pgex-6p-1 gst-rh-mnase  (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: