Journal: PLoS ONE

Article Title: HoxA-11 and FOXO1A Cooperate to Regulate Decidual Prolactin Expression: Towards Inferring the Core Transcriptional Regulators of Decidual Genes

doi: 10.1371/journal.pone.0006845

Figure Lengend Snippet: Chromatin immunoprecipiation was performed in differentiated hESC with either a control IgG, or antibodies to FOXO1A, p300, Pol-II, C/EBPβ, HoxA-11 or YY1. Results are shown as fold enrichment over IgG (after normalization to inputs). n = 6 replicate experiments, means±SEM. *, P<0.05; ***, P<0.001 (t-test compared to IgG).

Article Snippet: The nuclear lysate was precleared with protein G magnetic beads and incubated over night at 4°C with protein G magnetic beads, and 2 µg of either the antibodies against FOXO1A, p300, Pol-II, CEBPβ, HoxA-11 and Y11, or with the rabbit IgG as negative control (all from Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Nuclear S6K1 Enhances Oncogenic Wnt Signaling by Inducing Wnt/β-Catenin Transcriptional Complex Formation

doi: 10.3390/ijms232416143

Figure Lengend Snippet: S6K1 affects the formation of the Wnt/β-catenin transcriptional complex. ( a , b ) S6K1 knockdown reduces the interaction between β-catenin and TCF4. Co-immunoprecipitation assay was performed using TCF4 ( a ) and β-catenin ( b ) antibodies and 293T cells treated with LiCl (25 mM) for 8 h after transient transfection with S6K1 siRNA (48 h). ( c , d ) Inhibition of S6K1 disrupts formation of Wnt/β-catenin transcriptional complex. Each co-immunoprecipitation assay was performed with nuclear extracts from 293T cells treated with DMSO or PF-4708671 (10 µM) followed by LiCl treatment for 8 h, using Pygo2 ( c ) and p300 ( d ) antibody, respectively.

Article Snippet: Anti-p70 S6K1 (Santa Cruz Biotechnology, mouse SC-8418, rabbit SC-230, Dallas, TX, USA and Cell Signaling Technology, rabbit 9202, Danvers, MA, USA), anti-phospho-p70 S6K1 T389 (Cell Signaling Technology, rabbit 9205), anti-Flag (Sigma-Aldrich, F3165, Darmstadt, Germany), anti-c-Myc (Santa Cruz Biotechnology, SC-40), anti-β-catenin (Santa Cruz Biotechnology, mouse SC-7963 and rabbit SC-7199, and BD Transduction Laboratories, 610153, Franklin Lakes, NJ, USA), anti-acetyl-β-catenin K49 (Cell Signaling Technology, rabbit 9030, 9534), anti-TCF4 (Santa Cruz Biotechnology, mouse SC-166699, rabbit SC-13027), anti-BCL9 (Cell Signaling Technology, rabbit 15096), anti-Pygopus2 (Santa Cruz Biotechnology, mouse SC-390506), anti-p300 (Santa Cruz Biotechnology, mouse SC-48343 and rabbit SC-384), anti-cyclin D1 (Santa Cruz Biotechnology, SC-8396), anti-S6 (Cell Signaling Technology, rabbit 2217), anti-histone H3 (Santa Cruz Biotechnology, rabbit SC-10809), anti-lamin A/C (Cell Signaling Technology, rabbit 2032), anti-α-tubulin (Santa Cruz Biotechnology, SC-32293), anti-actin (Merck Millipore, Mab-1501, Temecula, CA, USA), and anti-IgG (Santa Cruz Biotechnology, mouse SC-2025 and rabbit SC-2027) were used.

Techniques: Co-Immunoprecipitation Assay, Transfection, Inhibition

Journal: Nature Communications

Article Title: Structural insights into p300 regulation and acetylation-dependent genome organisation

doi: 10.1038/s41467-022-35375-2

Figure Lengend Snippet: a Domain structure of p300 and b BRD4-NUT constructs. c IUPred, prediction of intrinsic disorder; FOLD, folding prediction using the foldindex (blue), PLAAC (Prion-Like Amino Acid Composition; orange) and PAPA (Prion Aggregation Prediction Algorithm; grey) analysis, NCPR (Net Charge Per Residue) with a sliding window of ten residues of the BRD4-NUT protein. d SEC-MALLS analysis of the TAZ2 domain of p300 alone (black), NUT4 alone (blue) and the complex between TAZ2 and NUT4 (red). SDS-PAGE analysis of the peak fractions of the TAZ2-NUT4 complex is shown. The experiment was repeated twice with consistency. e Competition analysis: Cos7 cells were transfected with constructs expressing GFP-BRD4-NUT (green) alone or co-transfected with RFP-NUT-F1c and RFP-NUT4 (red). Immunofluorescence with anti-p300 antibody was used to visualize endogenous p300. Scale bars, 10 μm. The experiment was repeated three times with consistency.

