability reporters, the short isoform of BRD4 (BRD4(S)) (Twist Bioscience), BRD2 (Addgene plasmid #65376, a gift from K. Miller50) or BRD3 (Addgene plasmid #65377, a gift from K. Miller50) were cloned into a pRRL lentiviral vector, fused to a 3×V5 tag and mTagBFP, and coupled to mCherry for normalization. For knockout and rescue studies, DCAF16 open reading frame cDNA (Twist Bioscience) was synonymously mutated to remove the sgRNA protospacer adjacent motif and seed sequence, coupled to a Flag tag and cloned into a pRRL lentiviral vector expressing iRFP670 for flow-cytometric detection. All plasmids and sgRNAs used in this study are shown in Extended Data Table 1, and the CRL-focused sgRNA libraries used for FACS-based and viability-based CRISPR–Cas9 screens are shown in Supplementary Tables 2 and  4, respectively.

Cell culture

HEK293, HCT-116, HeLa and MV4;11 cell lines, originally sourced from ATCC, were provided by the MRC PPU reagents facility at the University of Dundee. KBM7 iCas9 cells were a gift from J. Zuber. HEK293, HeLa, Lenti-X 293 T lentiviral packaging cells (Clontech) and HCT-116 were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher), 100 U ml−1 penicillin-streptomycin (Thermo Fisher) and 2 mM l-glutamine (Thermo Fisher). MV4;11 and KBM7 cells were cultured in IMDM (Gibco), supplemented with the same additives as above. All cell lines were grown in a humidified incubator at 37 °C and 5% CO2 and routinely tested for mycoplasma contamination. All cell lines were authenticated by short tandem repeat profiling.

Lentivirus production and transduction

Semiconfluent Lenti-X cells were co-transfected with lentiviral plasmids, the lentiviral pCMVR8.74 helper (Addgene plasmid #22036, a gift from D. Trono) and pMD2.G envelope (Addgene plasmid #12259, a gift from D. Trono) plasmids using polyethylenimine (PEI) transfection (PEI MAX MW 40,000, Polysciences) as previously described. Virus containing supernatant was clarified by centrifugation. Target cells were infected at limiting dilutions in the presence of 4 μg ml−1 polybrene (Santa Cruz Biotechnology).

CRISPR–Cas9 DCAF15-knockout cell line generation

The HCT-116 DCAF15-knockout cell line was generated via ribonuclear protein (RNP) transfection using sgRNAs (IDT) targeting DCAF15 exon 2 and exon 4 (Extended Data Table 1), spCas9 Nuclease V3 (IDT) and TransIT-X2 (Mirus Bio). Following transfection for 48 h, cells were trypsinized and re-plated in 96-well plates at low density and allowed to grow for >2 weeks. Single colonies were isolated and expanded and verified for DCAF15 knockout via western blotting, using an optimized RBM39 degradation assay as well as via genomic DNA sequencing.

CRISPR–Cas9 HiBiT and BromoTag knock-in cell line generation

HiBiT BRD2, BRD3 and BRD4 cell lines were generated via RNP transfection of single-stranded DNA oligonucleotides (IDT) as the ssODN donor templates, spCas9 (Sigma-Aldrich) and target-specific sgRNA (IDT) (Extended Data Table 1). HEK293 cells were resuspended in buffer R (Thermo Fisher), along with the RNP complex and ssODN template, and electroporated using a 10 µl Neon Electroporation System cuvette tip (Thermo Fisher). Immediately following electroporation, cells were added to pre-warmed DMEM supplemented with 10% FBS and 100 U ml−1 penicillin–streptomycin added for BromoTag cell lines only. Edited pools were analysed for HiBiT insertion by assaying for luminescence on a PHERAstar spectrophotometer (BMG Labtech) 48–72 h post-electroporation. Successful knock-in of HiBiT three days post-electroporation was first established using HiBiT lytic assay (Promega) on the mixed cell population. Following identification of luminescent signal these cells underwent single cell sorting using an SH800 cell sorter (Sony Biotechnology). Single cells were sorted into 3× 96-well plates per experiment in 200 μl of 50% filtered preconditioned media from healthy cells and 50% fresh DMEM. After two weeks, all visible colonies were expanded, validated using the HiBiT lytic assay.

BromoTag cell lines were generated in HEK293 cells via simultaneous transfection of two vectors at a 4:1 reagent:DNA ratio with FuGENE 6 (Promega). The first vector was a pMK-RQ vector containing 500-bp homology arms on either side of either an eGFP-IRES-BromoTag or eGFP-IRES-HiBiT-BromoTag sequence for integration into MCM4 and BRD4, respectively (Extended Data Table 1). The second vector was a custom pBABED vector harbouring U6-sgRNA, Cas9 and puromycin expression cassettes. Following transfection, cells were repeatedly washed with PBS and then treated with 1 µg ml−1 puromycin for one week before FACS sorting. Single cell clones were generated by FACS sorting of single GFP+ cells using an SH800 cell sorter and sorting between 2 to 10 96-well plates in 200 μl of 50% filtered preconditioned media from healthy cells mixed with 50% fresh media.

siRNA-mediated knockdown

Cells were transfected for 48 h using ON-TARGETplus SMARTPool siRNAs for DCAF15, DCAF16, DDB1, RBX1, CUL4A and CUL4B (all from Dharmacon) and RNAiMAX (Invitrogen) following the manufacturer’s instructions, with 35 pmol of siRNA per well in 6-well plates. When simultaneously targeting two genes, half the amount of siRNA was used for each gene.

Cell viability assay

MV4;11, HCT-116 or KBM7 cells were plated in 96-well plates at a density of 0.5 × 106 (MV4;11 and HCT-116) or 0.1 × 106 (KBM7) cells per ml in 50 µl cell suspension per well. The following day, 2× stocks of compounds were added for a final volume of 100 µl. Cells were treated for 24 h (MV4;11), 72 h (KBM7) or 96 h (HCT-116) in a humidified incubator at 37 °C and 5% CO2. CellTiterGlo (G7570, Promega) or CellTiterGlo 2.0 reagent (G924A, Promega) was added to the plates per manufacturer instructions, before shaking the plate for 3–20 min at 300 rpm and measuring the luminescence using a PHERAstar (BMG Labtech) operated on PHERAstar software (firmware v1.33) or VICTOR X3 (PerkinElmer) multilabel plate reader operated on PerkinElmer 2030 software (v4.0). The results were normalized to DMSO controls and analysed using Graphpad Prism (v9.5.1) to derive EC50 values by four-parameter non-linear regression curve fitting or interpolation of a sigmoidal standard curve.

Degradation assays and western blotting

HEK293 and HCT-116 cells were plated in 6-well plates at varying densities (0.2 to 0.6 × 106 cells per ml) depending on experimental setup. In all experiments, media was changed prior to compound treatment. Stock solutions of compounds were prepared in DMSO at a concentration of 10 mM and stored at −20 °C. Working dilutions were made fresh using DMEM media and added dropwise to 6-well plates. For competition assays, cells were pre-treated with 10 µM of the competition compounds, 3 µM MLN4924 or 50 µM MG132 for 1 h, before treating with IBG1 at 10 nM for 2 h.

