Ser-ADPr on H3S10 Prevents the Efficient Incorporation of H3K9 Acetylation and H3S10 Phosphorylation
We conducted reciprocal experiments based on our above findings, this time modifying histone H3 peptide first with PARP1/HPF1 complex (Figure S5), then subsequently incubating the reaction products in acetylation, phosphorylation, and methylation reaction mixtures using the purified catalytic domain of p300, the activated fragment of Aurora B kinase (Baronase; to phosphorylate H3S10) and Dim5 methyltransferase (Nunes Bastos et al., 2013, Moonat et al., 2013, Zhang et al., 2003). We detected the acetylated products of Ser-ADPr H3 peptides using a specific H3K9ac antibody and observed that K9ac is effectively prevented if the peptide is previously ADPr (Figure 4A, lane 5). To control for any possible interference of ADPr with western blot detection, we incubated the Ser-ADPr H3 peptide with p300, stopped the reaction, and removed Ser-ADPr from the peptide using ARH3. This assay showed only a negligible amount of H3K9Ac (Figure 4A, lane 6). In a similar experiment, we saw that Ser-ADPr of H3 peptide prevented subsequent H3S10 phosphorylation (Figure 4B, lane 5). Finally, we incubated an Ser-ADPr H3 peptide in an Lys methylation reaction and found that H3S10ADPr did not preclude the incorporation of H3K9me3, although it did substantially reduce the efficiency of the reaction compared to the unmodified H3 peptide (Figure 4C, lane 3 versus lane 5).
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.