Selectivity issues related to antibodies, especially those targeting histone PTMs, is by no means a new problem. Common concerns include cross-reactivity with other PTMs or states of methyl-lysine, and recent studies have begun characterizing how neighboring PTMs influence antibody recognition in a similar manner to effector protein binding9,15,16,22
. With so many biological methods relying on antibodies for detection and enrichment, thorough characterization of these reagents is paramount. Our results demonstrate a previously uncharacterized property of histone H4 site-specific acetyl antibodies, an inherent preference for poly-acetylated chromatin signatures. By standard criteria, these antibodies would be judged selective for their intended PTM. They all recognize a single protein band by immunoblot, can be competed with a peptide antigen, do not cross-react with other singly acetylated epitopes, and even have been shown to lose reactivity when probing for histones mutated at the target lysine (– and data not shown). Using our peptide arrays as a new criterion for antibody characterization, our findings suggest H4K5ac, H4K8ac and H4K12ac antibodies may not recognize their intended target, but instead, all recognize the same poly-acetylated histone signature.
Our unexpected finding regarding the property of H4 acetyl-specific antibodies raises new questions with the interpretation of genome-wide mapping studies of H4 acetylation events across species and cell lines. In general, genomic studies find a high correlation for the positioning of H4K5ac, K8ac, and K12ac across genomes23,24,25
. However, it is entirely possible that these findings represent the preference of these antibodies for H4 poly-acetylation – a chromatin signature that is present in vivo
and at levels that these antibodies would compete for over their individually acetylated counterparts. Given the problematic nature of these antibodies, the true genomic locations and relative distributions of the individual H4K45ac, H4K8ac and H4K12ac marks are likely still unknown. Since our mass spectrometry results show high species conservation and significant abundance of singly acetylated H4 tails, we suggest that the individual H4K5, H4K8 and H4K12 acetylation events might have non-overlapping genomic distributions and functions. Future studies involving improved site-specific H4 acetyl antibodies will be needed to test this hypothesis.
A counter argument to the concern of recognizing poly-acetylation signatures are classic genetic studies in budding yeast showing that mutation of H4 lysines 5, 8, and 12 individually have indistinguishable changes in gene expression profiles or growth defects, while only mutation of lysine 16 has a unique gene expression signature23,26
. Combing multiple H4 lysine mutations results in a cumulative effect on gene expression and defective growth - suggesting H4K5, H4K8 and H4K12 acetylation events are functionally redundant and cumulative. However, parallel studies have not yet been performed in more complex organisms where individual acetylation events might play a more significant role. With a new Drosophila melanogaster
histone replacement model now available, important and interesting questions such as these can be tested27
One question remaining is why might these antibodies strongly detect poly-acetylated histones in the first place? The fact that H3 acetyl antibodies do not have strong poly-acetyl preference suggests this problem is specific towards histone H4. A potential explanation may lie in the repetitive sequence surrounding the K5, K8 and K12 acetylation sites on the H4 tail. The GKG motif that surrounds these lysines is repeated on the histone H4 tail. Lysine 16, however, differs, from this pattern (AKR), and the H4K16ac antibody coincidently has the least enhanced preference for poly-acetylated H4. Regardless of the reasons, one would ideally want the H4 acetyl antibodies to behave more similarly to the H3 acetyl antibodies tested, recognizing singly modified acetyl-lysines similarly to poly-acetylated epitopes.
Beyond histones, we note that high-resolution mass spectrometry studies have recently identified thousands of acetylation events across multiple species28,29,30,31
. Site-specific acetyl antibodies will undoubtedly be developed for studying biological functions associated with these non-histone acetylation events. Importantly, the same GKG consensus motif that may pose a problem for H4 acetyl antibodies surrounds a number of identified acetylated lysines on non-histone proteins32,33
. It remains to be determined whether similar antibody-based detection issues will apply for these non-histone PTMs.
In conclusion, we describe a new and concerning property of site-specific acetyl antibodies that has been previously missed in all other forms of characterization. This re-defined property has implications for past data interpretation and represents a formidable challenge for future studies. This paper therefore serves to encourage more thorough validation of the next generation of acetyl antibodies for the biological community at large.