For cell collection, cells were washed once with ice-cold PBS before lysis for 15 min on ice with RIPA buffer supplemented with benzonase (1:1,000, Sigma or Millipore 70746) and cOmplete EDTA-free Protease Inhibitor Cocktail (11873580001, Roche). Following clearance via centrifugation, protein concentration of lysates was determined using the Pierce BCA Protein Assay (23225, Fisher Scientific) and 20–30 µg of lysate was prepared using 4× LDS sample buffer (Thermo Fisher) and 10% 2-mercaptoethanol or 50 mM dithiothreitol (DTT) and run on NuPAGE 4–12% bis-tris gels (Thermo Fisher). Proteins were transferred to nitrocellulose membranes, blocked for 1 h in 5% milk TBS-T at room temperature, before incubating with primary antibodies overnight at 4 °C. The following primary antibodies were used: BRD2 (1:1,000, no. Ab139690, Abcam), BRD3 (1:2,000, Ab50818, Abcam), BRD4 (1:1,000, E2A7X, 13440, Cell Signaling Technology and Ab128874, Abcam), BromoTag (1:1,000, NBP3-17999, Novus Biologicals), CUL4A (1:2,000, A300-738A, Bethyl Laboratories), CUL4B (1:2,000, 12916-1-AP, Proteintech), DDB1 (1:1,000, A300-462A, Bethyl Laboratories), MCM4 (1:1,000, ab4459, Abcam) RBM39 (1:1,000, HPA001591, Atlas Antibodies), RBX1 (1:1,000, D3J5I, 11922, Cell Signalling Technology), DCAF11 (1:2,000, A15519, ABclonal), cleaved caspase-3 (1:1,000, D3E9, 9579, Cell Signalling Technology), PARP1 (1:1,000, 9542, Cell Signalling Technology), MYC (1:500, D84C12, 5605, Cell Signalling Technology), β-actin (1:10,000, AC-15, A5441, Sigma-Aldrich), α-tubulin (1:500, DM1A, T9026, Sigma-Aldrich). Membranes were then washed in TBS-T and incubated with fluorescent or horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature, before further washes and imaging on a ChemiDoc Touch imaging system (Bio-Rad) operated on Image Lab software (v2.4.0.03). Secondary antibodies used were HRP anti-rabbit IgG (1:2500, 7074, Cell Signaling Technology), HRP anti-mouse IgG (1:5,000, 7076, Cell Signaling Technology), IRDye 680RD anti-mouse (1:5,000, 926-68070, Li-Cor), IRDye 800CW anti-rabbit (1:5,000, 926-32211, Li-Cor), StarBright blue 520 goat anti-mouse (1:5,000, 12005866, Bio-Rad) and hFABTM rhodamine anti-tubulin (1:5,000, 12004165, Bio-Rad). Western blots were quantified using Image Lab software (v6.1 build 7).

HiBiT degradation assays

Endogenously tagged HiBiT cells were plated in 96-well plates (PerkinElmer) at a density of 0.5 × 106 cells per ml, with 50 µl of cell suspension per well. The following day, 2× stocks of compounds were added for a final volume of 100 µl. Cells were treated for 5, 6 or 24 h as indicated in the respective figure legends before lysis using the HiBiT lytic assay buffer (Promega) per manufacturer instructions. Plates were then read on a BMG Pherastar plate reader for luminescence detection. Treated wells were normalized to a DMSO-only control and analysed using GraphPad Prism (v9.3.1) via fitting of non-linear regression curves for extraction of DC50 and maximal degradation (DMAX) values.

Kinetic ubiquitination and degradation assays

For kinetic ubiquitination assays, HiBiT-tagged HEK293 cells were seeded in 6-well plates at a density of 8 × 106 cells per ml in 2 ml volume. After 5 h, LgBiT and Halo-Ub cDNA (Promega) were transfected using FuGENE HD (Promega) with 1 µg of each plasmid at a 3:1 transfection reagent:plasmid ratio. The following day, cells were trypsinized and resuspended in phenol red-free OptiMEM (Gibco) supplemented with 4% FBS and seeded in 96-well plates at a density of 3.5 × 105 cells per ml in the presence or absence of 0.1 mM HaloTag NanoBRET ligand (Promega). Following overnight incubation, media was removed from the wells and replaced with 90 µl OptiMEM (4% FBS) with a 1:100 dilution of Vivazine substrate. The plates were incubated at 37 °C for 1 h before 10× stocks of experimental compounds were added and the plates were analysed on a GloMax Discover microplate reader (software v4.0.0, firmware v4.92; Promega) in kinetic mode for NanoBRET ratio metric (460 nm donor and 618 nm acceptor emissions) signal detection for 6 h, with measurements taken every 3–5 min. Data was processed by subtracting NanoBRET ligand-free controls before plotting NanoBRET signal versus time in GraphPad Prism (v9.3.1).

Kinetic degradation assays were performed as previously described51, using the HiBiT-tagged cells with exogenous LgBiT transfection as described above for the kinetic ubiquitination assays. Cells were incubated in Endurazine substrate (1:100) for 2.5 h at 37 °C prior to 10× compound addition, with luminescence measurements taken on a GloMAX Discover microplate reader (Promega) every 15 min for 24 h. Data were normalized to DMSO-only controls and plotted for luminescence signal versus time in GraphPad Prism (v9.3.1).

NanoBRET bromodomain confirmational sensor assay

Transient transfection of the dual NanoLuc and Halo-Tagged tagged BRD4Tandem plasmid (Promega) was performed as described previously51. In brief, 0.02 µg of plasmid and 2 µg of carrier DNA were combined with FuGENE HD (Promega) at a 3:1 ratio and added per well of a 6-well plate seeded with 70% confluent HEK293 cells. The following day, cells were trypsinized and resuspended in phenol red-free OptiMEM (Gibco) supplemented with 4% FBS and 100 µl were seeded per well in 96-well plates at a density of 2 × 105 cells per ml in the presence or absence of 0.1 mM HaloTag NanoBRET ligand (Promega). The following morning, the media was aspirated and replaced with phenol red-free media containing MG132 (10 µM final concentration) for 1 h, before cells were incubated with test compounds for 3 h. For cell lysis and detection, 100 µl of 2× NanoBRET substrate solution was added per well, the plate was incubated in darkness while shaking at 400 RPM for 3 min, before reading on a BMG Pherastar plate reader equipped with a NanoBRET filter (618/460 nm). Wells lacking Halo ligand were subtracted from wells containing Halo ligand, and the fold increase in signal compared to DMSO was plotted using GraphPad Prism (v9.3.1).

