S.B.R. of histones and can influence chromatin-based events including transcription, DNA replication, DNA repair, and dosage compensation1,4. One mechanism by which lysine acetylation influences chromatin function is usually by removing positive charges from lysine side chains, NECA thus making local chromatin structure more permissive to specific protein NECA machineries5. Lysine acetylation can also function by serving as a docking site for bromodomain-containing proteins, often found as subunits of histone acetyltransferases (HATs), NECA ATP-dependent chromatin remodelers, and transcriptional coactivators6,7. Significantly, recent studies show that bromodomain-containing proteins preferentially recognize poly-acetylated chromatin signatures7,8,9. These studies lend further support to the histone code hypothesis, which suggests that histone PTMs NECA function in a combinatorial fashion to regulate chromatin architecture and DNA-templated cellular processes10,11. Direct investigations of biological functions associated with specific histone PTMs have been facilitated by genetic and biochemical methods, and often depend on antibodies to monitor these PTMs. Furthermore, large scale epigenomics efforts, like the ENCODE and modENCODE projects, rely on these antibodies to map the genomic distribution of chromatin signatures12,13,14. Therefore, antibody specificity is usually of utmost importance for accurate data interpretation. The standard criteria for characterizing antibody specificity typically involves primary reactivity with a single species from cell extracts by immunoblotting that is diminished in the absence or mutation of epitope, and that can be competed with recombinant or synthetic antigen9,15,16. Extended criteria often involve characterizing the ability of antibodies to perform in biological assays, like chromatin immunoprecipitation (ChIP), immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and immunoblots. Recent studies from our lab and others demonstrate that neighboring FLI1 PTMs often enhance or perturb the ability of histone antibodies to recognize their intended target9,15,16. Furthermore, these studies have found that histone antibodies often have specific difficulties in recognizing their appropriate epitopes, either due to the inability to distinguish methyl-lysine says (mono-, di-, and tri-methylation) or to recognize off-target PTMs. In addition, studies from the modENCODE consortium have found that > 25% of commercial histone antibodies fail basic quality control measures17. Here, we uncover a novel house of histone H4 antibody-antigen recognition (preferential detection of poly-acetylated chromatin signatures) that presents a significant concern with the use of these reagents. Our findings caution interpreting results to date that employ these site-specific acetyl antibodies and suggest more thorough validation of antibodies is needed before they can be labeled as specific. Results Site-specific H4 acetyl antibodies prefer poly-acetylated substrates NECA To interrogate the interactions of chromatin-associated proteins and antibodies with combinatorial histone PTMs, we recently developed a peptide microarray platform where > 250 unique biotinylated histone peptides, made up of 0C8 possible PTMs, were immobilized on streptavidin-coated glass slides (Supplemental Table 1)9,16. These peptide arrays were probed with a number of commonly used commercial histone acetyl-specific antibodies (Supplemental Table 2) to discern their specificities. We found that acetyl-specific antibodies directed against H3 lysines 9 and 14 (H3K9ac and H3K14ac) generally performed as expected, in that they showed no discernable conversation with unmodified histones, and detected their intended PTM alone and in the context of adjacent acetylation events with similar signal intensity (Fig. 1a and Supplemental Fig. 1). Of note, H3S10 phosphorylation (H3S10p) perturbed the recognition of H3K9ac (see peptides 37, 41, 144, and 148 in Supplemental Fig. 1), but had little effect on H3K14ac recognition. H3S10p, enriched on mitotic chromatin18, has been shown to exist on the same histone tail as H3K9ac in cells19,20,21. Our array analysis therefore suggests this H3S10 phosphorylated population of H3K9 acetylated histone tails may be underrepresented in biological assays using this antibody. We also detected weak cross-reactivity of these antibodies with H4 and H2A acetylated peptides (Supplemental Fig. 1). Open in a separate window Physique 1 (a-c) Heat maps summarizing peptide array results for H3 and H4 acetyl antibodies.For each array, the most intense series of peptide spots (12 individual spots per peptide) is assigned a value of 1 1 (blue), and all values are normalized to this peptide. Values 0.1 are colored red in panel C to enable interpretation of low signal intensities. Each conversation is presented as an averaged normalized intensity from at least two impartial arrays (r2 > 0.9). See Rothbart in comparison to the single mark by mass spectrometry (Fig 2a). Importantly, a tetra-acetylated H4 peptide was able to compete the H4K12ac antibody at a concentration 10-fold lower than an H4K12ac peptide (Fig 2b). Comparable results were seen with an H4K5ac antibody (Fig 2c). Collectively,.