plasmid pgex 6p 1 brd4 full length  (Addgene inc)

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) Cryo-EM map and (b) cartoon representation of the BRD4-S/nucleosome complex showing how the BRD4 BD1 (pink) interacts with the nucleosome. The modeled histone H4 tail residues 11-16 are shown as a thicker gold line.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) representative motion-corrected micrographs, (b) representative 2D classes, (c) angular distribution of particles used to generate the cryo-EM map, (d) cryo-EM map of the BRD4/nucleosome complex colored by estimated local resolution determined with FSC = 0.143 cutoff in cryoSPARC, (e) conical Fourier shell correlation (cFSC) curve of the BRD4-nucleosome structure at 2.89 Å resolution, calculated between two independent half-maps using a conical mask with a specified half-angle and axis in Fourier space in cryoSPARC. Lines and arrows indicate the axis of rotation between successive views, (f) unmasked (red) and masked (blue) Fourier shell correlation (FSC) curves between two independent half-maps determined in cryoSPARC.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: Schematic representation of the cryo-EM data processing pipeline for the BRD4/nucleosome complex, as described in the Methods section.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) cryo-EM map of viewed from the side to show interaction of histone H4 tail with BRD4 BD1, (b) cryo-EM map prepared from subset of particles shows extra density (purple) beyond the C-terminus of BRD4 BD1 (top view on left, side view on right).

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) Time-resolved FRET binding assay results for BRD4-S binding to unmodified (blue), H4 tailless = H4(24-102) (green) and H4 K12acK16ac nucleosomes (pink) in 70 mM NaCl. (b) Effect of NaCl concentration on BRD4-S binding to unmodified (blue) or H4 K12acK16ac nucleosomes (pink) as assayed by TR-FRET.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) top: cartoon representation of BRD4 constructs, bottom: individual TR-FRET unnormalized fluorescence (not normalized to maximum fluorescence) binding results of for wild-type BRD4-S binding to H4 K12acK16ac nucleosomes and average of 3 measurements for BRD4 BD1 and BD2 binding to H4 K12acK16ac nucleosomes, (b) TR-FRET binding curves for BRD4-S binding to unmodified (blue) or H4 K12acK16ac (pink) nucleosomes in 100, 125 and 150 mM NaCl.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Construct, Fluorescence, Binding Assay

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) BRD4-S domains and basic patches highlighted in cartoon and primary sequence (left) and identity of BRD4-S basic patch mutations studied (right), (b) TR-FRET dissociation constants for BRD4-S basic patch mutants binding to H4 K12acK16ac nucleosomes in 70 mM NaCl, (c) TR-FRET dissociation constants for BRD4-S basic patch mutants binding to H4 K12acK16ac nucleosomes in 150 mM NaCl, (d) effect of salt concentration on select BRD4-S basic patch mutations on binding to H4 K12acK16ac nucleosomes.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Sequencing, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet:

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques:

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) BRD4 BD1 interactions with the nucleosome with key regions highlighted, (b) TR-FRET dissociation constants for BRD4-S bromodomain mutants binding to H4 K12acK16ac nucleosomes, (c) model for how BRD4 basic region 1 could interact with nucleosome DNA minor groove with C ⍺ positions of the 5 basic residues shown in blue spheres, (d) effect of BRD4-S BD1 mutations on BRD4-S/nucleosome complex mobility in gel mobility shift assay, (e) NMR structure of HMG-I(Y) AT-hook Arg-Gly-Arg region binding to DNA (PDB ID 2EZD), protein residues outside of the Arg-Gly-Arg region not shown, (f) TR-FRET dissociation constants for BRD4-S basic patch 1 mutants binding to H4 K12acK16ac nucleosomes.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Binding Assay, Mobility Shift

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: Acidic patch nucleosomes contain the H2A(E61A,E64A,D90A,E92A) quadruple mutation. BRD4-S binds wild-type nucleosomes (blue) and acidic patch nucleosomes (red) with similar affinity, but the RCC1 chromatin factor shown to use an arginine anchor to bind to the nucleosome acidic patch is adversely affected by the nucleosome acidic patch mutations.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) the BRD4-S m12/H4 K12acK16ac nucleosome complex (blue) elutes earlier than the BRD4-S WT/H4 K12acK16ac nucleosome complex, (b) the BRD4-S m12 protein elutes at similar time as the BRD4 WT protein.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques:

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: The BRD4 residues R68, K72, K76 observed to interact with nucleosomal DNA and the BRD2, BRD3 and BRDT equivalent BD1 and BD2 residues are highlighted in yellow.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques:

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: Superposition of ZL0590/BRD4 BD1 crystal structure (PDB 6U0D) with BRD4/nucleosome structure (this work) via BRD4 BD1.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques:

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Chemical structures of the parental BET protein family inhibitor (JQ1) and its derivative (ZZ1) featuring an appended chemical tag conferring degrader activity. b) HiBiT-BRD4 assay results for Jurkat cells pre-treated with the indicated inhibitors for 1 h, followed by treatment with ZZ1 for 5 h. c) Ubiquitin-proteasome system (UPS)-focused CRISPR screen for BRD4 BD1 -eGFP stability in K562-Cas9 cells treated with 1 µM ZZ1 for 16 h. d) Cartoon of the UBE2H∼Ub-bound YPEL5-GID/CTLH E3 ligase catalytic assembly highlighting its functional modules. e) In vitro ubiquitylation assay of fluorescently labeled *BRD4 BD1 (asterisk denotes an N-terminal FAM label) determining the minimal catalytic assembly sufficient for ZZ1-dependent activity. The examined GID/CTLH E3 ligases comprised the catalytic core (catalytic and scaffolding modules) alone or assembled with substrate regulatory subunits WDR26 and YPEL5. Reactions were quenched after 45 min. See for details of the GID/CTLH E3 ligase architecture and assays with a full suite of GID/CTLH E3 assemblies. f) Native gel mobility shift assay probing ZZ1-induced *BRD4 BD1 engagement by the WDR26 dimer or WDR26-YPEL5. Transparent regions in the cartoon represent truncated WDR26 domains. The truncations prevent higher-order oligomerization).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Activity Assay, Ubiquitin Proteomics, CRISPR, Functional Assay, In Vitro, Ubiquitin Assay, Labeling, Scaffolding, Mobility Shift