FACS-based CRISPR–Cas9 BRD4 stability screens

For pooled FACS-based CRISPR–Cas9 BRD4 protein stability screens, a CRL-focused sgRNA library49 was packaged in lentivirus using polyethylenimine (PEI MAX MW 40,000, Polysciences) transfection of Lenti-X cells and the lentiviral pCMVR8.74 helper (Addgene plasmid #22036, a gift from D. Trono) and pMD2.G envelope (Addgene plasmid #12259, a gift from D. Trono) plasmids. The virus containing supernatant was cleared of cellular debris by filtration through a 0.45-µm polyethersulfone filter and used to transduce KBM7 BRD4–BFP reporter cells harbouring a doxycycline-inducible Cas9 allele (iCas9) at a multiplicity of infection of 0.05 and 1,000-fold library representation. Library-transduced cells were selected with G418 (1 mg ml−1, Gibco) for 14 days, expanded and Cas9 expression was induced with doxycycline (0.4 µg ml−1, PanReac AppliChem).

Three days after Cas9 induction, 25 million cells per condition were treated with DMSO (1:1,000), MZ1 (10 nM), IBG1 (1 nM), GNE-0011 (1 µM), IBG3 (0.1 nM) or IBG4 (100 nM) for 6 h in 2 biological replicates. Cells were washed with PBS, stained with Zombie NIR Fixable Viability Dye (1:1,000, BioLegend) and APC anti-mouse Thy1.1 (also known as CD90.1) antibody (1:400, 202526, BioLegend) in the presence of Human TruStain FcX Fc Receptor Blocking Solution (1:400, 422302, BioLegend), and fixed with 0.5 ml methanol-free paraformaldehyde 4% (Thermo Scientific Pierce) for 30 min at 4 °C, while protected from light. Cells were washed with and stored in FACS buffer (PBS containing 5% FBS and 1 mM EDTA) at 4 °C overnight. The next day, cells were strained trough a 35-µm nylon mesh and sorted on a BD FACSAria Fusion (BD Biosciences) operated on BD FACSDiva software (v8.0.2) using a 70-µm nozzle. Aggregates, dead (Zombie NIR positive), Cas9-negative (GFP) and sgRNA library-negative (THY1.1–APC) cells were excluded, and the remaining cells were sorted based on their BRD4–BFP and mCherry levels into BRD4high (5–10% of cells), BRD4mid (25–30%) and BRD4low (5–10%) fractions. For each sample, cells corresponding to at least 1,500-fold library representation were sorted per replicate.

Next-generation sequencing (NGS) libraries of sorted cell fractions were prepared as previously described48. In brief, genomic DNA was isolated by cell lysis (10 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 0.1% SDS), proteinase K treatment (New England Biolabs) and DNAse-free RNAse digest (Thermo Fisher Scientific), followed by two rounds of phenol extraction and 2-propanol precipitation. Isolated genomic DNA was subjected to several freeze–thaw cycles before nested PCR amplification of the sgRNA cassette.

Barcoded NGS libraries for each sorted population were generated using a two-step PCR protocol using AmpliTaq Gold polymerase (Invitrogen). The resulting PCR products were purified using Mag-Bind TotalPure NGS beads (Omega Bio-tek) and amplified in a second PCR introducing the standard Illumina adapters. The final Illumina libraries were bead-purified, pooled and sequenced on HiSeq 3500 or NovaSeq 6000 platforms (Illumina).

Screen analysis was performed as previously described48. In brief, sequencing reads were trimmed using fastx-toolkit (v0.0.14), aligned using Bowtie2 (v2.4.5) and quantified using featureCounts (v2.0.1). The crispr-process-nf Nextflow workflow is available at https://github.com/ZuberLab/crispr-process-nf/tree/566f6d46bbcc2a3f49f51bbc96b9820f408ec4a3. For statistical analysis, we used the crispr-mageck-nf Nextflow workflow, available at https://github.com/ZuberLab/crispr-mageck-nf/tree/c75a90f670698bfa78bfd8be786d6e5d6d4fc455. To calculate gene-level enrichment, the sorted populations (BRD4high or BRD4low) were compared to the BRD4mid populations in MAGeCK (0.5.9)52, using median-normalized read counts.

Viability-based CRISPR–Cas9 screen

The ubiquitin–NEDD8 system CRISPR-knockout library (Supplementary Table 4) was generated using the covalently closed circular-synthesized (3Cs) technology, as previously described53,54. The library contained 3,347 gRNAs cloned under the U6 promoter in a modified pLentiCRISPRv2-puromycin vector containing a modified gRNA scaffold sequence starting with GTTTG. Each gene was represented by four gRNAs selected with the Broad Institute CRISPick tool55,56,57. Additionally, the library included a set of essential genes, non-targeting as well as AAVS1-targeting control sgRNAs.

HCT-116 cells were transduced with the ubiquitin–NEDD8 system lentiviral CRISPR–Cas9 library at a multiplicity of infection of 0.5 and a coverage of 500. Cells were selected with 1 μg ml−1 puromycin for 12 days. Eight million selected cells per condition were then plated in T175 flasks. Cells were treated with DMSO or IBG1 (58 nM), corresponding to 4 times the IC50 value for 3 days, followed by replating and treatment for additional 3 days. After a total of 6 days of treatment, cells were trypsinized, washed three times with PBS, followed by genomic DNA isolation. Sequencing libraries were prepared via PCR as previously described54 and purified via GeneJET Gel Extraction Kit (Thermo Fisher Scientific).

Raw sequencing data were demultiplexed with bcl2fastq v2.20.0.422 (Illumina) to generate raw fastq files. To determine the abundance of individual gRNAs per samples, the fastq files were trimmed using cutadapt (v2.8) to retain only the putative gRNA sequences. These sequences were then aligned to the original gRNA library with Bowtie2 (v2.3.0) and only perfect matches were counted. Statistical analysis was performed via MAGeCK52, using median or total read count normalization and removal of gRNAs with zero counts in the control samples. Genes with a log2-transformed fold change (LFC) > 1 or P value 

Flow-cytometric BRD4 reporter assay

KBM7 iCas9 cells were transduced with lentivirus expressing wild-type, mutated or truncated versions of the SFFV–BRD4(S)–mTagBFP–P2A–mCherry reporter to generate stable reporter cell lines. For evaluation of reporter degradation, cells were treated with DMSO (1:1,000), IBG1 (1 nM), dBET6 (10 nM), IBG3 (0.1 nM) or IBG4 (100 nM) for 6 h before flow cytometry analysis on an LSR Fortessa (BD Biosciences) operated on BD FACSDiva software (v9.0).

To quantify the influence of genetic perturbations on compound-induced reporter degradation, stable BRD4(S) or BRD4Tandem reporter cell lines were transduced with lentiviral sgRNA (pLenti-U6-sgRNA-IT-EF1αs-THY1.1-P2A-NeoR) and/or transgene expression vectors (pRRL-SFFV-3xFlag-DCAF16-EF1αs-iRFP670) to 30–50% transduction efficiency. Cas9 expression was induced with doxycycline (0.4 µg ml−1) for 3 days, followed by 6 h of degrader treatment. Cells were stained for sgRNA expression with an APC-conjugated anti-mouse Thy1.1 antibody (202526, BioLegend; 1:400) and human TruStain FcX Fc receptor blocking solution (422302, BioLegend; 1:400) for 5 min in FACS buffer (PBS containing 5% FBS and 1 mM EDTA) at 4 °C. Cells were washed and resuspended in FACS buffer and analysed on an LSR Fortessa (BD Biosciences).

