H2A.X N-terminal acetylation is a newly identified NAA40-mediated modification that is responsive to UV irradiation

Abstract

Background: N-terminal acetylation (Nt-Ac), mediated by N-terminal acetyltransferases (NATs) is one of the most abundant protein modifications occurring approximately in 80% of all eukaryotic proteins. In contrast to the broad spectrum NATs, the human N-alpha-acetyltransferase 40 (NAA40) is highly specific, currently known to Nt-acetylate only the two histone proteins H4 and H2A, which share an Ser(1)-Gly(2)-Arg(3)-Gly(4) N-terminal sequence. Previous work from our lab and others has highlighted the biological and clinical relevance of this NAA40-mediated modification.

Results: In this study, by performing in silico analysis of protein sequences combined with biochemical assays we identify the histone variants H2A.X and H2A.J and the chromatin remodeler SMARCD2 as new potential substrates of human NAA40. Subsequently, focusing on H2A.X, we show for the first time by mass spectrometry analysis that H2A.X is N-terminally acetylated (Nt-acH2A.X) within human cells. Next, we demonstrate that NAA40 specifically interacts and N-terminally acetylates histone H2A.X, in vitro and within cells. Finally, we provide evidence that H2A.X N-terminal acetylation is responsive to Ultraviolet B (UVB)-induced DNA damage and its associated enzyme NAA40 affects the survival of cells exposed to UVB irradiation.

Conclusion: Our findings identify H2A.X as a novel bona fide substrate of NAA40. Moreover, the responsiveness of H2A.X N-terminal acetylation to UV-induced DNA damage indicates that this is a dynamic modification with potential biological functions.

Keywords: Acetylation; Chromatin; DNA damage; Epigenetics; H2A.X; Histone; NAA40; Variant.

Fig. 1

Identification of potential novel human NAA40 substrates. A Human NAA40 is known to acetylate the N-terminal end of proteins starting with ‘SGRG’, occurring on its two reported substrates H2A and H4; B Computational screening for potential NAA40 substrates based on the presence of N-terminal SGRG/MSGRG sequence; C Proteins containing N-terminal SGRG/MSGRG motif found in single species; D Schematic presenting the evolutionary conservation of the ‘SGRG’ motif of H4, H2A, H2A.X, H2A.J, and SMARCD2 across various species; E Quantification of Nt-acetylation of H4, H2A.X, H2A.J and SMARCD2 peptides by recombinant human wild type NAA40 (NAA40^WT) or catalytically inactive NAA40 (NAA40^E139Q) using a Fluorescence assay. F Michaelis constant (Km) and G Catalytic constant (kcat) values of NAA40 against H4, H2A.X, H2A.J and SMARCD2 peptides. H Catalytic efficiency (kcat/Km) of NAA40 against H4, H2A.X, H2A.J and SMARCD2 peptides. Statistical analyses were performed using unpaired two-tailed Student’s t test (*p < 0.05, **p < 0.01). Images created with BioRender.com

Fig. 2

Highly correlated expression of NAA40 with H2A.X. A Plot depicting co-expression score between NAA40 and 17,759 human genes across 1391 datasets relating to cancer. Red circles indicate H2A variants, blue H2A isoforms, pink H4 isoforms, and the green symbol SMARCD2; B Co-expression measured by Pearson correlation for NAA40-H2A.X (left panel); C NAA40-H2A.J (middle panel) and D NAA40-SMARCD2 (right panel) across all samples in the TCGA dataset; E Co-expression measured by Pearson correlation for NAA40-H2AX (left panel); F NAA40-H2A.J (middle panel) and G NAA40-SMARCD2 (right panel) across DepMap cancer cell line panel

Fig. 3

NAA40 interacts with and modifies H2A.X in vitro. A Peptide pull down assay investigating the direct interactions of GST-NAA40^WT and GST-NAA40^E139Q with peptides corresponding to H4, Nt-acetylated H4 (left panel), and H2A.X and Nt-acetylated H2A.X (right panel). Samples were analyzed with Western blot using GST antibody. Dot Blot was used for peptide loading detection. The numbers below each blot indicate densitometry analysis of protein levels after normalization to the peptide loading control; B Quantification of Nt-acetylation of full-length H4, H2A and H2A.X recombinant proteins by the wild type NAA40 (NAA40^WT) or catalytically inactive NAA40 (NAA40^E139Q). H2B was used as a negative control; C Michaelis constant (Km) and D Catalytic constant (kcat) values of NAA40 against H4, H2A and H2A.X recombinant proteins; E Catalytic efficiency (kcat/Km) of NAA40 against H4, H2A and H2A.X recombinant proteins. Statistical analyses were performed using unpaired two-tailed Student’s t test (*p < 0.05, **p < 0.01)