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Western blots showing BRD4 and BRD3 degradation in MOLT-4 cells after 5 h treatment with ZZ1. b) Quantitative proteome-wide mass spectrometry in MOLT-4 cells after 3 h treatment with 1 µM of ZZ1. c) Identifying of the BRD4 region required for ZZ1-induced degradation with a cellular fluorescent reporter assay. The examined reporters were either the isolated BRD4 bromodomains (BD1 or BD2) or a tandem construct comprising BD1 and BD2 connected by the intervening native sequence (BRD4 BD1+BD2 ).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Western Blot, Mass Spectrometry, Reporter Assay, Isolation, Construct, Sequencing

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Color-coded guide to the GID/CTLH E3 subunits and their reported functions. b) Schematic illustrating the architecture of the GID/CTLH E3 ligases, which share a common catalytic core that associates with divergent auxiliary subunits enabling substrate targeting. WDR26 acts as a supramolecular assembly factor by connecting two copies of the catalytic core (either alone or bound to GID4-ARMC8) into a singular giant oval structure with a large hollow center. Each WDR26 homodimer in the supramolecular assembly can bind a single copy of YPEL5, yielding the YPEL5-GID/CTLH E3. Subunits are colored according to the guide in (a). c) Identifying E3 ligase leveraged by ZZ1 with in vitro ubiquitylation assays. The suite of GID/CTLH E3 assemblies shown in (b) was tested for activity towards fluorescent BRD4 bromodomain substrates, either in isolation (BRD4 BD1 and BRD4 BD2 ) or in tandem (BRD4 BD1+BD2 ). Asterisk denotes the fluorescent FAM label appended to substrates’ N-termini. All reactions were quenched after 45 min. d) Western blots showing BRD4 degradation in WT or YPEL5-KO Jurkat cells treated with the indicated concentration of ZZ1 for 5 h. e) Western blots showing BRD4 degradation in WT or WDR26-KO HEK293T cells treated with the indicated concentration of ZZ1 for 5 h. f) Western blots showing BRD4 degradation in HEK293T (YPEL5 low ) or TC-71 (YPEL5 high ) cells treated with the 2 µM of ZZ1 for 5 h. g) Co-immunoprecipitation of FLAG-tagged BRD4 and YPEL5-WDR26-containing GID/CTLH E3 in the presence of ZZ1. FLAG-tagged BRD4 transfected cells were preincubated with the proteasomal pathway inhibitor (bortezomib) for 1 h to prevent BRD4 degradation. h) In vitro ubiquitylation assay as in (b) but performed with the endogenous NMNAT1 substrate to recapitulate its previously reported GID/CTLH E3-dependent regulation. In contrast to its essential role in ZZ1-induced BRD4 ubiquitylation, YPEL5 acts as an inhibitor of NMNAT1 targeting. Reactions were quenched after 45 min. i) Previous cryo-EM structures of NMNAT1- and YPEL5-bound GID/CTLH E3 assemblies (EMD-18175 and EMD-18170, respectively) fit with segmented focused-refined maps of relevant modules (EMD-18345 and EMD-18316, respectively) explaining the biochemically defined mode of NMNAT1 targeting: the hexameric NMNAT1 and YPEL5 are engaged by the overlapping binding sites on dimeric WDR26 modules. Consequently, YPEL5 sterically blocks WDR26-mediated NMNAT1 recruitment.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: In Vitro, Activity Assay, Isolation, Western Blot, Concentration Assay, Immunoprecipitation, Transfection, Ubiquitin Assay, Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Cryo-EM map of the neosubstrate recognition complex (YPEL5-GID/CTLH E3-ZZ1-BRD4 BD1 ) resolved to 12 Å and fit with prior structures (extracted from PDB: 7NSC , 8PJN , 8QBN , 3MXF ) and AlphaFold models of the constituent GID/CTLH modules. b) Map of the YPEL5-WDR26 module-ZZ1-BRD4 BD1 ternary complex resolved to 3.4 Å and sharpened with DeepEMhancer . Close-up highlights additional electron density at the interface of YPEL5 and BRD4 BD1 corresponding to the ZZ1 degrader. c) Atomic model of the ZZ1-induced ternary complex depicting the overall YPEL5-WDR26 receptor module architecture and its mode of BRD4 BD1 engagement. d) Close-up of YPEL5 in complex with ZZ1-BRD4 BD1 overlayed with lenalidomide-bound CRBN CTD (PDB: 4TZ4 ) illustrating the structural similarity of their ligand-binding domains. Both domains are stabilized by coordination of a zinc atom and contain a central groove that binds ligands. e) YPEL5 and CRBN (PDB: 5FQD ) MGD ternary complexes have divergent modes of degrader engagement and neosubstrate positioning.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep, Ligand Binding Assay

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Flowchart of the cryo-EM data processing workflow generating the focused-refined map of the ternary complex comprising ZZ1-SO 2 H c-Glue, YPEL5-WDR26 E3 receptor module and BRD4 BD1 neosubstrate. The scale bar in the motion-corrected representative micrograph corresponds to 300 Å. b) The final post-processed map color-coded to illustrate variations in its local resolution. c) Gold-standard Fourier shell correlation (FSC) plot. The dotted line represents 0.143 cut-off criterion for estimating nominal resolution.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) In vitro ubiquitylation assays testing specificity of ZZ1-induced ubiquitylation towards bromodomains of BET protein family members that all bind the parental substrate-recruiting JQ1 handle of ZZ1. b) Superposition of the YPEL5 structure with that of the thalidomide binding domain of CRBN (CRBN CTD ) engaging an Immunomodulatory Drug (IMiD) MGD (PDB: 4TZ4). The close-up highlights CRBN residues forming the IMiD-engaging hydrophobic site (the “tri-Trp pocket”) and the corresponding YPEL5 residues (shown as sticks). Despite adopting a homologous fold, YPEL5 does not possess two out of three residues critical for IMiD binding. c) Examining ligand-binding preference of YPEL5 by performing in vitro BRD4 BD1 ubiquitylation assay with ZZ1 and CRBN-based PROTACs employing the BRD4 ligand JQ1. d) Analysis of BRD4 BD1 -eGFP degradation in K562 stability reporter cells treated with indicated compounds or compound combinations (co-treatment with equimolar mixtures).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: In Vitro, Binding Assay, Ligand Binding Assay, Ubiquitin Assay