Flow-cytometric data analysis was performed in FlowJo v10.8.1. BFP and mCherry mean fluorescence intensity values for were normalized by background subtraction of the respective values from reporter-negative KBM7 cells. BRD4 abundance was calculated as the ratio of background subtracted BFP to mCherry mean fluorescence intensity, and is displayed normalized to DMSO-treated, sgRNA and cDNA double-negative cells.

Quantitative proteomics

For unbiased identification of degrader target proteins, 50 × 106 KBM7 iCas9 cells per condition were treated with DMSO (1:1,000), IBG1 (1 nM) or dBET6 (10 nM) for 6 h in biological triplicates. Cells were collected via centrifugation, washed three times in ice-cold PBS and snap-frozen in liquid nitrogen. Cell pellets were lysed in 500 µl of freshly prepared lysis buffer (50 mM HEPES pH 8.0, 2% SDS, 1 mM PMSF and protease inhibitor cocktail (Sigma-Aldrich)). Samples incubated at room temperature for 20 min before heating to 99 °C for 5 min. DNA was sheared by sonication using a Covaris S2 high-performance ultrasonicator. Cell debris was removed by centrifugation at 16,000g for 15 min at 20 °C. Supernatant was transferred to fresh tubes and protein concentration determined using the BCA protein assay kit (Pierce Biotechnology). Filter-aided sample preparation was performed using a 30 kDa molecular weight cut-off centrifugal filters (Microcon 30, Ultracel YM-30, Merck Millipore) as previously described58. In brief, 200 µg of total protein per sample was reduced by the addition of DTT to a final concentration of 83.3 mM, followed by incubation at 99 °C for 5 min. Samples were mixed with 200 μl freshly prepared 8 M urea in 100 mM Tris-HCl (pH 8.5) (UA solution) in the filter unit and centrifuged at 14,000g for 15 min at 20 °C to remove SDS. Residual SDS was washed out by a second wash step with 200 μl UA. Proteins were alkylated with 100 µl of 50 mM iodoacetamide in the dark for 30 min at room temperature. Thereafter, three washes were performed with 100 μl of UA solution, followed by three washes with 100 μl of 50 mM TEAB buffer (Sigma-Aldrich). Proteolytic digestion was performed using trypsin (1:50) overnight at 37 °C. Peptides were recovered using 40 μl of 50 mM TEAB buffer followed by 50 μl of 0.5 M NaCl. Peptides were desalted using Pierce Peptide Desalting Spin Columns (Thermo Scientific). TMTpro 16plex Label Reagent Set was used for labelling according to the manufacturer’s instructions (Pierce). After the labelling reaction was quenched, the samples were pooled, the organic solvent removed in a vacuum concentrator, and the labelled peptides purified by C18 solid phase extraction.

For offline fractionation via reverse phase high-performance liquid chromatography (HPLC) at high pH as previously described59, tryptic peptides were re-buffered in 10 mM ammonium formate buffer (pH 10). Peptides were separated into 96 time-based fractions on a Phenomenex C18 reverse phase column (150 × 2.0 mm Gemini-NX, 3 µm C18 110 Å, Phenomenex) using an Agilent 1200 series HPLC system fitted with a binary pump delivering solvent at 50 µl min−1. Acidified fractions were consolidated into 36 fractions via a concatenated strategy as previously described59. After removal of solvent in a vacuum concentrator, samples were reconstituted in 0.1% TFA prior to liquid chromatography–mass spectrometry (LC–MS/MS) analysis.

Mass spectrometry analysis was performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer coupled to a Dionex Ultimate 3000 RSLCnano system (via a Nanospray Flex Ion Source) (all Thermo Fisher Scientific) interface and operated via Xcalibur (v4.3.73.11) and Tune (v3.4.3072.18). Peptides were loaded onto a trap column (PepMap 100 C18, 5 μm, 5 × 0.3 mm, Thermo Fisher Scientific) at a flow rate of 10 μl min−1 using 0.1% TFA as loading buffer. After loading, the trap column was switched inline with an Acclaim PepMap nanoHPLC C18 analytical column (2.0 µm particle size, 75 µm internal diameter × 500 mm, 164942, Thermo Fisher Scientific). The column temperature was maintained at 50 °C. Mobile phase A consisted of 0.4% formic acid in water, and mobile phase B consisted of 0.4% formic acid in a mixture of 90% acetonitrile and 10% water. Separation was achieved using a 4-step gradient over 90 min at a flow rate of 230 nl min−1. In the liquid junction setup, electrospray ionization was enabled by applying a voltage of 1.8 kV directly to the liquid being sprayed, and non-coated silica emitter was used. The mass spectrometer was operated in a data dependent acquisition (DDA) mode using a maximum of 20 dependent scans per cycle. Full MS1 scans were acquired in the Orbitrap with a scan range of 400−1,600 m/z and a resolution of 120,000 at 200 m/z. Automatic gain control (AGC) was set to ‘standard’ and a maximum injection time (IT) of 50 ms was applied. MS2 spectra were acquired in the Orbitrap at a resolution of 50,000 at 200 m/z with a fixed first mass of 100 m/z. To achieve maximum proteome coverage, a classical tandem MS approach was chosen instead of the available synchronous precursor selection (SPS)-MS3 approach. To minimize TMT ratio compression effects by interference of contaminating co-eluting isobaric peptide ion species, precursor isolation width in the quadrupole was set to 0.5 Da and an extended fractionation scheme applied. Monoisotopic peak determination was set to ‘peptides’ with inclusion of charge states between 2 and 5. Intensity threshold for MS2 selection was set to 2.5 × 104. Higher energy collision induced dissociation (HCD) was applied with a normalized collision energy (NCE) of 34%. Normalized AGC was set to 200% with a maximum injection time of 86 ms. Dynamic exclusion for selected ions was 90 s.

The acquired raw data files were processed using Proteome Discoverer (v.2.4.1.15), via the TMT16plex quantification method. Sequest HT database search engine and the Percolator validation software node were used to remove false positives with FDR 1% at the peptide and protein level. All MS/MS spectra were searched against the human proteome (Canonical, reviewed, 20 304 sequences) and appended known contaminants and streptavidin, with a maximum of two allowable miscleavage sites. The search was performed with full tryptic digestion with or without deamidation on amino acids asparagine, glutamine, and arginine. Methionine oxidation and protein N-terminal acetylation, as well as methionine loss and protein N-terminal acetylation with methionine loss were set as variable modifications, while carbamidomethylation of cysteine residues and tandem mass tag (TMT) 16-plex labelling of peptide N termini and lysine residues were set as fixed modifications. Data were searched with mass tolerances of ±10 ppm and ±0.025 Da for the precursor and fragment ions, respectively. Results were filtered to include peptide spectrum matches with Sequest HT cross-correlation factor (Xcorr) scores of ≥1 and high peptide confidence assigned by Percolator. MS2 signal-to-noise (S/N) values of TMTpro reporter ions were used to calculate peptide or protein abundance values. Peptide spectrum matches with precursor isolation interference values of ≥70% and average TMTpro reporter ion S/N ≤ 10 were excluded from quantification. Both unique and razor peptides were used for TMT quantification. Correction of isotopic impurities was applied.