Article Snippet: After saturation of the membrane in 1X PBS (#14200-067, Gibco), 0.2% Tween 20 (P7549, Sigma) and 5% milk (Regilait) or Bovine albumin Serum (BSA, #04-100-812-C, Avantor) during 1 h at RT, membranes were incubated at 4 °C overnight with an anti-p300 antibody (1:1000, Santa-Cruz, sc-585), anti-GFP (1:5000, Covance, MMS-118R), anti-H4 acetylated (1:1000, Millipore, #06-598), anti-Lysine-acetylated (1:1000, Cell Signalling, #9441), anti-HDAC1 (1:1000, Santa-Cruz #sc-7872), anti-HA (1:4000, Abcam, ab9110).

Techniques: Construct, SDS Page, Transfection, Expressing, Immunofluorescence

Journal: Nature Communications

Article Title: Structural insights into p300 regulation and acetylation-dependent genome organisation

doi: 10.1038/s41467-022-35375-2

Figure Lengend Snippet: a Representative Biolayer Interferometry (BLI) profiles of p300 TAZ2-NUT binding from which the steady-state data were derived. Colours indicate different concentrations of TAZ2 used. b Steady state analysis of p300 TAZ2 interaction with wild-type (black) and NUT mutants using BLI. Data are presented as mean values +/− SD. N = 3 independent technical replicate experiments were done. c Sequences of the NUT AD and mutants (in bold red) used. Top: Skylign plot showing NUT AD sequence conservation; Bottom: predicted secondary structure. d GST pulldown analysis of TAZ2 and NUT8 (396-470) wild type and mutants 1 and 2. The experiment was repeated three times with consistency. e AlphaFold prediction of NUT (396-482) structure. f Left: AlphaFold prediction of the TAZ2-NUT complex structure. Right: mutated residues in NUT mutant 1 (blue) and 2 (green) are shown.

Article Snippet: After saturation of the membrane in 1X PBS (#14200-067, Gibco), 0.2% Tween 20 (P7549, Sigma) and 5% milk (Regilait) or Bovine albumin Serum (BSA, #04-100-812-C, Avantor) during 1 h at RT, membranes were incubated at 4 °C overnight with an anti-p300 antibody (1:1000, Santa-Cruz, sc-585), anti-GFP (1:5000, Covance, MMS-118R), anti-H4 acetylated (1:1000, Millipore, #06-598), anti-Lysine-acetylated (1:1000, Cell Signalling, #9441), anti-HDAC1 (1:1000, Santa-Cruz #sc-7872), anti-HA (1:4000, Abcam, ab9110).

Techniques: Binding Assay, Derivative Assay, Sequencing, Mutagenesis

Journal: Nature Communications

Article Title: Structural insights into p300 regulation and acetylation-dependent genome organisation

doi: 10.1038/s41467-022-35375-2

Figure Lengend Snippet: a AlphaFold prediction of p300 core-CH3 (1048-1838) structure. The structure is coloured according to domain structure. The TAZ2 domain is positioned in the HAT active site thus autoinhibiting substrate access. The TAZ2 C-terminal helix deletion (Δ1813-1838) is coloured in magenta b NUT AD binding displaces the TAZ2 domain from the HAT active site thus enabling allosteric HAT activation. c p300s or d ,p300 core were incubated with [ 14 C]Acetyl-CoA in the presence or absence (C: p300 alone) of increasing concentrations of wild type or mut1 NUT8 (396-470). e Increasing concentrations of GST-E1a we incubated with p300s. f Increasing concentrations of NCPs were incubated with p300s or p300 core. Panels c-f: Top two images: Analysis by SDS–PAGE followed by Coomassie staining. Bottom two images: 14 C phosphor imaging. Experiments c-f were repeated independently three times with consistency. Representative data are shown. Source data are provided as a Source Data file. g Cos7 cells were transfected with the HA-tagged p300 WT (top) or p300 Δ1813-1838 (bottom) and analysed by anti HA immunofluorescence. Four representative nuclei are shown. The percentage of cells showing condensates ( n = 100 cells) is indicated. Experiments were repeated twice with consistency. Scale bars, 5 μm.

