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. 2018 Sep 25;24(13):3488-3502.e5.
doi: 10.1016/j.celrep.2018.08.092.

Interplay of Histone Marks With Serine ADP-Ribosylation

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Free PMC article

Interplay of Histone Marks With Serine ADP-Ribosylation

Edward Bartlett et al. Cell Rep. .
Free PMC article

Abstract

Serine ADP-ribosylation (Ser-ADPr) is a recently discovered protein modification that is catalyzed by PARP1 and PARP2 when in complex with the eponymous histone PARylation factor 1 (HPF1). In addition to numerous other targets, core histone tails are primary acceptors of Ser-ADPr in the DNA damage response. Here, we show that specific canonical histone marks interfere with Ser-ADPr of neighboring residues and vice versa. Most notably, acetylation, but not methylation of H3K9, is mutually exclusive with ADPr of H3S10 in vitro and in vivo. We also broaden the O-linked ADPr spectrum by providing evidence for tyrosine ADPr on HPF1 and other proteins. Finally, we facilitate wider investigations into the interplay of histone marks with Ser-ADPr by introducing a simple approach for profiling posttranslationally modified peptides. Our findings implicate Ser-ADPr as a dynamic addition to the complex interplay of modifications that shape the histone code.

Keywords: DNA damage; HPF1; PARP1; histone code; histone crosstalk; nucleosome; serine ADP-ribosylation; tyrosine ADP-ribosylation.