Data were normalized to total peptide abundance and scaled ‘to all average’. Abundances were compared to DMSO-treated cells and protein ratios were calculated from the grouped protein abundances using an ANOVA hypothesis test. Adjusted P values were calculated using the Benjamini–Hochberg method. Proteins with less than three unique peptides detected were excluded from downstream analysis.

Protein construction, expression and purification

His6–TEV–BRD4 bromodomain 1 (BRD4BD1) (amino acids 44–178) and His6–TEV–BRD4 bromodomain 2 (BRD4BD2) (amino acids 333–460) were expressed in Escherichia coli BL21(DE3) and purified as described previously60. In brief, proteins were purified by nickel affinity chromatography and SEC. His6 tag cleavage and reverse nickel affinity was performed prior to SEC for some applications, for others the tag was left on. Purified proteins in 20 mM HEPES, 150 mM sodium chloride, 1 mM DTT, pH 7.5 were aliquoted and flash frozen in liquid nitrogen and stored at −80 °C.

His6–SUMO–TEV–BRD4Tandem (residues 1–463) was prepared as previously described51. In brief, protein was expressed in E. coli BL21(DE3) and purified sequentially by nickel affinity on a HisTrap HP 5 ml column (Cytiva), His6 tag cleavage by SENP1 followed by reverse nickel affinity, cation exchange on a HiTrap SP HP 5 ml column (Cytiva), and size exclusion on a HiLoad 16/600 Superdex 200 pg column (Cytiva). Purified protein in 20 mM HEPES, 100 mM sodium chloride, 1 mM TCEP, pH 7.5 was aliquoted and flash frozen in liquid nitrogen then stored at −80 °C.

BRD4Tandem (residues 43–459) was cloned into pRSF-DUET or a modified pGEX4T1 with an N-terminal His10 tag and HRV3C cleavage site or a His12-GST tag and TEV cleavage site, respectively.

His10−3C-BRD4Tandem (residues 43–459) was transformed into E. coli BL21(DE3) and overnight expression at 18 °C was induced with 0.35 mM IPTG at OD600 ~ 0.8–1. Cells were collected by centrifugation and pellets were resuspended in ice-cold PBS then spun down again. Supernatant was removed and pellets were flash frozen in liquid nitrogen and stored at −80 °C. Cells were thawed and resuspended in lysis buffer (50 mM HEPES, 500 mM NaCl, 0.5 mM TCEP, pH 7.5) supplemented with 2 mM magnesium chloride, DNAse and cOmplete EDTA-free Protease Inhibitor Cocktail (Roche, 1 tablet per litre initial culture volume) and lysed at 30,000 psi using a CF1 Cell Disruptor (Constant Systems). The lysate was cleared by centrifugation at 20,000 rpm for 30 min at 4 °C then syringe-filtered using a 0.45-μm filter. The lysate was supplemented with 40 mM imidazole and loaded on to a 5 ml HisTrap HP column (Cytiva) equilibrated in lysis buffer with 40 mM imidazole, washed at 60 mM imidazole and eluted with a gradient up to 100% elution buffer (50 mM HEPES, 500 mM NaCl, 0.5 mM TCEP, 500 mM imidazole, pH 7.5). The prep was split as required for tag cleavage or for purification of the His10–3C-tagged form. For tag cleavage, the sample was buffer exchanged into lysis buffer on a HiPrep 26/10 Desalting column and HRV3C protease was added to cleave the tag overnight at 4 °C. Imidazole was added to 20 mM to the cleaved BRD4Tandem and the sample was run on a 5 ml HisTrap HP column equilibrated in lysis buffer with 20 mM imidazole and washed with the same imidazole concentration. The flow-through and wash containing BRD4Tandem were pooled and, along with uncleaved His10–3C–BRD4Tandem, were concentrated in 10,000 MWCO Amicon centrifugal filter units (Merck Millipore). The proteins were each loaded separately onto a HiLoad 26/600 Superdex 200 pg column (GE LifeSciences) equilibrated in 20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, pH 7.5. Fractions containing either pure BRD4Tandem or His10–3C–BRD4Tandem were confirmed by SDS–PAGE, then pooled, concentrated and aliquoted for storage at −80 °C until use.

For use in cryo-electron microscopy (cryo-EM) with DCAF16 and IBG1, His12–GST–TEV–BRD4Tandem (residues 43–459) expression in E. coli BL21(DE3) cells was induced at OD600 = 2 with 0.5 mM IPTG at 20 °C for 16 h. Cells were collected by centrifugation and resuspended in lysis buffer (50 mM HEPES, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, pH 7.5) (10 ml g−1 pellet weight) supplemented with DNAse and 1 cOmplete EDTA-free Protease Inhibitor Cocktail tablet (Roche) per 2 l of culture. Cells were lysed at 30,000 psi using a CF1 Cell Disruptor (Constant Systems) and lysate was clarified by centrifugation. Lysate was filtered through a BioPrepNylon Matrix Filter (BioDesign) then incubated with 1 ml Ni-NTA resin per litre culture for 1 h. The lysate–resin slurry was poured into a Bio-Rad Econo-column and resin was washed with >10 column volumes lysis buffer. Bound protein was eluted with elution buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 500 mM imidazole, 0.5 mM TCEP) then incubated with 1 ml glutathione agarose resin per litre culture for 30 min. The mixture was poured into an Econo-column and resin was washed with 20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, pH 7.5. TEV protease was added to the resin slurry for on-bead cleavage and the column was incubated overnight on a roller at 4 °C. Protein was eluted from the column then concentrated and run on a HiLoad 16/600 Superdex 75 pg column equilibrated in 20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, pH 7.5. Fractions containing protein were pooled, concentrated and aliquoted then flash frozen in liquid nitrogen then stored at −80 °C until use.

