Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies

Abstract

Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins.

An Interactive Database for the Assessment of Histone Antibody Specificity

“…Consistent with our previous observations (Rothbart et al, 2012e), microarray analysis shows that site-specific H4 acetyl antibodies preferentially bind epitopes with iterative increases in acetylation content (Figure 2C). For example, all of the H4K5ac antibodies screened show enhanced signal on peptides containing H4K5ac and one or more H4Kac sites, with iterative increases in acetylation content (up to 4 sites on a single peptide) being preferred epitopes for the majority of H4 acetyl antibodies screened.…”

“…This platform allows for robust and comprehensive characterization of the behavior of histone-interacting proteins, including the specificity of histone antibodies (Figure 1A) (Cai et al, 2013; Fuchs et al, 2011; Rothbart et al, 2013; Rothbart et al, 2012a; Rothbart et al, 2012e). Notably, antibody reactivity with differing modification states (e.g., mono-, di- and trimethyl lysine) and the influence of epitope recognition by neighboring histone PTMs can be determined with high precision.…”

“…Indeed, large-scale epigenomics ‘roadmap’ efforts like the Encyclopedia of DNA Elements (ENCODE) projects depend on these antibodies for genome-wide mapping of histone PTM distribution (Ernst et al, 2011; Gerstein et al, 2010; Kharchenko et al, 2011). However, recent studies have made some surprising and alarming observations regarding the behavior of histone PTM antibodies, including off-target recognition, strong influence by neighboring PTMs, and an inability to distinguish the modification state on a particular residue (e.g., mono-, di-, or tri-methyl lysine) (Bock et al, 2011; Egelhofer et al, 2011; Fuchs et al, 2011; Fuchs and Strahl, 2011; Hattori et al, 2013; Nishikori et al, 2012; Rothbart et al, 2012e). Consequently, poor antibody choice can lead to misinformed conclusions regarding the location and function of the histone PTM being queried (Baker, 2015).…”

Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry

“…The histone H4 4 -17 peptide contains four lysine residues that may be acetylated. Multiple acetylation has been observed in many different organisms in vivo and is strongly correlated with active transcription (33,34). Fig.…”

Histone-binding domains: Strategies for discovery and characterization

“…In an analysis of several commonly used antibodies, many antibodies were found to lack the advertised specificity [25, 47–49]. Inconsistency exists in the quality of modification-specific antibodies that are critical for understanding chromatin biology; therefore, care must be taken when interpreting data using these reagents.…”