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Structure of the ternary complex illustrating electrostatically-driven interactions between the negatively charged sulfinic acid moiety of ZZ1-SO 2 H and the basic bottom of the YPEL5 binding groove (represented as an electrostatic potential surface). b) Intact mass spectrometry demonstrates conversion of the sulfonyl fluoride moiety of ZZ1 (middle) to sulfinic acid (right) after incubation in a DTT-containing buffer. The position of the transformed group is indicated in the degrader’s chemical structure (left). Peaks corresponding to lower molecular weight species correspond to ZZ1 and ZZ1-SO 2 H derivatives formed upon hydrolysis of their tert -butyl ester. c) Stimulation of YPEL5-GID/CTLH E3-dependent in vitro ubiquitylation of *BRD4 BD1 . ZZ1, its sulfinic acid derivative, (ZZ1-SO 2 H) and its sulfonic acid derivative (ZZ1-SO 3 H) were tested. d) FP assay quantifying the propensity of ZZ1-SO 2 H to induce the ternary complex formation. Binding of *BRD4 BD1 to the YPEL5-WDR26 module upon degrader titration results in a dose-dependent increase of fluorescence polarization. Fitting polarization values to the “[agonist] vs. response” model yielded the half-maximal effective concentration (EC 50 ). e) HPLC analysis of intracellular levels of ZZ1 and its acidic metabolites. Jurkat cells were treated with 5 µM ZZ1 for 5 h.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Binding Assay, Mass Spectrometry, Incubation, Transformation Assay, Molecular Weight, In Vitro, FP Assay, Titration, Fluorescence, Concentration Assay

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Close-up of electron density (gray transparent) in the cryo-EM structure of the ternary complex (shown in ) corresponding to the ZZ1-SO 2 H chemical tag and the surrounding YPEL5 residues, along with their atomic coordinates (sticks). Absence of continuous density between the sulfinic acid and neither YPEL5 nucleophilic amino acid side chains suggests the non-covalent ZZ1 mode-of-action. b) Intact mass spectrometry analysis testing formation of potential ZZ1-induced covalent adducts between YPEL5 (within the YPEL5-WDR26 subcomplex) and BRD4 BD1 . c) Real-time FP assay probing kinetics of ternary complex formation in the DTT-containing buffer induced by ZZ1, ZZ1-SO 2 H, or ZZ1 pre-incubated in the FP buffer. Polarization signal was measured over time after combining the protein mix (*BRD4 BD1 and YPEL5-WDR26) prepared in the DTT-containing buffer with the degrader compounds. d) Schematic of the proposed mechanism of sulfonyl fluoride conversion to sulfinic acid triggered by nucleophilic thiol groups of reducing agents, such as DTT ( in vitro ) or GSH (in cells). e) Real-time FP assay testing effect of DTT on the rate of the ZZ1-SO 2 H-induced ternary complex formation. The varying DTT concentrations during the experiment were controlled by the composition of the buffer used for preparing the protein mix (*BRD4 BD1 and YPEL5-WDR26) prior to degrader addition. f) HiBiT-BRD4 assay results for Jurkat cells pre-treated with 100 µM GSH for 2 h, followed by treatment with the indicated compounds for 5 h.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep, Mass Spectrometry, FP Assay, Incubation, In Vitro

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Close-up of cryo-EM density corresponding to the YPEL5-engaging chemical tag of ZZ1-SO 2 H (compound coordinates shown as sticks). b) Molecular details of the ternary complex interface highlighting the constellation of YPEL5 residues engaging ZZ1-SO 2 H and those involved in direct interactions with BRD4 BD1 . The hydrogen bonds are depicted as gray dashes. c) In vitro ubiquitylation assay probing YPEL5 residues shown in (b) involved in: (1) anchoring the sulfinic acid moiety (T63, K95), (2) contacts with the degrader phenyl ring and BRD4 hydrophobic sidechains (L62), and (3) direct interactions with BRD4 (R41). Reactions were quenched after 30 minutes. SDS-PAGE gels were imaged by a fluorescence scan and stained with Coomassie to visually inspect YPEL5 levels (bottom). d) Cellular BRD4 degradation assay testing the structurally visualized binding mode. The impact of YPEL5 mutations on ZZ1 potency is illustrated as the ratio of DC 50 values between mutant and WT YPEL5-expressing Jurkat cells. Plots used for DC 50 measurements are presented in . e) Competitive FP assay probing cooperativity within the ZZ1-SO 2 H-induced ternary complex. BRD4 BD1 -bound fluorescent JQ1* tracer was displaced by titration of unlabeled competitors reducing fluorescence polarization. The extent of cooperativity was determined by calculating the ratio of IC 50 values (estimated by fitting polarization values to the “[inhibitor] vs. response” model) in the absence and presence of excess YPEL5-WDR26 (apparent cooperativity factor α app ).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep, In Vitro, Ubiquitin Assay, SDS Page, Fluorescence, Staining, Degradation Assay, Binding Assay, Mutagenesis, Expressing, FP Assay, Titration