Fig. 4

Histone H2A.X is N-terminally acetylated by NAA40 within cells. A Co-Immunoprecipitation of NAA40 tagged with V5 using an antibody against V5. Nuclear lysates from HCT116 cells in which the expressed NAA40 carries a V5-tag were used for IP, while empty vector (EV) cells, were used as negative control. 10% Input shows expression and loading of proteins. V5-IP shows immunoprecipitated proteins. IgG was used as an additional negative control. Samples were analyzed by Western blotting with the indicated antibodies; B Schematic showing the approach used in mass-spec (MS) analysis to distinguish the N-terminal peptides derived from H2A and H2A.X; C MS/MS spectrum of the (N-ac)SGRGK(ac)TGGK peptide corresponding to the N-terminal end of H2A.X after Lys-C digestion. Charged y (red) and b (blue) ions used to match the peptide sequence with N-terminal acetylation are annotated."b ions"were generated through fragmentation of the peptide bond from the N terminus, and"y ions"were generated through fragmentation of the peptide bond from the C terminus; D Quantitative real-time PCR (qRT-PCR) analysis of NAA40 mRNA levels normalized to β-actin performed in HCT116 cell line expressing doxycycline-inducible scramble shRNA (SCR), or shNAA40. The relative values of NAA40 transcripts represent the mean ± s.d. of three independent experiments; E Western blot analysis of cell extracts derived from dox-treated SCR or NAA40-KD HCT116 cells using antibodies against NAA40 and Actin as loading control; F MS analysis of (Nt-ac)SGRGK(ac)TGGK relative levels in HCT116 SCR and NAA40-KD cells. Data were normalized to the SCR control condition; G MS analysis of (Nt-ac)SGRGK(ac)TGGK levels in two breast cancer cell lines (MDA-MB-231 and SUM159) and two non-cancerous breast epithelial lines (MCF10A and MCF12). Statistical analyses were performed using unpaired two-tailed Student’s t test (*p < 0.05, **p < 0.01)

Fig. 5

Nt-acH2A.XK5ac is reduced upon UVB irradiation and NAA40 regulates resistance to this treatment. A Western blot analysis of γH2A.X in non-treated (NT) or UVB-treated HCT116 cells. Cells were irradiated with UVB (10 mJ/cm²) and allowed to recover for 4 h. Antibodies against total histone H2A.X and Actin were used as loading controls; B MS quantification of (N-ac)SGRGK(ac)TGGK relative levels in HCT116 non-treated (NT) and UVB-treated cells. Data were normalized to the NT control condition; C qRT-PCR analysis of NAA40 mRNA normalized to β-actin in HCT116 NT and UVB-treated cells. Values represent mean ± SD (n = 3); D Western blot analysis of NAA40 in HCT116 NT and UVB-treated cells. Actin served as loading control; E Quantification of colony formation assay in HCT116 SCR and NAA40-KD cells NT or treated with different UVB doses. Survival rates were normalized to untreated cells. Data represent the mean ± SD (n = 3); F Representative images of colony formation assay in HCT116 SCR and NAA40-KD cells, either NT or treated with different UVB doses; G MS analysis of γH2A.X relative levels in HCT116 SCR and NAA40-KD non-treated and UVB-treated cells. Results appear as a ratio of UVB versus non-treated cells; H MS quantification of (N-ac)SGRGK(ac)TGGK relative levels in NAA40-V5 overexpressing (OE) non-treated and UVB-treated cells. Data were normalized to the NT control condition; I Quantification of colony formation assay in HCT116 NAA40-V5 OE and EV cells NT or treated with different UVB doses. Survival rates were normalized to untreated cells. Data represent the mean ± SD (n = 3); J Representative images of colony formation assay in HCT116 NAA40-V5 OE and EV cells, either NT or treated with different UVB doses; K MS analysis of γH2A.X relative levels in HCT116 NAA40-V5 OE and EV cells non-treated and UVB-treated cells. Results appear as a ratio of UVB versus non-treated cells. Statistical analyses were performed using unpaired two-tailed Student’s t test (*p < 0.05, **p < 0.01)