A synthetic non-histone substrate to study substrate targeting by the Gcn5 HAT and sirtuin HDACs

Abstract

Edited by John M. Denu Gcn5 and sirtuins are highly conserved histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes that were first characterized as regulators of gene expression. Although histone tails are important substrates of these enzymes, they also target many nonhistone proteins that function in diverse biological processes. However, the mechanisms used by these enzymes to choose their nonhistone substrates are unknown. Previously, we used SILAC-based MS to identify novel nonhistone substrates of Gcn5 and sirtuins in yeast and found a shared target consensus sequence. Here, we use a synthetic biology approach to demonstrate that this consensus sequence can direct acetylation and deacetylation targeting by these enzymes in vivo. Remarkably, fusion of the sequence to a nonsubstrate confers de novo acetylation that is regulated by both Gcn5 and sirtuins. We exploit this synthetic fusion substrate as a tool to define subunits of the Gcn5-containing SAGA and ADA complexes required for nonhistone protein acetylation. In particular, we find a key role for the Ada2 and Ada3 subunits in regulating acetylations on our fusion substrate. In contrast, other subunits tested were largely dispensable, including those required for SAGA stability. In an extended analysis, defects in proteome-wide acetylation observed in ada3⌬ mutants mirror those in ada2⌬ mutants. Altogether, our work argues that nonhistone protein acetylation by Gcn5 is determined in part by specific amino acids surrounding target lysines but that even optimal sequences require both Ada2 and Ada3 for robust acetylation. The synthetic fusion substrate we describe can serve as a tool to further dissect the regulation of both Gcn5 and sirtuin activities in vivo.

Targeting the SAGA and ATAC Transcriptional Coactivator Complexes in MYC-Driven Cancers

“…GCN5 KAT activity and specificity is augmented by association with the Alteration/Deficiency in Activation (ADA) proteins [1,5], and independent ADA-GCN5 complexes have been identified in yeast and metazoans [6]. GCN5 requires association with both ADA2 and ADA3 for robust acetylation of histone and nonhistone targets [7]. GCN5 is associated with two forms of ADA2 in metazoans, termed ADA2A and ADA2B [8].…”

HDAC6 inhibitor, ACY1215 suppress the proliferation and induce apoptosis of gallbladder cancer cells and increased the chemotherapy effect of gemcitabine and oxaliplatin

“…HDACs can regulate gene expression through histone deacetylation, and some HDACs subtypes can also influence the function of non-histones and regulate a variety of cellular pathways (Lee et al, 2014). HDACs inhibitors can affect a variety of cellular effects, inhibit angiogenesis, and induce cell apoptosis (Na et al, 2010;Rossl et al, 2019). As an anti-tumor agent, HDACs inhibitors have attracted wide attention.…”

Inhibition of histone acetyltransferase GCN5 by a transcription factor FgPacC controls fungal adaption to host-derived iron stress

“…Based on the sequence of ZnF3 domain from different PacC homologues in Pezizomycotina , we identified a highly conserved fragment ZnF3 (133–143) corresponding to amino acid residues 133–143 of FgPacC (Figure 7D ). Previous studies in yeast have shown that Gcn5 can target the sequences with an S-x-K-K/R-P motif ( 71 ). Notably, this putative Gcn5-binding motif (SFKRP, amino acid residues 137–141) is localized in the ZnF3 (133–143) fragment of FgPacC30.…”