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) HiBiT-BRD4 assay results for WT and mutant YPEL5-expressing Jurkat cells treated with the indicated compounds for 5 h. The plots comparing the estimated DC 50 values are presented in . b) FP assay for establishing the competitive ligand displacement experiment probing cooperative ternary complex formation . Polarization values upon BRD4 BD1 titration to the fluorescent JQ1 tracer (FAM-JQ1) were fit to the one-site binding model to estimate the BRD4 BD1 -JQ1 affinity (equilibrium dissociation constant, K D ). c) Quantitative proteome-wide mass spectrometry in MOLT-4 cells after 3 h treatment with 1 µM ZZ2. d) Intact mass spectrometry demonstrates conversion of the sulfonyl fluoride moiety of ZZ2 (left) to sulfinic acid (right) after incubation in a DTT-containing buffer. Peaks corresponding to lower molecular weight species correspond to ZZ2 and ZZ2-SO 2 H derivatives formed upon hydrolysis of their tert -butyl ester. e) Close-up of ternary complex structures induced by ZZ1-SO 2 H and ZZ2-SO 2 H highlighting their common binding mode. YPEL5 and BRD4 residues involved in protein-protein and/or protein-degrader contacts are shown as sticks. Spheres represent water molecules, while dashes denote hydrogen bonds.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Mutagenesis, Expressing, FP Assay, Titration, Binding Assay, Mass Spectrometry, Incubation, Molecular Weight

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Electrostatic potential surface of the ZZ1-SO 2 H-bound YPEL5 groove showcasing a vacant basic pocket (outlined with a red dash) adjacent to the unsubstituted position of the degrader’s chemical tag (indicated by an arrow). b) Chemical structure of ZZ2 highlighting the incorporated chloro group (red) at the second ortho -position of ZZ1’s chemical tag. c) HiBiT-BRD4 assay results for Jurkat cells treated with the indicated compounds for 5 h. d) Qualitative comparison of ZZ1-SO 2 H and ZZ2-SO 2 H ability to trigger in vitro YPEL5-GID/CTLH E3-catalyzed *BRD4 BD1 ubiquitylation. ZZ2-SO 2 H was generated by pre-incubation of ZZ2 in the DTT-containing buffer . e) FP assay quantifying propensity of ZZ1-SO 2 H and ZZ2-SO 2 H to promote ternary complex formation. Note that polarization values obtained upon ZZ2-SO 2 H titration fit the “[agonist] vs. response” model but its superior MGD activity precludes accurate estimation of EC 50 . f) Real-time FP assay testing rates of ternary complex formation triggered by ZZ1 and ZZ2 as well as their acidic derivatives. Fluorescence polarization was monitored over time upon mixing *BRD4 BD1 and YPEL5-WDR26 with different versions of the degraders. g) Close-up of the ZZ2-SO 2 H-induced ternary complex cryo-EM structure, resolved to 3.4 Å and sharpened with DeepEMhancer. The images highlight the overall fit of the degrader’s chemical tag (left) and the filling of the vacant YPEL5 basic pocket by the introduced chloro group according to the structure-based design (right).

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Comparison, In Vitro, Generated, Incubation, FP Assay, Titration, Activity Assay, Fluorescence, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display

doi: 10.1101/2024.09.24.614843

Figure Lengend Snippet: a) Flowchart of the cryo-EM data processing workflow generating the focused-refined map of the ternary complex comprising the improved ZZ2-SO 2 H c-Glue, YPEL5-WDR26 E3 receptor module and BRD4 BD1 neosubstrate. The scale bar in the motion-corrected representative micrograph corresponds to 300 Å. b) The final post-processed map color-coded to illustrate variations in its local resolution. c) Gold-standard Fourier shell correlation (FSC) plot. The dotted line represents 0.143 cut-off criterion for estimating nominal resolution.

Article Snippet: The cDNA of full-length BRD4 was a gift from Peter Howley (Addgene #14447; http://n2t.net/addgene:14447 ; RRID:Addgene_14447), whereas those of BRD2 and BRD3 were acquired from an in-house human cDNA library (Max Planck Institute of Biochemistry).

Techniques: Cryo-EM Sample Prep

Journal: Molecular Biology of the Cell

Article Title: Interplay of condensation and chromatin binding underlies BRD4 targeting

doi: 10.1091/mbc.e24-01-0046

Figure Lengend Snippet: FIGURE 1: Chromatin binding influences BRD4 condensate distribution in living cells. (A) Schematic of BRD4 full-length (FL) and deltaN-terminus (∆N) constructs, as well as PONDR predicted disorder score. (B) From top: Immunofluorescence of endogenous BRD4 protein in cultured human U2OS cells without and with addition of 1 µM JQ1. Exogenous live expression of BRD4FL-mCherry and BRD4∆N-mCherry in the same cell before (−JQ1) and after (+JQ1) addition of 1 µM JQ1. (C) Panel of images of U2OS cells with increasing expression level of BRD4FL-mCherry (same cell −/+ JQ1) or BRD4∆N-mCherry at similar expression levels. (D) Quantification of number and size of BRD4 condensates from immunofluorescence of endogenous, or live expression of BRD4FL-mCherry or BRD4∆N-mCherry, −/+ JQ1. Points represent averages of three biological replicates of 25 cells each within the expression level gate defined in 1C, error bars SEM. Statistical test one-way ANOVA, ****p < 0.0001. (E) Estimated condensate volume measured in the same set of cells expressing BRD4FL-mCherry before (x-axis) and after (y-axis) disruption of chromatin binding through addition of 1 µM JQ1. If condensate volume is not affected, points should lie on the diagonal, indicated by shading.

Article Snippet: The full-length BRD4 DNA fragment was amplified by PCR from pcDNA4-TO-HA-BRD4FL (Addgene plasmid #31351) (Rahman et al., 2011) incorporated into linearized FM5 lentiviral vectors containing 12 | A. R. Strom et al. Molecular Biology of the Cell standardized linkers (generously provided by David Sanders) using the In-Fusion HD cloning kit (Takara Bio, 638910).