Figures

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Figure 1
Figure 1
Modifiers of Serine-ADP-Ribosylation of Histone Peptides (A) Autoradiogram showing ADPr, and subsequent ARH3-mediated glycohydrolysis of H3 1–20aa, H3 27–45aa, H2A 1–17aa, and H4 1–23aa peptides. Coomassie staining of the SDS-PAGE is included and represents the loading control. (B) Autoradiogram showing PARP1/2 + HPF1-mediated ADPr of H3 peptide with Lys9 substituted by Ala and Arg, and Ser10 substituted by Ala. Coomassie staining of the SDS-PAGE is included. (C) 293T cells were transfected with the same amount of empty vector (EV) or plasmid expressing WT, K9A, K9R, or S10A FLAG-tagged histone H3 protein and treated for 10 min with H2O2. Inputs (A) and FLAG-IPs (B) were analyzed by western blotting.
Figure 2
Figure 2
Discovery of Tyrosine as a Target Residue for ADPr (A) Autoradiogram showing ADPr of H3 peptide (1–20aa) with Ser10 substituted by Ala, Thr, Tyr, Glu, and Asp, alongside Lys9 substituted by Arg and Ala. Coomassie staining of the SDS-PAGE is included. (B) High-resolution ETD fragmentation spectrum of an HPF1 peptide modified by ADP-ribose on tyrosine 238. The chemical structure of ADP-ribose is depicted (see also Figure S2B). 1, peaks corresponding to co-isolated species in their original charge state. Multiple species in charge states 2–5 passed through the quadrupole and could not be completely deconvoluted. (C) High-resolution ETD fragmentation spectrum of an HPF1 peptide modified by ADP-ribose on serine 97. The chemical structure of ADP-ribose is depicted. (D) Autoradiogram showing a panel of PARPs incubated with HPF1 protein. Reaction with mono(ADP-ribosyl)ating PARP1 E988Q (EQ) mutant enhances detection of the HPF1 ADPr. Coomassie staining of the SDS-PAGE is included. (E) Autoradiogram showing PARP1 E988Q-mediated ADPr of HPF1 WT, HPF1 R239A, and GST-HPF1 proteins. Coomassie staining of the SDS-PAGE is included. (F) 293T cells were transfected with the same amount of EV or plasmid expressing WT, S97A, or Y238A FLAG-tagged HPF1 protein and left untreated or treated for 10 or 120 min with H2O2. Inputs and FLAG-IPs were analyzed by western blotting. CMV, cytomegalovirus. (G) 293T parental or PARP1 KO cells were transfected with the same amount of EV or plasmid expressing WT FLAG-tagged HPF1 protein and left untreated or treated for 10 or 120 min with H2O2. Inputs and FLAG-IPs were analyzed by western blotting.
Figure 3
Figure 3
Canonical H3 Histone Marks Reduce the Efficiency of H3S10ADPr on H3 Peptide (A) Autoradiogram showing PARP1/2 + HPF1-mediated ADPr of H3 peptide with WT, K9ac, K9me1, and S10ph modifications. Coomassie staining of the SDS-PAGE is included. (B) As in (A), except PARP1 and HPF1 only, with H3 (1–20aa) WT, K9ac, K9me1, K9me2, and K9me3 peptides. Coomassie staining of the SDS-PAGE is included. (C) As in (B), except with H3 (1–20aa) WT, K4ac, K4me3, K9ac, K9me1, K9me3, S10ph, K14ac, K18ac, and K18me3 peptides. Coomassie staining of the SDS-PAGE is included. (D) As in (B), except with H3 (21–44aa) WT, K27ac, K27me1, K27me2, K27me3, and WT peptides. Coomassie staining of the SDS-PAGE is included.
Figure 4
Figure 4
H3S10ADPr Reduces the Efficiency of Subsequent H3K9 Acetylation and H3S10 Phosphorylation (A) Western blot showing PARP1/HPF1 ADPr of H3 (1–20aa) peptide and subsequent p300-mediated acetylation. One reaction was stopped after p300 incubation, then supplemented with ARH3 to remove ADPr before signal detection. Membrane probed with H3K9ac antibody, with H3K9ac peptide included as a positive marker. (B) Western blot showing PARP1/HPF1 ADPr of H3 (1–20aa) peptide and subsequent Baronase-mediated phosphorylation. Control sample excludes NAD from the PARP1/HPF1 reaction. Membrane probed with H3S10ph antibody, with H3S10ph peptide included as a positive marker. (C) Western blot showing PARP1/HPF1 ADPr of H3 (1–20aa) peptide and subsequent Dim5-mediated methylation. Control sample excludes NAD from the PARP1/HPF1 reaction. Membrane probed with H3K9me3 antibody, with H3K9me3 peptide included as a positive marker.
Figure 5
Figure 5
A Technique to Rapidly Analyze ADP-Ribosylated Peptides (A) Schematic representation of the approach to rapidly and easily analyze the modification status of positively charged histone tail peptides. (B) Imperial stained gel showing ADPr of H3 (1–21aa) peptides after addition of PARP1/HPF1 during a 6-hr time course. The upward band shift denotes ADPr of the H3 peptide. (C) Imperial stained gel showing ADPr of H3 (1–21aa) peptides after addition of PARP1/HPF1 and subsequent addition of ARH3. The upward band shift denotes ADPr of the H3 peptide. (D) Imperial stained gel showing H3 (1–21aa) WT, K4me1, K4me2, K4me3, R8me1, R8me2a, K9ac, K9me1, K9me2, K9me3, S10ph, T11ph, and K14ac peptides and subsequent ADPr following addition of PARP1 and HPF1. The upward band shift denotes ADPr of the H3 peptide. (E) A schematic showing a map of histone H3 1–20aa with histone marks that interfere with Ser-ADPr on H3 peptide in vitro.
Figure 6
Figure 6
H3K9ac and S10ADPr Are Mutually Exclusive Histone Marks in Human Cells (A) Autoradiogram showing PARP1 mediated ADPr in the presence of absence of HPF1, with either WT or K9ac human recombinant nucleosomes. Coomassie staining of the SDS-PAGE is included. (B) Western blot showing PARP1/HPF1 ADPr of recombinant human nucleosome and subsequent p300-mediated acetylation. One reaction includes Baronase incubation instead of ADPr reaction, before p300 acetylation reaction. Membrane probed with H3K9ac antibody, with commercially obtained recombinant human H3K9ac nucleosome included as a positive marker. (C) High-resolution ETD fragmentation spectrum of a H3 peptide modified by methyl on lysine 9 and ADP-ribose on serine 10 obtained from Leidecker et al. (2016). The chemical structure of methyl and ADP-ribose are depicted. (D) High-resolution ETD fragmentation spectrum of a H3 peptide modified by acetylation on lysine 9 and lysine 14 obtained from Leidecker et al. (2016). The chemical structure of acetylation is depicted. 1, Peak corresponding to an unfragmented co-eluting, co-isolated +2 precursor deconvoluted into the +1 state. 2, Peak corresponding to an unfragmented co-eluting, co-isolated +3 precursor deconvoluted into the +1 state. (E) Schematic summary of canonical histone H3 marks and their interactions with Ser-ADPr based on the mass spectrometry (MS) analysis of U2OS cell extracts from Leidecker et al. (2016). The marks depicted on the top are H3 marks that can coexist with Ser10 or Ser28 ADPr in vivo, while the H3 marks depicted on the bottom are mutually exclusive with ADPr on Ser10 or Ser28.
Figure 7
Figure 7
Histone Mark Response to DNA Damage with PARP Inhibition and Persistent Ser-ADPr (A) 293T cells were pretreated with DMSO or olaparib and treated with H2O2. Western blotting analysis of the changes in histone H3 K9ac, K9me3, S10P, K14ac, K27ac, K27me3, and K36ac, as well as total pan-Kac histone acetylation and cell-cycle protein levels was performed at the indicated times after the induction of DNA damage. (B) U2OS WT and ARH3 KO cells were treated with H2O2. The levels of H3K9ac, H3K9me3, and pan-Kac were examined by western blotting at the indicated time points.

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