A DCAF15 construct lacking the proline-rich region (amino acids 276–380; DCAF15Δpro) with N-terminal His6-TEV-Avi tag, DDB1(ΔBPB) (residues 396–705 replaced with a GNGNSG linker), and full-length DDA1 coding sequences were cloned into a pFastBacDual vector. Bacmid was generated using the Bac-to-Bac baculovirus expression system (Thermo Fisher Scientific). Baculovirus was generated via an adapted single-step protocol61,62. In brief, bacmid (1 µg ml−1 culture volume) was mixed with 2 µg PEI 25 K (Polysciences) per µg bacmid in 200 µl warm PBS and incubated at room temperature for 30 min. The mixture was added to a suspension culture of Sf9 cells at 1 × 106 cells per ml in Sf-900 II SFM (Gibco) and incubated at 27 °C with shaking at 110 rpm. Viral supernatant (P0) was collected after 4–6 days. For expression, Spodoptera frugiperda cells (Sf9) were grown to densities between 1.9 to 3.0 × 106 cells per ml in Sf-900 II SFM (Gibco) and infected with a total virus volume of 1% per 1 × 106 cells per ml. Cells were incubated at 27 °C in 2 l Erlenmeyer flasks (~500 ml culture per flask) with shaking at 110 rpm for 48 h. Cells were spun at 1,000g for 10 min and supernatant was discarded. Pellets were resuspended in lysis buffer (50 mM HEPES, 200 mM NaCl, 2 mM TCEP, pH 7.5) with magnesium chloride (to 2 mM), benzonase (to 1 µg ml−1) and cOmplete EDTA-free Protease Inhibitor Cocktail (Roche, 2 tablets per litre initial culture volume). The suspension was frozen and stored at −80 °C, and then thawed. Cell suspensions were sonicated and lysates were centrifuged at 40,000 rpm for 30 min. The supernatant was incubated with 1.5 ml Ni-NTA agarose resin (Qiagen) on a roller at 4 °C for 1.5 h. The lysate–resin slurry was loaded into a glass bench top column. Supernatant was allowed to flow through then the resin was washed with wash buffer (50 mM HEPES, 200 mM NaCl, 2 mM TCEP, 20 mM imidazole, pH 7.5). Bound protein was eluted with elution buffer (50 mM HEPES pH 7.5, 200 mM NaCl, 2 mM TCEP, 500 mM imidazole). TEV protease was added to protein and dialysed with buffer (50 mM HEPES, 200 mM NaCl, 2 mM TCEP, pH 7.5). Cleaved protein was run over 1.5 ml Ni-NTA agarose resin and the flow-through and washes with binding buffer were collected and pooled. Protein was diluted with buffer (25 mM HEPES, 2 mM TCEP, pH 7.5) to adjust the NaCl concentration to 50 mM, then loaded onto a HiTrap Q HP 5 ml column (Cytiva). The column was washed with IEX buffer A and bound protein was eluted with a 0–100% IEX buffer B (25 mM HEPES, 1 M NaCl, 2 mM TCEP, pH 7.5) gradient. Fractions containing protein were pooled and concentrated to ~1–2 ml then run on 16/600 Superdex 200 pg column in GF buffer (25 mM HEPES, 300 mM NaCl, 1 mM TCEP, pH 7.5). Fractions containing the purified protein complex were pooled, concentrated and aliquoted then flash frozen in liquid nitrogen for storage at −80 °C.

The coding sequences for full-length DCAF16 or DCAF11 with TEV-cleavable N-terminal His6-tags were cloned into a pFastBacDual vector under the control of the polh promoter. Coding sequences for full-length DDB1 or DDB1(ΔBPB) and full-length DDA1 were cloned into a pFastBacDual vector under the control of polh and p10 promoters, respectively. Bacmid was generated using the Bac-to-Bac baculovirus expression system (Thermo Fisher Scientific). Baculovirus was generated as described above and viral supernatant (P0) was collected after 5–7 days. For expression, Trichoplusia ni High Five cells were grown to densities between 1.5 to 2 × 106 cells per ml in Express Five SFM (Gibco) supplemented with 18 mM l-glutamine and infected with a total virus volume of 1% per 1 × 106 cells per ml, consisting of equal volumes of DCAF16/DCAF11 and DDB1 + DDA1 baculoviruses. Cells were incubated at 27 °C in 2 l Erlenmeyer flasks (~600–650 ml culture per flask) with shaking at 110 rpm for 72 h. Cells were spun at 1,000g for 20 min and supernatant was discarded. Pellets were resuspended in 25 ml binding buffer (50 mM HEPES, 500 mM NaCl, 1 mM TCEP, pH 7.5), flash frozen in liquid nitrogen and stored at −80 °C. Pellets were thawed and diluted with binding buffer to ~100 ml l−1 original culture volume. Tween-20 (to 1% (v/v)), magnesium chloride (to 2 mM), benzonase (to 1 µg ml−1) and cOmplete EDTA-free Protease Inhibitor Cocktail (Roche, 2 tablets per litre initial culture volume) were added to the cell suspension and stirred at room temperature for 30 min. Cell suspensions were sonicated, and lysates were centrifuged at 23,000 rpm for 60 min. Supernatants were filtered through 0.45-µm filters and supplemented with 10 mM imidazole then incubated with 2 ml cobalt agarose resin per litre culture on a roller at 4 °C for 1 h. The lysate–resin slurry was loaded into a glass bench top column. Supernatant was allowed to flow through then the resin was washed with wash buffer (50 mM HEPES, 500 mM NaCl, 1 mM TCEP, 15 mM imidazole, pH 7.5). Bound protein was eluted with elution buffer (50 mM HEPES, 500 mM NaCl, 1 mM TCEP, 250 mM imidazole, pH 7.5) and buffer exchanged on a 26/10 HiPrep Desalting column (Cytiva) into Binding Buffer. TEV protease was added to protein and incubated for 2 h at room temperature then 4 °C overnight. Imidazole was added to the cleaved protein to a concentration of 10 mM and the sample was run over cobalt agarose resin. Flow-through and washes with binding buffer supplemented with 10 mM imidazole were collected and pooled. Protein was buffer exchanged into ion exchange (IEX) buffer A (50 mM HEPES, 50 mM NaCl, 1 mM TCEP, pH 7.5) on a 26/10 HiPrep Desalting column then loaded onto a HiTrap Q HP 5 ml column (Cytiva). The column was washed with IEX buffer A and bound protein was eluted with a 0–100% IEX buffer B (50 mM HEPES, 1 M NaCl, 1 mM TCEP, pH 7.5) gradient. Fractions containing protein were pooled and concentrated then run on 16/600 Superdex 200 pg column in equilibrated in 20 mM HEPES, 150 mM NaCl, 1 mM TCEP, pH 7.5. Fractions containing the purified protein complex were pooled and concentrated then aliquoted and flash frozen in liquid nitrogen for storage at −80 °C.

Sulfo-Cy5 NHS ester labelling

For DCAF16 labelling, sulfo-Cy5 NHS ester (Lumiprobe) in DMF was prepared to a final concentration of 800 µM with DCAF16–DDB1(ΔBPB)–DDA1 (100 µM) and sodium bicarbonate (100 mM). For DCAF11 labelling, sulfo-Cy5 NHS ester (Lumiprobe) in DMF was prepared to a final concentration of 1 mg ml−1 with DCAF11–DDB1(ΔBPB)–DDA1 (1 mg ml−1) and sodium bicarbonate (100 mM). The solutions were protected from light and shaken for 1 h at room temperature. The solutions were spun down at 15,000g for 5 min then run on a Superdex 200 10/300 GL column (Cytiva) to remove free dye and aggregated protein. Fractions containing the sulfo-Cy5-labelled protein were pooled and concentrated, the degree of labelling was calculated to be greater than 100% for each batch of labelled protein. Labelled protein was aliquoted then flash frozen in liquid nitrogen and stored at −80 °C.

