Effects of Oncohistone Mutations and PTM Crosstalk on the N-Terminal Acetylation Activities of NatD

Abstract

Acetylation at the α-N-terminus (Nα) is the most abundant modification detected on histone H4 and H2A, which is catalyzed by N-terminal acetyltransferase D (NatD or NAA40). Histone H4 and H2A contain an identical N-terminal SGRGK sequence that is enriched with post-translational modifications (PTMs) and frequently occurred oncogenic mutations known as "oncohistone" mutations. However, there is a lack of information on how oncohistone mutations and other PTMs affect NatD-catalyzed acetylation. Herein, we determined how the local chemical environment on the N-terminal SGRGK sequence impacts NatD-catalyzed Nα-acetylation on histone H4/H2A. Our studies indicate that all oncohistone mutations at SGRG suppressed NatD-catalyzed acetylation. Meanwhile, H4 Ser1 phosphorylation and Arg3 methylation negatively impact the NatD-mediated acetylation, but the Lys5 acetylation only has a marginal effect. This work reveals the impacts of oncohistone mutations on NatD activity and unravels the crosstalk between NatD and PTMs, implying potential regulatory mechanism of NatD and highlighting different avenues to interrogate the NatD-mediated pathway in the future.

Figure 1.

(A) Nα-acetylation catalyzed by NatD. (B) Mutations of oncohistone H4 and H2A and chemical modifications at residues Ser1, Arg3, and Lys5 on the H4 substrate. Modifications include phosphorylation (p), monomethylation (me), asymmetric dimethylation (me2a), symmetric dimethylation (me2s), and acetylation (ac). Mutations detected on H4 and H2A: yellow circle; mutations detected only on H4: green circle; mutations detected only on H2A: blue circle.

Figure 2.

Effect of C-terminal truncations of hNatD-catalyzed acetylation. (A) Steady-state parameters of AcCoA, C-terminal truncated H4, H2A, and SMARCD2 peptides. Fold changes of (B) k_cat and (C) k_cat/K_m normalized to full-length histone H4 (FL H4).

Figure 3.

Effects of oncohistone mutations on N-terminal acetylation of H4/H2A peptides. (A) Kinetic profiles of oncohistone H4/H2A pentapeptides. The mutation count of H4 and H2A was extracted from a processed cBioPortal dataset. Fold change of (B) k_cat and (C) k_cat/K_m normalized to wild-type H4-5.

Figure 4.

Effects of PTMs on H4-5 peptide (SGRGK) histone on Nα-acetylation. (A) Steady-state parameters of chemically modified H4 pentapeptides. Fold changes of (B) k_cat and (C) k_cat/K_m normalized to wild-type H4-5.

Figure 5.

Inhibitory effects of H4/H2A peptides carrying either oncohistone mutation or PTM on Nα-acetylation by hNatD. The competitive inhibition assay was conducted with a 3-fold serial dilution of mutant and modified peptides starting from 300 μM in the presence of 6 μM of H4-5 and 0.6 μM of AcCoA. The activity was normalized to the activity in the absence of competing peptides.

Figure 6.

Impacts of H4/H2A peptides carrying oncohistone mutations or PTMs on NatD-catalyzed acetylation. (A) NatD-catalyzed acetylation on various peptides. PTMs include phosphorylation (p), monomethylation (me), asymmetric dimethylation (me2a), symmetric dimethylation (me2s), and acetylation (ac). Mutations were detected on H4 and H2A (yellow), only on H4 (green), only on H2A (blue). The charge status contains neutral (0 within in a gray circle), positive (+ within in a blue circle), and negative (– within in a red circle). Solid red line with blunt end = over 100-fold decrease, red dotted line with blunt end =10–100-fold decrease, and black dotted line = less than 10-fold decrease. (B) Inhibitory effects on NatD-catalyzed acetylation of H4-5 (SGRGK) in a competitive inhibition assay.