Article Snippet: After saturation of the membrane in 1X PBS (#14200-067, Gibco), 0.2% Tween 20 (P7549, Sigma) and 5% milk (Regilait) or Bovine albumin Serum (BSA, #04-100-812-C, Avantor) during 1 h at RT, membranes were incubated at 4 °C overnight with an anti-p300 antibody (1:1000, Santa-Cruz, sc-585), anti-GFP (1:5000, Covance, MMS-118R), anti-H4 acetylated (1:1000, Millipore, #06-598), anti-Lysine-acetylated (1:1000, Cell Signalling, #9441), anti-HDAC1 (1:1000, Santa-Cruz #sc-7872), anti-HA (1:4000, Abcam, ab9110).

Techniques: Binding Assay, Activation Assay, Incubation, SDS Page, Staining, Imaging, Transfection, Immunofluorescence

Journal: Nature Communications

Article Title: Structural insights into p300 regulation and acetylation-dependent genome organisation

doi: 10.1038/s41467-022-35375-2

Figure Lengend Snippet: a Cos7 cells were transfected with GFP-BRD4-NUT variants or GFP alone as the control. Co-immunoprecipitation (IP) experiments were performed using anti-GFP antibody and immuno-blotted (IB) for p300, GFP and Acetylated-Lysine. Input samples were loaded as a control. b Purified GST-NUT8 wild type and mutants 1 and 2 were incubated with Cos7 cells extract for 4 h. The complexes were pull-down by Glutathione Sepharose beads and analysed by SDS-PAGE followed by Coomassie staining (bottom) or by IB for p300 (top). Source data are provided as a Source Data file. Experiments in a-b we repeated independently at least three times. Representative data are shown. c Cos7 cells were transfected with the indicated GFP-BRD4-NUT and RFP-p300 constructs and analysed by fluorescence microscopy, BRD4-NUT (green) and p300 (red). Scale bars, 10 μm. Quantification of foci formation of different BRD4-NUT variants and their co-localization with p300 are shown in Supplementary Tables , . d Cos7 cells were transfected with the indicated GFP-BRD4-NUT constructs and treated with DMSO (control), 1,6-hexanediol, p300 HAT inhibitor A485, pan-BET inhibitor JQ1, HDAC inhibitor TSA or p300 bromodomain inhibitor CBP30. Scale bars, 10 μm. Quantification of foci formation in presence of CBP30 is shown in Supplementary Table . e Schematic representation of GFP-labelled BRD4 mutants and Alexa Fluor 594 labelled reconstituted chromatin containing non-acetylated (12x NCP) or acetylated (Ac 12X NCP) 12 Nucleosome Core Particles. Fluorescence microscopy images of non-acetylated or acetylated 12x NCP chromatin incubated with GFP-BD1-BD2. Experiments in c-e were repeated independently at least three times with consistency. Scale bars, 10 μm.

Article Snippet: After saturation of the membrane in 1X PBS (#14200-067, Gibco), 0.2% Tween 20 (P7549, Sigma) and 5% milk (Regilait) or Bovine albumin Serum (BSA, #04-100-812-C, Avantor) during 1 h at RT, membranes were incubated at 4 °C overnight with an anti-p300 antibody (1:1000, Santa-Cruz, sc-585), anti-GFP (1:5000, Covance, MMS-118R), anti-H4 acetylated (1:1000, Millipore, #06-598), anti-Lysine-acetylated (1:1000, Cell Signalling, #9441), anti-HDAC1 (1:1000, Santa-Cruz #sc-7872), anti-HA (1:4000, Abcam, ab9110).