Fluorescence polarization assay

Stock solutions of reaction components including DCAF15(Δpro)–DDB1(ΔBPB)–DDA1, DCAF16–DDB1(ΔBPB)–DDA1, His6–BRD4BD1, His6–BRD4BD2, BRD4Tandem (residues 43–459), and FITC-sulfonamide probe7 were prepared in FP assay buffer (25 mM HEPES pH 7.5, 300 mM NaCl, 1.0 mM TCEP). DCAF15(Δpro)–DDB1(ΔBPB)–DDA1, DCAF16–DDB1(ΔBPB)–DDA1, BRD4BD1, BRD4BD2 and BRD4Tandem were titrated 1:3 in FP assay buffer. Components were added to Corning 384-Well solid black polystyrene microplates to a final volume of 15 µl. Final concentration of 20 nM for FITC-sulfonamide probe was used while DCAF15(Δpro)–DDB1(ΔBPB)–DDA1, DCAF16–DDB1(ΔBPB)–DDA1, BRD4BD1, His6–BRD4BD2 and BRD4Tandem were titrated from 4 µM to 5.5 nM. Background subtraction was performed with 20 nM FITC-sulfonamide probe and no protein constructs. Components were mixed by spinning down plates at 50g for 1 min and the plate was covered and incubated at room temperature for 1 h, before analysis on a PHERAstar FS (BMG LABTECH) with fluorescence excitation and emission wavelengths of 485 and 520 nm, respectively, with a settling time of 0.3 s.

AlphaLISA displacement assay

The alphaLISA assays were performed as described previously51 using His6–BRD4BD1, His6–BRD4BD2 or His10–BRD4Tandem and the biotinylated JQ1 probe. Assay conditions in the present work used were as follows: 100 nM bromodomain protein, 10 nM Bio-JQ1 probe, 25 µg ml−1 acceptor (nickel chelate) and donor (anti-His–europium; both PerkinElmer). All components were diluted to working concentrations in alphaLISA buffer (50 mM HEPES, 100 mM NaCl, 0.1% BSA, 0.02% CHAPS, pH 7.5). Bromodomain protein was co-incubated with test compounds using 384-well AlphaPlates (PerkinElmer) in the absence or presence of DCAF16 (1 µM) for 1 h, before adding the acceptor and donor beads simultaneously in a low light environment and incubating the plate at room temperature for a further 1 h. The plate was then read on a BMG Pherastar equipped with an alphaLISA module. Data were normalized to a DMSO control and expressed as % bound vs log[concentration] of compound and analysed by non-linear regression, with extraction of binding affinity values (IC50) from the curves. Where applicable, Kd values were calculated from a titration of bromodomain protein on the same assay plate alone into the probe, as described previously63.

TR-FRET proximity assay

Stock solutions of reaction components including sulfo-Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1, sulfo-Cy5-labelled DCAF11–DDB1(ΔBPB)–DDA1, His6–BRD4BD1, His10–BRD4BD2, His10–BRD4Tandem, experimental compounds and LANCE Eu-W1024 Anti-His6 donor (PerkinElmer) were prepared in TR-FRET assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM TCEP, 0.05% Tween-20). Two types of TR-FRET assay were performed: titration of compound into protein (complex-formation assay) and titration of sulfo-Cy5-labelled DCAF into BRD4 vs BRD4–compound (complex-stabilization assay). For the former, compounds were titrated 1:4 into 100 nM BRD4 and 100 nM Cy5-DCAF to a PerkinElmer OptiPlate-384 (white) to a final well volume of 16 μl. For the complex-stabilization assay, sulfo-Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 or DCAF11–DDB1(ΔBPB)–DDA1 were titrated 1:4 and 1:3 respectively in TR-FRET assay buffer. Components were added to PerkinElmer OptiPlate-384 (white) to a final well volume of 16 μl. Final concentrations of 100 or 200 nM for BRD4 constructs and 0.5 µM or 1 µM for IBG1 respectively were used. LANCE Eu-W1024 anti-His6 donor and DMSO concentrations were kept constant across the plate for both assay formats at 2 nM and 0.5%, respectively. Background subtraction was performed with using concentration matched samples containing sulfo-Cy5-labelled DCAF complexes but not BRD4. Components were mixed by spinning down plates at 50g for 1 min and plates were covered and incubated at room temperature for 30 min. Plates were read on a PHERAstar FS (BMG LABTECH) with fluorescence excitation and dual emission wavelengths of 337 and 620/665 nm, respectively, with an integration time between 70 and 400 μs. Data were processed in GraphPad Prism (v9.3.1), curve fitting for the IBG1 curve was performed by setting the maximum as DMSO-only 5 µM sulfo-Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 datapoint.

Analytical SEC

For DCAF16 experiments, DCAF16–DDB1(ΔBPB)–DDA1, BRD4Tandem (residues 1–463), BRD4BD1 (His6 tag removed), BRD4BD2 (His6 tag removed), and IBG1 were incubated alone and in various combinations in buffer (20 mM HEPES, 150 mM NaCl, 1 mM TCEP, 2% DMSO, pH 7) on ice for 50 min. Final concentrations used for Fig. 4a and Extended Data Fig. 4a were 10 µM DCAF16–DDB1(ΔBPB)–DDA1, 5 µM BRD4Tandem, 25 µM IBG1 in 250 µl reaction volumes. Final concentrations used for Fig. 4b were 5 µM DCAF16–DDB1(ΔBPB)–DDA1, 5 µM BRD4Tandem, 5 µM BRD4BD1, 5 µM BRD4BD2, 12.5 µM IBG1 in 200 µl reaction volumes. Samples were run on a Superdex 200 Increase 10/300 gl column in 20 mM HEPES, 150 mM NaCl, 1 mM TCEP, pH 7.

For DCAF11 experiments, DCAF11–DDB1(ΔBPB)–DDA1, BRD4Tandem (residues 43–463) and IBG4 were incubated alone and in various combinations in buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, 2% DMSO, pH 7.5) at final concentrations of 5 µM, 5 µM and 10 µM, respectively. Samples were run on a Superdex 200 Increase 10/300 gl column in 20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, pH 7.5.

For BRD4 intramolecular dimerization experiments, BRD4Tandem (residues 43–463) and compounds were incubated in buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, 2% DMSO, pH 7.5) at final concentrations of 5 µM and 10 µM, respectively. Samples were run on a Superdex 200 Increase 10/300 gl column in 20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, pH 7.5.

