Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet:

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet: The BRD4 tandem bromodomains stably associate with H4 poly-acetylated nucleosomes in biolayer interferometry. ( A-D ) Biolayer interferometry binding assays of ( A ) BD1-2 with H4 poly-acetylated nucleosomes ([H4K5acK8acK12acK16ac] 2 ), ( B ) BD1-2 with unacetylated nucleosomes, ( C ) BD1 alone with H4 poly-acetylated nucleosomes, and ( D ) BD1 alone with unacetylated nucleosomes. Protein concentrations are indicated at the right of each plot. Note the difference in y-axis scale between ( A ) 1 to -0.5 nm and ( B-D ) 0.3 to -0.3 nm. Octet fitting of BD1-2 binding curves to H4 poly-acetylated nucleosomes (12.5, 6.25, and 3.12 μM) using a 2:1 heterogeneous ligand model produced R 2 values >0.98 (Fig. S2). Experiments were performed in triplicate.

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Stable Transfection, Binding Assay, Produced

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet: AlphaFold model suggesting a plausible mode of BRD4-S engagement with an H4 K12 acetylated nucleosome ([H4K12ac] 2 ). ( A ) Top-ranked AlphaFold model prediction of BRD4-S (blue) in complex with an H4K12ac nucleosome (H2A, golden; H2B, tomato; H3, green; H4, violet, Widom 601 DNA, grey). BD1 and BD2 are shaded in darker blue for clarity. ( B ) Rotated (45°) view of the modeled BRD4-S-H4K12ac nucleosome complex. ( C ) Zoom-in of the BD1 acetyl-lysine binding pocket highlighting residues Y97 and N140 positioned near H4K12ac. ( D ) Zoom-in of the BD2 acetyl-lysine binding pocket highlighting residues Y390 and N433 positioned near H4K12ac. This view also shows the relevant placement of the C-terminal BRD4-S region including the BID, ET, and phosphoregulatory sites, in the model.

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

doi: 10.64898/2026.04.22.719657

Figure Lengend Snippet: Bromodomain binding pocket mutations in the BRD4-S disrupt breast cancer growth and migration phenotypes. ( A ) Cell growth assay following BRD4-S depletion and complementation with the indicated 3xFLAG-BRD4-S constructs (mean ± SEM; n≥5 independent experiments). Statistical analysis as described in Methods. ( B ) Transwell migration assay following BRD4-S depletion and complementation with the indicated 3xFLAG-BRD4-S constructs (mean ± SEM; n ≥ 3 independent experiments). Statistical significance was calculated by two-tailed t-tests (***p<0.0005, ****p<0.0001). ( C ) Western blots of endogenous BRD4-S depletion and rescue by 3xFLAG-tagged BRD4-S (1-719) constructs in doxycycline-treated MDA-MB-231 cells. Blots were probed with an antibody raised for the N-terminus of BRD4 (BRD4-S; for endogenous ∼100 kDa), M2 FLAG (FLAG), and β-actin (Actin) ( D ) Graphical illustration of putative model for BRD4-S chromatin engagement and regulation of cancer cell growth and migration (BioRender, https://BioRender.com/7dfa8yg ). This model shows multiple possible binding modes of BRD4-S on poly-acetylated nucleosomes that would be difficult to distinguish or determine in biochemical and structural studies, yet could all contribute to recruitment of regulatory complexes and transcriptional control of nearby genes including those for cancer growth and migration.

Article Snippet: Luminex nucleosome panels consisted of the following 601 147 bp DNA-biotinylated nucleosomes (all from EpiCypher): unmodified (rNuc), 16-0006; H4K12 acetylated ([H4K12ac] 2 ), 16-0312; H4 poly-acetylated ([H4K5acK8acK12acK16ac] 2 ), 16-0313; H3 poly-acetylated ([H3K4acK9acK14acK18ac] 2 ), 16-0336; H4 N-terminal tail truncated ([H4ΔN15] 2 ), 16-0018; and trypsin-digested tailless, 16-0027.

Techniques: Binding Assay, Migration, Growth Assay, Construct, Transwell Migration Assay, Two Tailed Test, Western Blot, Control

Journal: bioRxiv

Article Title: Mitochondrial uracil DNA glycosylase contributes to nuclear base excision repair

doi: 10.64898/2026.04.30.721890

Figure Lengend Snippet: A) Schematic of the UNG locus in the human genome and the regions targeted for disruption with CRISPR/Cas9. B) Validation of UNG knockouts in 293T cells by immunoblotting and in vitro uracil excision assay [S, uracil-containing 43-mer ssDNA substrate; P, cleaved 30-mer ssDNA product; (-) = HED buffer only (negative control); (+) = 5 Units of E. coli uracil-DNA glycosylase (positive control for uracil excision)]. C) Fluorescence microscopy showing that UNG1/2 double-KO is necessary for full inhibition of nuclear UNG activity (scale=200μm; corresponding flow analysis in Supplementary Figure S4C ).

Article Snippet: Negative and positive controls with HED buffer and recombinant E. coli uracil-DNA glycosylase (New England Biolabs M0280S, lot 10272153), respectively, were included.

Techniques: Disruption, CRISPR, Biomarker Discovery, Western Blot, In Vitro, Excision Assay, Negative Control, Positive Control, Fluorescence, Microscopy, Inhibition, Activity Assay