Techniques: Binding Assay, Construct, Immunofluorescence, Cell Culture, Expressing, Disruption

Journal: Molecular Biology of the Cell

Article Title: Interplay of condensation and chromatin binding underlies BRD4 targeting

doi: 10.1091/mbc.e24-01-0046

Figure Lengend Snippet: FIGURE 2: Thermodynamic effects of BRD4 chromatin binding are measured the Corelet synthetic oligomerization system. Representative images of BRD4FL Corelets before and after light activation of a nucleus without (A) or with (B) 1µM JQ1. (C) Quantification of the number and size of BRD4FL Corelet condensates per nucleus induced with light activation −/+ JQ1 in the same set of nuclei. Error bars represent SEM across four trials of 25 cells each, Student’s t test ***p = 0.0002 in Count, *** p = 0.001 in Area. (D) Condensate volume comparison in −/+JQ1 conditions in the same set of cells with BRD4FL (pink) or BRD4∆N (black) Corelets. BRD4∆N Corelet condensate volume is unaffected by the addition of JQ1, as can be seen from points largely along the diagonal (shaded pink), while BRD4FL Corelet condensate volume is much lower after addition of JQ1. Lines are linear fit. (E, F) Phase diagram and logistic regression of BRD4FL measured with the Corelet system in the absence (E) and presence (F) of JQ1. Phase diagrams are constructed from the same cells expressing BRD4FL −/+ JQ1 demonstrating the shifted valence between −JQ1 (gray) and +JQ1 (pink).

Article Snippet: The full-length BRD4 DNA fragment was amplified by PCR from pcDNA4-TO-HA-BRD4FL (Addgene plasmid #31351) (Rahman et al., 2011) incorporated into linearized FM5 lentiviral vectors containing 12 | A. R. Strom et al. Molecular Biology of the Cell standardized linkers (generously provided by David Sanders) using the In-Fusion HD cloning kit (Takara Bio, 638910).

Techniques: Binding Assay, Activation Assay, Comparison, Construct, Expressing

Journal: Molecular Biology of the Cell

Article Title: Interplay of condensation and chromatin binding underlies BRD4 targeting

doi: 10.1091/mbc.e24-01-0046

Figure Lengend Snippet: FIGURE 3: A coarse-grained model for simulating BRD4 Corelet condensation. (A) Coarse-grained simulations contain representations of the chromatin polymer (blue) and Corelet oligomerization platform (gold) with attached BRD4 molecules, each composed of two spheres representing the N-terminus (capable of interacting with chromatin) and C-terminus (capable of self-interaction). (B) The valence-dependent phase diagram in the absence of chromatin, obtained via direct-coexistence simulation (representative snapshot on left), shows a critical valence-1 of 1/6. C. A phase diagram showing the presence of chromatin-Corelet condensates in simulations with strong chromatin binding (40% acetylated histone tails) predicts an apparent critical valence-1 of 1/2. Representative snapshots are shown (on right) for the indicated simulation conditions. Related simulation data at lower acetylation levels are provided in Supplemental Figure S2F.

Article Snippet: The full-length BRD4 DNA fragment was amplified by PCR from pcDNA4-TO-HA-BRD4FL (Addgene plasmid #31351) (Rahman et al., 2011) incorporated into linearized FM5 lentiviral vectors containing 12 | A. R. Strom et al. Molecular Biology of the Cell standardized linkers (generously provided by David Sanders) using the In-Fusion HD cloning kit (Takara Bio, 638910).

Techniques: Polymer, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: Interplay of condensation and chromatin binding underlies BRD4 targeting

doi: 10.1091/mbc.e24-01-0046

Figure Lengend Snippet: FIGURE 4: BRD4 condensate nucleation is seeded on chromatin. (A) Schematic of two modes of condensate nucleation: substrate-seeded (heterogeneous) and not seeded (homogeneous). Without seeding, the free energy of protein clustering increases until the cluster reaches its critical radius (rcrit), at which point it becomes energetically favorable to grow. A seed can lower the energetic barrier to reach rcrit. (B) Seeded nucleation is expected to have a shorter delay time before droplet formation, and increased nucleation rate (slope) compared with nonseeded nucleation. (C) Quantification of the number of condensates nucleated over time in the same cell before and after JQ1 treatment demonstrates the expected changes in delay time and slope. (D) Representative images of a 4 by 4 micron square nuclear area as light-induced BRD4FL-mCh-sspB Corelet condensates nucleate rapidly in untreated conditions (2 s, top), but are delayed in the same cell after JQ1 treatment (10 s, bottom). (E) Quantification of the nucleation rate (slope), delay time, and number density for 9 cells of similar expression level before and after JQ1 treatment. (F) Repeated activation-deactivation cycles of BRD4∆N and overlay of condensate positions in subsequent cycles shows whether nucleation occurs repeatedly in the same nuclear locations. (G) Overlay of condensate positions in subsequent cycles of activation for BRD4FL −/+ JQ1. (H) PCC quantification and difference in PCC (deltaPCC) of overlaid images of 33 cells in subsequent activation cycles. Negative controls are PCCs between images of different cells. ***p = 0.0015 by Mann– Whitney exact, two-tailed t test. (I) Short-term (2 min) 200 nM JQ1 treatment disperses BRD4 condensates (JQ1), yet they form again quickly after washing out JQ1 (Recovery). Segmented masks of identified BRD4 puncta within nuclear outline, overlay of JQ1 and recovery timepoints (green) with pretreatment condensate positions (magenta). (J) Quantification of condensate dissolution during JQ1 treatment (shaded area) and recovery after washout. Error bars represent SD of three biological trials of 10 cells each. (K) Overlay of images before/during JQ1 treatment and before treatment/after JQ1 washout (left). PCC of 30 cells before/during JQ1, and before treatment/after JQ1 washout.

Article Snippet: The full-length BRD4 DNA fragment was amplified by PCR from pcDNA4-TO-HA-BRD4FL (Addgene plasmid #31351) (Rahman et al., 2011) incorporated into linearized FM5 lentiviral vectors containing 12 | A. R. Strom et al. Molecular Biology of the Cell standardized linkers (generously provided by David Sanders) using the In-Fusion HD cloning kit (Takara Bio, 638910).

