Mutation mimicking H3 K56Ac can promote histone disassembly from promoters during their induction, but not from bulk chromatin.

<p><b>A.</b> ChIP analysis of YFP from cells expressing wild type H3.3-YFP or H3.3 K56Q-YFP or H3.3 K56R-YFP in MCF7 cells following addition of estradiol. Data are normalized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155409#pone.0155409.g001" target="_blank">Fig 1</a>. Data are the average and standard deviation of three independent experiments. * indicates significant changes from time 0, p<0.01 measured by the Student’s unpaired t-test <b>B</b>. FRAP analysis of YFP tagged H3.3 in MCF7 vs MCF10A cells (average and SD are shown for 30 cells). <b>C</b>. FRAP analysis of YFP tagged H3.1 in MCF7 vs MCF10A cells (average and SD are shown for 30 cells). <b>D</b>. FRAP analysis of H3.3-YFP or H3.3 K56Q-YFP or H3.3 K56R-YFP in MCF7 cells. <b>E</b>. FRAP analysis of H3.1-YFP or H3.1 K56Q-YFP or H3.1 K56R-YFP in MCF7 cells.</p

The Commercial Antibodies Widely Used to Measure H3 K56 Acetylation Are Non-Specific in Human and <i>Drosophila</i> Cells

<div><p>Much of our understanding of the function of histone post-translational modifications in metazoans is inferred from their genomic localization and / or extrapolated from yeast studies. For example, acetylation of histone H3 lysine 56 (H3 K56Ac) is assumed to be important for transcriptional regulation in metazoan cells based on its occurrence at promoters and its function in yeast. Here we directly assess the function of H3 K56Ac during chromatin disassembly from gene regulatory regions during transcriptional induction in human cells by using mutations that either mimic or prevent H3 K56Ac. Although there is rapid histone H3 disassembly during induction of some estrogen receptor responsive genes, depletion of the histone chaperone ASF1A/B, which is required for H3 K56 acetylation, has no effect on chromatin disassembly at these regions. During the course of this work, we found that all the commercially available antibodies to H3 K56Ac are non-specific in human cells and in <i>Drosophila</i>. We used H3-YFP fusions to show that the H3 K56Q mutation can promote chromatin disassembly from regulatory regions of some estrogen responsive genes in the context of transcriptional induction. However, neither the H3 K56R nor K56Q mutation significantly altered chromatin disassembly dynamics by FRAP analysis. These results indicate that unlike the situation in yeast, human cells do not use H3 K56Ac to promote chromatin disassembly from regulatory regions or from the genome in general. Furthermore, our work highlights the need for rigorous characterization of the specificity of antibodies to histone post-translational modifications <i>in vivo</i>.</p></div

Commercial H3 K56Ac antibodies are non-specific in human cells.

<p><b>A.</b> Peptide competition analysis using the indicated peptides, of acid extracted histones from HeLa and 293 cells that were treated with or without sodium butyrate (NaBu) to inhibit class I and II HDACs. The antibody used in the western is indicated on the right. Duplicate samples are loaded for each condition. The sizes on the left indicate kD size of Biorad Dual Color Standards molecular weight markers that were transposed onto the film from the membrane. <b>B</b>. Western blot analysis of acid extracted histones from 293 cells that were wild type (WT) or stably expressed H3.1c-FLAG or H3.1c K56R-FLAG, or from H1299 cells that stably expressed H3.1c-FLAG or H3.1c K56R-FLAG. * indicates a proteolytic degradation product of H3 generated by cleavage of the N-terminus that occurs upon generating the total protein extracts. ** indicates a smaller H3 degradation product seen in the wild type sample only, due to over handling of the extract. Data is shown for the Active Motif, the Epitomics and the Cell signalling H3 K56Ac antibodies. <b>C</b>. IHC analysis of the Active Motif mAb, on breast cancer tissue samples shows non-specific cytoplasmic staining instead of distinct nuclear staining. Green arrows point to cytoplasm and black arrows point to nuclei. Both slides are also stained with haematoxylin and eosin (H&E) stain, which stains nuclei blue and eosinophilic structures pink.</p

Commercial H3 K56Ac antibodies are non specific in flies.

<p>The top schematic explains the mosaic nature of the wild type histone expression, indicated by GFP staining. All cells express H3 K56R, but the black patches express no endogenous wild type histones. Below are shown wing imaginal discs from flies with clones of cells expressing only H3 K56R (indicated by white arrows in the top row) that are marked by lack of GFP expression. Staining with the indicated H3 K56Ac antibodies showed no difference between H3 K56R mutant clones or the adjacent wild type clones, indicating that the antibodies are non specific. Representative images are shown.</p

H3 K56Ac is not required for chromatin disassembly gene regulatory regions of estrogen responsive genes.

<p><b>A</b>. Dot blot analysis of the indicated commercial H3 K56Ac antibodies, the indicated peptides. 10 μl of each peptide at 300 μM concentration was spotted on the top row, followed by 10 fold dilutions below. A short and longer exposure are shown. <b>B</b>. The top shows RT-PCR analysis of mRNA induction at the indicated times after addition of estradiol, normalized to GAPDH. Data are the average and standard deviation of three independent experiments. Below is shown a ChIP analysis of histone H3 occupancy at the indicated time points after estradiol addition from the same time course as the mRNA analysis, at the indicated EREs in the pS2 (<i>TFF1)</i>, <i>GREB1</i>, and <i>PGR</i> promoters. Each data point was normalized to the input and a telomeric control region at the same time point. At the bottom is shown a ChIP analysis of histone H3 K56Ac levels normalized to H3 occupancy. Each data point was normalized to the input and a telomeric control region at the same time point. Shown are the average and standard deviation of three independent experiments. The H3 K56Ac data for the <i>PGR</i> gene regions are shown again in the inset with the y-axis expanded, to enable visualization of the very low signal. * indicates significant changes from time 0, p<0.05 measured by the Student’s unpaired t-test. <b>C</b>. ChIP analysis of H3 and H3 K56Ac levels normalized as in B, during the indicated shRNA knockdowns. Shown are the average and standard deviation of three independent experiments. * indicates significant changes from time 0, p<0.05 measured by the Student’s unpaired t-test. <b>D</b>. Western blot analysis of ASF1A, ASF1B, H3K56 Acetylation, and tubulin alpha from samples taken from the experiment shown in C. Protein extracts were made with RIPA buffer from MCF7 cells that were infected with lentivirus of non-silencing (control), ASF1A shRNA, ASF1B shRNA, ASF1A/B shRNA. The Active Motif polyclonal antibody was used for experiments shown in B-D.</p

MOESM1 of Development of novel cellular histone-binding and chromatin-displacement assays for bromodomain drug discovery

Additional file 1. Six supplementary figures and three supplementary tables