Isothermal titration calorimetry

Titration experiments were performed with an ITC200 instrument (Malvern) in 100 mM Bis-tris propane, 50 mM NaCl, 0.5 mM TCEP, pH 7.5 at 298 K. Protein samples were prepared by dialysing in buffer in D-Tube Dialyzer Midi, MWCO 6–8 kDa (Millipore). BRD4Tandem (residues 43–459) was pre-incubated alone, or with either IBG1 or IBG3 at a 1:1.1 molar ratio for 30 min at room temperature prior to titrations at a DMSO concentration of 2% (v/v). DCAF16–DDB1(ΔBPB)–DDA1 at 2% DMSO (v/v) was titrated into either BRD4Tandem alone, pre-complexed BRD4Tandem–IBG1 or pre-complexed BRD4Tandem–IBG3. The titration consisted of 0.4 μl initial injection (discarded during data analysis) followed by 19 injections of 2 μl at 180 s intervals between injections. Data were fitted using a one-set-of-site binding model to obtain dissociation constant (Kd), binding enthalpy (ΔH) and stoichiometry (N) using MicroCal PEAQ-ITC Analysis Software1.1.0.1262.

Cryo-EM sample and grid preparation

Protein complexes for cryo-EM were prepared by first co-incubating BRD4Tandem (residues 43–459) with IBG1 in 20 mM HEPES, 50 mM NaCl, 0.5 mM TCEP-HCl, 2% (v/v) DMSO, pH 7.5 for 10 min at room temperature. DCAF16–DDB1(ΔBPB)–DDA1 was added to the mixture to give final concentrations of 14 µM BRD4Tandem, 14 µM DCAF16–DDB1(ΔBPB)–DDA1 and 35 µM IBG1 in a final reaction volume of 200 µl and incubated on ice for 50 min. The sample was loaded onto a Superdex 200 Increase 10/300 GL column in 20 mM HEPES, 50 mM NaCl, 0.5 mM TCEP-HCl, pH 7.5. Due to incomplete complex formation and to avoid monomeric proteins, only the earliest eluting fraction containing the ternary complex was taken and concentrated to 4.8 µM. Quantifoil R1.2/1.3 Holey Carbon 400 mesh gold grids (Electron Microscopy Sciences) were glow discharged for 60 s with a current of 35 mA under vacuum using a Quorum SC7620. The complex (3.5 µl) was dispensed onto the grid, allowed to disperse for 10 s, blotted for 3.5 s using blot force 3, then plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) with the chamber at 4 °C and 100% humidity.

Cryo-EM data acquisition

Cryo-EM data were collected on a Glacios transmission electron microscope (Thermo Fisher) operating at 200 keV. Micrographs were acquired using a Falcon4i direct electron detector, operated in electron counting mode. Movies were collected at 190,000× magnification with the calibrated pixel size of 0.74 Å per pixel on the camera. Images were taken over a defocus range of –3.2 µm to −1.7 µm with a total accumulated dose of 12.7 e− Å−2 using single-particle EPU (Thermo Fisher Scientific, v3.0) automated data software. A total of 2,075 movies were collected in EER format and after cleaning up for large motion and poor contrast transfer function (CTF) a total of 1,896 movies were used for further processing. Cryo-EM data collection, refinement and validation statistics are presented in Extended Data Table 2.

Cryo-EM image processing

Movies were imported into cryosparc64 (v4.1.2) and the EER movie data was fractionated into 8 fractions to give a dose of 1.59 e− Å−2 per fraction. Movies were processed using patch motion correction and CTF correction then manually curated to remove suboptimal movies. Manual picking of 153 particles was performed on 20 micrographs, which were used for blob tuner with minimum and maximum diameters of 70 and 130 Å, respectively. 12,579 particles were picked by blob tuner, extracted with a box size of 324 pix (240 Å) and run through initial 2D classification. Good classes with diverse views were selected and used as templates for template picking on 1,895 movies. Picks were inspected and curated, and 1.35 million particles were extracted with box size 324 pix and used for 2D classification. Particles from the well-resolved, diverse classes were used for ab initio reconstruction with 3 classes. One class contained primarily empty DDB1(ΔBPB) and a second class contained biased views upon testing of the particle set with 2D re-classification, leading to smeared maps. The third class unambiguously contained density corresponding to DDB1(ΔBPB), two bromodomains, and density likely corresponding to DCAF16 between them. Particles belonging to the second and third class were run through heterogenous refinement. The best class yielded a map into which DDB1(ΔBPB) and two bromodomains could be placed with confidence. To improve the resolution, movies were re-imported in cryosparc and fractionated into 18 fractions to give a lower dose of ~0.7 e− Å−2 per fraction. 50 templates for particle picking were generated using the create templates job with the input map from the previous heterogeneous refinement. The templates were used in the template picker to pick particles from 1,132 curated movies with a minimum CTF fit resolution cut-off of 3.5. Picks were curated with thresholds of NCC score > 0.4, local power >368 and 4.

Cryo-EM model building

DDB1(ΔBPB), BRD4BD1 and BRD4BD2 extracted from PDB entries 5FQD27, 3MXF65 and 6DUV, respectively, were manually placed into the map in WinCoot66 (v0.9.8.1) by rigid body fitting. Despite co-purifying with DCAF16 and DDB1(ΔBPB), we did not see density for DDA1, as was observed in another DDB1-substrate receptor structure from a recent publication67. Correct placement of each bromodomain was aided by manual inspection of residues Asn93 and Gly386 in equivalent positions in the ZA loops of BD1 and BD2, respectively. In one bromodomain, this position was facing solvent while in the other it was at a protein–protein interface with density corresponding to DCAF16. Given that mutation of Gly386 to Glu prevents degradation of BRD4 by IBG1 (Fig. 3i), BD2 was placed in the position where Gly386 was adjacent to the DCAF16 density. The BD2 ZA loop is three residues longer than the BD1 ZA loop, further confirming the correct positioning of each domain based on the map around these positions. Both bromodomains were joined onto a single chain designation. Initial restraints for IBG1 were generated using a SMILES string with eLBOW (in Phenix v1.20.1-4487)68, then run through the GRADE webserver (Grade2 v1.3.0). IBG1 was fitted into density by overlaying the JQ1 moiety with its known binding mode in either the BRD4BD1 or BRD4BD2. Positioning the ligand in BD2 was compatible with electron density, whereas positioning in BD1 caused a clash with DCAF16 due to the rigid linker. DCAF16 was built using a combination of models from ColabFold69,70 (v1.3), ModelAngelo71 (v0.2.2) and manual building in Coot (v0.9.8.1). ColabFold correctly predicted the α5 and α6 helices that bind the DDB1 central cavity while ModelAngelo correctly built the 4-helical bundle of α3, 4, 7 and 8, as well as α6 in the DDB1 cavity. Correctly built parts of the models were combined, and the structure was refined with rounds of model building in Coot, fitting with adaptive distance restraints in ISOLDE72 (v1.6) and refinement with Phenix (v1.20.1-4487) real-space refinement73,74. Figures were generated in ChimeraX75 (v1.6) and The PyMOL Molecular Graphics System76 (v2.5.2, Schrödinger, LLC).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.