Techniques: Expressing, Activation Assay, MANN-WHITNEY, Two Tailed Test, Dissolution

Journal: Molecular Biology of the Cell

Article Title: Interplay of condensation and chromatin binding underlies BRD4 targeting

doi: 10.1091/mbc.e24-01-0046

Figure Lengend Snippet: FIGURE 5: Simulations of BRD4FL Corelet condensation quantify enhancement of chromatin-seeded nucleation. (A) Schematic of a typical nucleation event. τnucl is the time required for cluster size to reach the threshold to form a stable nucleus (red line). τdelay is the time required for the nucleated cluster to grow via diffusion-limited growth to reach a size that is observable under the microscope (green line), which is estimated to be 15 times larger than the stable nucleus size in our simulations. (B) Example of a heterogeneous (on-chromatin) nucleation event, in which the largest cluster (highlighted in red) interacts with the chromatin polymer. (C) Example of a homogeneous (off-chromatin) nucleation event. (D) Probability of on-chromatin (orange) or off-chromatin (blue) nucleation events for endogenous (top) and Corelet (bottom) simulations at two BRD4-histone tail interaction strengths (representing +JQ1 and −JQ1), and four acetylation fractions (0.1, 0.2, 0.3, 0.4). (E) Quantification of the nucleation rate in endogenous and Corelet simulations with (−JQ1) and without (+JQ1) strong BRD4-histone tail interactions. (F) Quantification of the delay time before observable condensates are formed in endogenous and Corelet simulation systems with (−JQ1) and without (+JQ1) strong BRD4-histone tail interactions. (E, F) Seven independent simulations for each measurement are run at an acetylation fraction of 0.4 for both +JQ1 and −JQ1. In the +JQ1 Corelet simulations, only two out of seven simulations achieved nucleation. Error bars represent the SE. ***p ≤ 0.001, ****p ≤ 0.0001 by Student’s t test.

Article Snippet: The full-length BRD4 DNA fragment was amplified by PCR from pcDNA4-TO-HA-BRD4FL (Addgene plasmid #31351) (Rahman et al., 2011) incorporated into linearized FM5 lentiviral vectors containing 12 | A. R. Strom et al. Molecular Biology of the Cell standardized linkers (generously provided by David Sanders) using the In-Fusion HD cloning kit (Takara Bio, 638910).

Techniques: Diffusion-based Assay, Microscopy, Polymer

Journal: Molecular Biology of the Cell

Article Title: Interplay of condensation and chromatin binding underlies BRD4 targeting

doi: 10.1091/mbc.e24-01-0046

Figure Lengend Snippet: FIGURE 6: Epigenetic acetylation level influences nucleation behaviors in endogenous and exogenous BRD4 systems. (A) Representative images and analysis examples of endogenous BRD4 puncta by immunofluorescence in cells treated with control media (Control), histone acetyltransferase inhibitor (A485), or histone deacetylase inhibitor (TSA). (B, C) Quantification of endogenous BRD4 condensate count per nucleus (B) and condensate average area (C) measured by immunofluorescence in drug-treated cells. n = 25 cells each, error is SD. ***p = 0.002, ****p = 0.0001 by one-way ANOVA. D. Representative images of BRD4FL-mCh puncta in living cells treated with control media, A485, or TSA at three timepoints: before addition of JQ1 (−JQ1), during JQ1 incubation (+JQ1) and after JQ1 washout and recovery. (E–F) Quantification of the number of condensates per nucleus in drug-treated cells before addition of JQ1 (E) and after washout and recovery (F). Expression range as defined in Supplemental Figure S4, D and E. n = 11, 11, 12 cells, error is SD, *p = 0.031, **p = 0.008 by one-way ANOVA. (G) Timecourse graph of the number of BRD4FL-mCh puncta per nucleus during JQ1 treatment (shaded area) and nucleation after washout. (H) Nucleation rate of BRD4FL-mCh puncta measured as slope after JQ1 washout. N = 11, 11, 12 for A485, control, TSA respectively. **p = 0.006, ****p = 0.001. (I) Model figure summarizing findings about on- and off-chromatin BRD4 nucleation.

Article Snippet: The full-length BRD4 DNA fragment was amplified by PCR from pcDNA4-TO-HA-BRD4FL (Addgene plasmid #31351) (Rahman et al., 2011) incorporated into linearized FM5 lentiviral vectors containing 12 | A. R. Strom et al. Molecular Biology of the Cell standardized linkers (generously provided by David Sanders) using the In-Fusion HD cloning kit (Takara Bio, 638910).

Techniques: Immunofluorescence, Control, Histone Deacetylase Assay, Incubation, Expressing

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 1. BRD4 NCC loss of function produces severe craniofacial phenotypes.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 2. BRD4 mutant mandibular cNCCs fail to properly differentiate to osteoblast lineages.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: Mutagenesis

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 3. Loss of BRD4 disrupts in vitro cNCCs osteoblast differentiation.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: In Vitro

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 4. BRD4 binds to proximal active enhancers to regulate osteogenic transcription.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 5. BRD4 directly regulates transcription of factors critical for osteoblast differentiation.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 6. BRD4 associates with RUNX2 to regulate osteoblast differentiation

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 7. Model of BRD4 function in CdLS craniofacial pathogenesis (created with

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation

doi: 10.1242/dev.202110

Figure Lengend Snippet: BRD4 NCC loss of function produces severe craniofacial phenotypes. (A) Schematic of BRD4 protein with locations of bromodomains (Bromo), extraterminal domain (ET), exon 5 coding sequence that is removed by Cre/LoxP and amino acid Y432 where point mutation produces Cornelia de Lange syndrome. (B-I) Lateral and side view images of E18.5 wild-type (WT) (B,C), Brd4 cW1KO (D,E), more severe Brd4 cS10KO (F,G) and less severe Brd4 cS10KO (H,I) embryos highlighting exencephaly (red arrows), mid-facial clefting (blue arrows) and open eye phenotypes (yellow arrows). All Brd4 cW1KO and Brd4 cS10KO embryos demonstrate severe anterior facial hypoplasia with smaller frontal, nasal and mandible regions. (J) Summary of Brd4 cW1KO and Brd4 cS10KO phenotypic frequencies ( N =5 and N =24, respectively). (K-P) Alizarin Red and Alcian Blue stain of bone and cartilage wholemount images of E18.5 WT and Brd4 cS10KO embryos with ventral wholemount view (K,L), dissected mandible (M,N) and ventral cranial base view (O,P) highlighting micrognathia (white arrows), basisphenoid bone (aqua arrows) and presphenoid bone formation (black arrow). Scale bars: 5 mm (K,L); 2 mm (M,N); 3 mm (O,P).