Techniques: Transfection, Immunoprecipitation, Purification, Incubation, SDS Page, Staining, Construct, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Structural insights into p300 regulation and acetylation-dependent genome organisation

doi: 10.1038/s41467-022-35375-2

Figure Lengend Snippet: a Example image of nuclear droplets. Red square: Time laps imaging of BRD4-NUT and p300 droplets; Scale bar, 4 μm. b Time laps imaging after addition of 3% [w/v] 1,6 Hexanediol. Scale bar, 12 μm. Experiments in a-b were repeated independently two times with consistency. c Top: Example image of droplets (red square) after photobleaching. Scale bar, 2 μm. Bottom: Quantification of the FRAP experiments. N = 3 independent technical replicates were done. Averages and standard error of mean (SEM; grey bars) are shown. d Quantification of the characteristic time of recovery rates for different GFP-BRD4-NUT mutants (green, top) and RFP-p300 (red, bottom). N = 3–4 technical replicate FRAP experiments were done for each mutant (40 to 65 foci measured). Averages recovery rates for each N replicate experiment are shown as dots (•). Bar height represents the mean recovery rate value. Statistical differences in recovery rates were determined by a two-sided Student’s t -test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; n.s. non-significant difference as compared to WT. e Half-bleach FRAP experiment. Top: The area before (left) and after (right) laser bleach are shown. The red box indicates the bleached area. Scale bar, 0.5 μm. Bottom: The change in fluorescence intensity along the A-B axis was analysed in the Kymograph. f Top: Plot of fluorescence recovery from the Kymograph for the unbleached area (orange), centre (blue) and periphery (grey) of the droplet. Bottom: cartoon of the internal and external exchange. Experiments in e , f were repeated twice with consistency.

Article Snippet: After saturation of the membrane in 1X PBS (#14200-067, Gibco), 0.2% Tween 20 (P7549, Sigma) and 5% milk (Regilait) or Bovine albumin Serum (BSA, #04-100-812-C, Avantor) during 1 h at RT, membranes were incubated at 4 °C overnight with an anti-p300 antibody (1:1000, Santa-Cruz, sc-585), anti-GFP (1:5000, Covance, MMS-118R), anti-H4 acetylated (1:1000, Millipore, #06-598), anti-Lysine-acetylated (1:1000, Cell Signalling, #9441), anti-HDAC1 (1:1000, Santa-Cruz #sc-7872), anti-HA (1:4000, Abcam, ab9110).

Techniques: Imaging, Mutagenesis, Fluorescence

Journal: Nature Communications

Article Title: Structural insights into p300 regulation and acetylation-dependent genome organisation

doi: 10.1038/s41467-022-35375-2

Figure Lengend Snippet: a p300 is maintained in the inactive state by HDACs. Activation requires TF binding which results autoacetylation and removal of autoinhibition. b In enhancer signalling, TFs interact through their ADs with the different protein interaction domains of p300 which results in scaffolding of dynamic TF assemblies. Local TF and histone acetylation allows binding of BD containing proteins including BRD4 thus establishing positive feed-back. HDACs are located in these condensates and enable dynamic turnover thus resulting in bistability and response to changes in TF signalling. c In BRD4-NUT NMC, the acetylation-dependent reaction drives formation of intra- and interchromosomal interactions resulting in the formation of a Megadomain. d TF binding and acetylation establish open Euchromatin. TFs compete for limiting p300 co-activators but TF-DNA binding outside enhancer clusters remains transient and, due to high HDAC activity, does not effectively establish an acetylation-dependent condensate. e Depletion and partitioning of p300 into euchromatic compartments results in hypoacetylation due to high HDAC activity. Hypoacetylated compartments become a substrate for heterochromatinization.

Article Snippet: After saturation of the membrane in 1X PBS (#14200-067, Gibco), 0.2% Tween 20 (P7549, Sigma) and 5% milk (Regilait) or Bovine albumin Serum (BSA, #04-100-812-C, Avantor) during 1 h at RT, membranes were incubated at 4 °C overnight with an anti-p300 antibody (1:1000, Santa-Cruz, sc-585), anti-GFP (1:5000, Covance, MMS-118R), anti-H4 acetylated (1:1000, Millipore, #06-598), anti-Lysine-acetylated (1:1000, Cell Signalling, #9441), anti-HDAC1 (1:1000, Santa-Cruz #sc-7872), anti-HA (1:4000, Abcam, ab9110).

Techniques: Activation Assay, Binding Assay, Scaffolding, Activity Assay