PMC:6172693 / 26539-33347 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6172693","sourcedb":"PMC","sourceid":"6172693","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6172693","text":"H3K9ac and S10ADPr Are Mutually Exclusive Histone Marks in Human Cells\nWe sought to assess whether the results generated using histone H3 peptides could be replicated with intact human nucleosomes in vitro. WT and H3K9ac recombinant human mononucleosomes were incubated with PARP1 in the presence and absence of HPF1. We observed a clear contrast between the WT and H3K9ac nucleosomes when incubated with PARP1/HPF1, with WT displaying a higher level of Ser-ADPr (Figure 6A). The H3K9ac nucleosomes were still significantly modified, albeit to a lower degree, presumably due to modifications of other previously observed histone tail sites, such as H3S28 and H2BS6 (Leidecker et al., 2016). Similarly, we performed an assay using a nucleosome substrate to test the reciprocal reactions, namely Ser-ADPr, and then acetylation of the nucleosome (Figure 6B). We saw that prior Ser-ADPr reduced subsequent H3K9 acetylation, as detected by the specific anti-H3K9ac antibody (Figure 6B). These results suggest that the interplay that we observe between Ser-ADPr and acetylation of neighboring Lys residues on the peptide level also occurs in the context of whole nucleosomes and in vivo. We also observed that prior H3S10 phosphorylation of the nucleosome also significantly reduced subsequent p300-mediated acetylation of H3K9 (Figure 6B).\nFigure 6 H3K9ac and S10ADPr Are Mutually Exclusive Histone Marks in Human Cells\n(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.\n(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.\n(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.\n(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.\n(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.\nTo compare the interplay observed in our in vitro system with that in cells, we analyzed U2OS cell extracts by high-resolution electron-transfer dissociation (ETD) mass spectrometry (Leidecker et al., 2016). We identified H3S10ADPr in the presence of mono-, di-, and trimethylation of H3K9 and with H3K14ac, but never with H3K9ac (Figures 6C and S6A–S6C). Any detection of H3K9ac was in the absence of H3S10ADPr, although we were able to detect H3K9ac in co-existence with marks other than H3S10ADPr, such as H3K14ac (Figure 6D). To test whether our failure to detect H3K9ac and H3S10ADPr together was due to technical limitations, we purified small amounts of H3K9acS10ADPr peptide generated from a highly inefficient reaction and analyzed it by mass spectrometry. We found that we were able to detect both histone marks on the same peptide (Figure S6D), further indicating that the apparent non-coexistence of these marks is due to the mutual exclusivity in vivo rather than our technical inability of detecting doubly modified H3K9ac/H3S10ADPr peptides. Our findings define two groups of histone H3 PTMs that can either coexist with or are mutually exclusive to Ser-ADPr (Figure 6E).\nTo further characterize the interplay between histone Ser-ADPr and other PTMs in vivo, we assessed the levels of H3S10ph, H3K9ac, H3K9me3, and several other PTMs around the H3S10ADPr site in 293T cells following DNA damage (Figure 7A). Our results confirmed previously published data showing reduction of H3K9ac in response to DNA damage (Tjeertes et al., 2009), because we also observed striking specific deacetylation of the H3K9 site after 120 min of treatment (Figure 7A). We also observed significant deacetylation of H3K14 under the same conditions (Figure 7A). DNA damage-induced deacetylation of both H3K9 and H3K14 was completely blocked by pre-treatment with a PARP inhibitor, olaparib. We did not observe DNA damage-induced deacetylation of H3K27ac or K36ac, or demethylation of H3K9me3 or H3K27me3, among others (Figure 7A). As evident from the patterns for the cell-cycle proteins cyclin A, B1, E1, Cdc2 T15P, PRC1 T481P, and p21 (Figure 7A), the cell cycle was unaffected by olaparib treatment in our experimental settings.\nFigure 7 Histone Mark Response to DNA Damage with PARP Inhibition and Persistent Ser-ADPr\n(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.\n(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.\nTo test our hypothesis that Ser-ADPr specifically affects these canonical histone marks, we performed similar experiments in ARH3 KO cells. We have previously demonstrated that these cells have chronically increased histone ADPr, including the Ser10 site (Fontana et al., 2017, Palazzo et al., 2018). DNA damage-induced deacetylation was more robust in these cells, which was especially obvious at 10 and 120 min post-DNA damage (Figure 7B). It is worth mentioning that some other acetylated proteins, detected by pan-acetylation antibody, displayed a different profile of increasing acetylation after DNA damage treatment (Figure 7B). These results combined suggest that interplay between histone ADPr and K9 acetylation and some other forms of histone modifications takes place in living cells. This knowledge can offer a framework for the further investigation of crosstalk between Ser-ADPr and other histone marks, and onward toward a wider understanding of the physiological function of Ser-ADPr as a histone mark and as a PTM.","divisions":[{"label":"Title","span":{"begin":0,"end":70}},{"label":"Figure caption","span":{"begin":1335,"end":2969}},{"label":"Figure caption","span":{"begin":5195,"end":5775}}],"tracks":[{"project":"2_test","denotations":[{"id":"30257210-27723750-20058719","span":{"begin":684,"end":688},"obj":"27723750"},{"id":"30257210-27723750-20058720","span":{"begin":2102,"end":2106},"obj":"27723750"},{"id":"30257210-27723750-20058721","span":{"begin":2316,"end":2320},"obj":"27723750"},{"id":"30257210-27723750-20058722","span":{"begin":2775,"end":2779},"obj":"27723750"},{"id":"30257210-27723750-20058723","span":{"begin":3171,"end":3175},"obj":"27723750"},{"id":"30257210-19407812-20058724","span":{"begin":4513,"end":4517},"obj":"19407812"},{"id":"30257210-28650317-20058725","span":{"begin":6048,"end":6052},"obj":"28650317"},{"id":"30257210-29480802-20058726","span":{"begin":6070,"end":6074},"obj":"29480802"}],"attributes":[{"subj":"30257210-27723750-20058719","pred":"source","obj":"2_test"},{"subj":"30257210-27723750-20058720","pred":"source","obj":"2_test"},{"subj":"30257210-27723750-20058721","pred":"source","obj":"2_test"},{"subj":"30257210-27723750-20058722","pred":"source","obj":"2_test"},{"subj":"30257210-27723750-20058723","pred":"source","obj":"2_test"},{"subj":"30257210-19407812-20058724","pred":"source","obj":"2_test"},{"subj":"30257210-28650317-20058725","pred":"source","obj":"2_test"},{"subj":"30257210-29480802-20058726","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93c8","default":true}]}]}}