Article Snippet: GST-tagged human BRD4 (Addgene plasmid #14447 ) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: Sequencing, Mutagenesis, Staining

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation

doi: 10.1242/dev.202110

Figure Lengend Snippet: BRD4 mutant mandibular cNCCs fail to properly differentiate to osteoblast lineages. (A-D) Brightfield and Rosa Tomato reporter fluorescence wholemount imaging of E11.5 Brd4 cS10Het control and Brd4 cS10KO embryos had similar Rosa Tomato + cNCC domains. Dashed white line in A depicts sectioning region for immunofluorescence at E11.5 in E-J. (E-J) Immunofluorescence within coronal sections of the E11.5 wild-type (WT) and Brd4 cS10KO first branchial arch for activated cleaved Caspase-3 (Casp.; E,F), bromodeoxyuridine (BrdU; G,H) incorporation and phosphorylated histone H3 serine 10 (pH3S10; I,J) along with DAPI (blue) demonstrated normal proliferation and lack of apoptosis in Brd4 cS10KO embryos. (K-N) Brightfield and Rosa Tomato reporter fluorescence at E13.5 illustrated loss of cNCC domains in Brd4 cS10KO embryos relative to Brd4 cS10Het controls. Dashed white line in K depicts sectioning region for immunofluorescence at E13.5 in O-T. (O-T) Immunofluorescence within coronal sections of the E13.5 WT and Brd4 cS10KO developing mandible for BRD4 (O,P), RUNX2 with type II collagen (COL2; Q,R), and RUNX2 with Osterix (OSX; S,T). Brd4 cS10KO RUNX2 + pre-osteoblasts fail to induce Osterix expression. All immunofluorescence images are overlaid with DAPI nuclear stain (blue). Scale bars: 1 mm (A-D,K-N); 200 µm (E-J,O-T).

Article Snippet: GST-tagged human BRD4 (Addgene plasmid #14447 ) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: Mutagenesis, Fluorescence, Imaging, Control, Immunofluorescence, Expressing, Staining

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation

doi: 10.1242/dev.202110

Figure Lengend Snippet: Loss of BRD4 disrupts in vitro cNCCs osteoblast differentiation. (A-D) BRD4 immunofluorescence in wild-type (WT), hypomorphic ( Brd4 hypo ) with trans-heterozygous frameshift (fs) mutations in exon3, or knockout ( Brd4 KO1 or Brd4 KO2 ) cNCC cell lines with trans-heterozygous fs mutations in exons 3 and 5. DAPI nuclear stain shown in blue. (E) Western blot of BRD4 hypomorphic and knockout cNCC lines demonstrated loss of BRD4 relative to nucleolin (NLN) loading control. (F) Western blot of osteochondral transcription factors SOX9 and RUNX2 are unaltered in BRD4 knockout cNCCs relative to NLN loading control. (G) Flow cytometry histogram of Cell Trace Far Red (CTFR) tracking dye demonstrated gradual dilution as WT cNCCs proliferate across 5 days of growth. (H,I) Cells labeled with similar levels of CTFR dye at onset (H) revealed slightly slower proliferation rates for BRD4 hypomorphic and BRD4 knockout cNCC lines compared with WT at day (D)3 of growth (I). (J-L) At D7 of osteogenic differentiation, compared with WT, Brd4 KO1 and Brd4 KO2 lines lack detectable alkaline phosphatase activity (Alk. Phos.). (M-O) At D10 of osteogenic differentiation, WT cNCCs exhibited robust alkaline phosphatase activity that was diminished in Brd4 KO1 and lost in Brd4 KO2 . (P-S) At D7 of differentiation, WT first branchial arch primary cNCCs (WT BA D7) exhibit similar alkaline phosphatase activity as O9-1 cell culture (WT cNCC D7); however, Brd4 cS10KO primary cNCCs (BA D7) fail to differentiate (S). Scale bars: 10 µm (A-D); 2 mm (J-O); 1 mm (P-S).

Article Snippet: GST-tagged human BRD4 (Addgene plasmid #14447 ) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: In Vitro, Immunofluorescence, Knock-Out, Staining, Western Blot, Control, Flow Cytometry, Labeling, Activity Assay, Cell Culture

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation

doi: 10.1242/dev.202110

Figure Lengend Snippet: BRD4 binds to proximal active enhancers to regulate osteogenic transcription. (A) Venn diagram plots of significantly altered (logFC≥1 or≤−1) expressed genes (WT RPKM≥1) from Brd4 KO1 or Brd4 KO2 compared with wild-type (WT) in day (D)0 undifferentiated cNCCs or at D3 and D6 of osteogenic differentiation. Both Brd4 KO1 and Brd4 KO2 lines demonstrated overlap of upregulated and downregulated genes. (B) UCSC genome browser tracks of BRD4 binding in ESCs (blue), BRD4 binding in D0 undifferentiated cNCCs or at D3 and D6 of osteogenic differentiation in WT (purple) or Brd4 KO2 (KO, black) cells. Also illustrated are enhancer histone modifications including H3K27ac (pink) in WT D0 undifferentiated cNCCs or at D3 and D6 of osteogenic differentiation and H3K4me2 accumulation (green) in WT D0 undifferentiated cNCCs. RUNX2 binding (red) in WT D0 cNCC, at D3 of osteogenic differentiation and MC3T3 pre-osteoblasts highlighted RUNX2 enrichment [osterix ( Sp7 ) and Adamts4 ] at BRD4 sites, reduction in Brd4 KO2 (D0 KO) and absence in ESC controls (black). H3K27me3 accumulation (red) in WT D0 undifferentiated cNCCs illustrated repressive chromatin regions. Gene loci of interest are osterix, Col1a1 , Adamts4 , Malat1 , and Hoxb gene loci. BRD4 is bound (red arrows) to active enhancers of target genes featuring high levels of H3K27ac, H3K4me2 and RUNX2 binding.

