Site specificity analysis of Piccolo NuA4-mediated acetylation for different histone complexes

Abstract

We have a limited understanding of the site specificity of multisubunit lysine acetyltransferase (KAT) complexes for histone-based substrates, especially in regards to the different complexes formed during nucleosome assembly. Histone complexes could be a major factor in determining the acetylation specificity of KATs. In the present study, we utilized a label-free quantitative mass spectrometry-based method to determine the site specificity of acetylation catalyzed by Piccolo NuA4 on (H3/H4)2 tetramer, tetramer bound DNA (tetrasome), and nucleosome core particle (NCP). Our results show that Piccolo NuA4 can acetylate multiple lysine residues on these three histone complexes, of which NCP is the most favorable, (H3/H4)2 tetramer is the second, and tetrasome is the least favorable substrate for Piccolo NuA4 acetylation. Although Piccolo NuA4 preferentially acetylates histone H4 (H4K12), the site specificity of the enzyme is altered with different histone complex substrates. Our results show that before nucleosome assembly is complete, H3K14 specificity is almost equal to that of H4K12 and DNA-histone interactions suppress the acetylation ability of Piccolo NuA4. These data suggest that the H2A/H2B dimer could play a critical role in the increase of acetylation specificity of Piccolo NuA4 for NCP. This demonstrates that histone complex formation can alter the acetylation preference of Piccolo NuA4. Such findings provide valuable insight into regulating Piccolo NuA4 specificity by modulating chromatin dynamics, and in turn manipulating gene expression.

Histone Acetylation Inhibits RSC and Stabilizes the +1 Nucleosome

“…Acetylation is especially abundant in the vicinity of the promoter (Kuo et al, 1998; Reinke and Horz, 2003), including the histones of the +1 nucleosome. Histone acetylation is catalyzed by a number of enzymes, including the Gcn5 subunit of the SAGA complex, which targets H3 residues K9 and K14, and the Esa1 subunit of the NuA4 complex, which preferentially acetylates H4K12, but which can acetylate other residues in H4, and in H2A, and H3 as well (Kuo et al, 2015). SAGA is required for the initiation of transcription (Baptista et al, 2017), and so is likely responsible for acetylation of the +1 nucleosome.…”

HDAC8 substrate selectivity is determined by long- and short-range interactions leading to enhanced reactivity for full-length histone substrates compared with peptides

“…The pH was then reduced for mass spectrometry (MS) by addition of 3.5 l of 10% formic acid. Tryptic peptides were analyzed by MS/MS analysis in the Andrews lab at Fox Chase Cancer Center, as described previously (45,66). Graphical analysis was done using Prism (GraphPad Software, Inc.).…”

“…Recently, substrate specificity for the Saccharomyces cerevisiae lysine acetyltransferase piccolo NuA4 has been measured using histone protein substrates, demonstrating acetylation of multiple lysine residues (45). However, HDAC substrate specificity studies to date have utilized acetylated peptides (13-15, 46 -50), predicated on the assumption that HDAC8 uses similar interactions to recognize peptide and full-length protein substrates.…”

Critical genomic regulation mediated by Enhancer of Polycomb

“…2a) (Boudreault et al 2003; Mitchell et al 2008; Rossetto et al 2014). Epl1, through its EPcA region, is critical for Esa1 acetyltransferase activity, especially toward nucleosomes in vitro, and further contributes to target specificity (Berndsen et al 2007; Chittuluru et al 2011; Huang and Tan 2013; Kuo et al 2015; Lalonde et al 2013; Selleck et al 2005). Most recently, the structure of the EPcA domain was solved in complex with the other piccolo-NuA4 subunits, and the first bypass mutant of EPL1 was identified using genetic suppression analysis that has been so powerful for the study of many critical proteins and biological processes (Hughes 2016; Prelich 1999; van Leeuwen et al 2017 ).…”