Article Snippet: GST-tagged human BRD4 (Addgene plasmid #14447 ) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: Binding Assay

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation

doi: 10.1242/dev.202110

Figure Lengend Snippet: BRD4 directly regulates transcription of factors that are crucial for osteoblast differentiation. (A) MSigDB canonical pathways that overlap with the top 500 day (D)3 and D6 osteogenic BRD4 targets ( Tables S2,S3, sheet 2). (B) MSigDB human phenotype ontogeny pathways that overlap with the top 500 D3 and D6 osteogenic BRD4 targets ( Tables S2,S3 , sheet 2). (C) Key for volcano plots in D,E. Significantly upregulated genes are colored green. Significantly downregulated genes are colored red if they do not change across osteogenic differentiation, blue if they increase in wild-type (WT) expression at D3 of osteogenic differentiation, purple if they increase in WT expression at D6 of osteogenic differentiation. BRD4 directly bound, downregulated targets have filled circles. (D) Volcano plot of log 2 fold change versus −log 10 false discovery rate (FDR) comparing D3 osteogenic WT expression with Brd4 KO2 . Only gene sets in common with Brd4 KO1 are color coded. (E) Volcano plot of log 2 fold change versus −log 10 false discovery rate comparing D6 osteogenic WT expression with Brd4 KO2 . Only gene sets in common with Brd4 KO1 are color coded. In D,E, BRD4 binds directly to regulate expression of large sets of genes that are crucial for osteoblast differentiation. (F) Numbers of BRD4 bound enhancers versus promoters for target genes ( Tables S1-S3 , sheet 2). (G) Comparison of super-enhancer frequency for BRD4 bound enhancers compared with annotated enhancers lacking BRD4 binding. (H) BRD4 downregulated direct targets at D3 and D6 of osteogenic differentiation ( Tables S2,S3 , sheet 2) were compared for overlap with downregulated genes in E13.5 Brd4 cS10KO embryonic cNCCs ( Table S4 ) and charted as percentage overlap. (I-L) Immunofluorescence on coronal sections of the E13.5 WT and Brd4 cS10KO developing mandible for RUNX2 with type I collagen (COL1A1; I,J), or FGFR2 (K,L) along with DAPI (blue). Brd4 cS10KO RUNX2 + pre-osteoblasts fail to induce COL1A1 and FGFR2 expression. Scale bars: 200 μm.

Article Snippet: GST-tagged human BRD4 (Addgene plasmid #14447 ) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: Expressing, Comparison, Binding Assay, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation

doi: 10.1242/dev.202110

Figure Lengend Snippet: BRD4 associates with RUNX2 to regulate osteoblast differentiation. (A) Enrichment of DNA transcription factor binding motifs were analyzed at day (D)3 or D6 BRD4 bound target osteogenic enhancers ( Tables S2,S3 , sheet 4) using the HOMER findMotifsGenome.pl program. RUNX2 motifs were enriched at both time points compared with BRD4 unbound enhancers. (B) D3 RUNX2 CUT&RUN relative read density was plotted at BRD4 stem cell enhancers ( Table S1 , sheet 4), D3 BRD4 osteogenic enhancers ( Table S2 , sheet 4), D6 osteogenic enhancers ( Table S3 , sheet 4) or enhancers not bound by BRD4. RUNX2 demonstrated enrichment at BRD4 osteogenic and stem cell enhancers. (C) Profile of counts per million mapped reads (CPM) normalized RUNX2 enrichment in wild-type (WT; blue) or Brd4 KO2 cells (red) at D0 stem cell enhancers (left) or D3 BRD4 osteogenic enhancers (right). CUT&RUN for RUNX2 in ESCs (black) served as a negative control due to lack of expression in these stem cells. (D) Structure of BRD4 protein with reference to short or long isoforms. (E) Flag-tagged BRD4 constructs were co-transfected into HEK293T with HA-tagged RUNX2 followed by immunoprecipitation (IP) on Flag antibody conjugated beads. The C-terminus of BRD4 encoded by the long isoform is responsible for protein IP of RUNX2. (F) Lentiviral transduction of the human BRD4 long isoform was capable of restoring Brd4 KO2 osteoblast differentiation after 10 days of differentiation, whereas BRD4 short isoform was not capable of supporting differentiation. Images depict alkaline phosphatase (Alk. Phos.) activity on substrate colorimetric reaction. (G) Lentiviral transduction and overexpression of RUNX2 was capable of restoring Brd4 KO2 osteoblast differentiation after 10 days of differentiation. Scale bars: 2 mm.

Article Snippet: GST-tagged human BRD4 (Addgene plasmid #14447 ) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: Binding Assay, Negative Control, Expressing, Construct, Transfection, Immunoprecipitation, Transduction, Activity Assay, Over Expression

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation

doi: 10.1242/dev.202110

Figure Lengend Snippet: Model of BRD4 function in Cornelia de Lange syndrome craniofacial pathogenesis. Our results indicate that BRD4 binds to enhancers to induce transcription of osteogenic genes and proper cNCC osteoblast differentiation. BRD4 is required for efficient RUNX2 recruitment to drive appropriate expression of the RUNX2 transcriptional program during osteogenic differentiation. Although BRD4 also associates with the NIPBL cohesin loading protein, the predominantly mutated factor in CdLS, the role of this association in osteogenic enhancer activity and craniofacial development is unknown. Figure created with BioRender.

Article Snippet: GST-tagged human BRD4 (Addgene plasmid #14447 ) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: Expressing, Activity Assay