Journal: bioRxiv

Article Title: Poly(ADP-ribose)-binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions

doi: 10.1101/2020.07.27.223131

Figure Lengend Snippet: (A) Western blot analysis of cell lines from with indicated antibodies. (B and C) KDM5A deficiency leads to DNA damage that is exacerbated by PARP inhibition. HCT116 WT or HCT116 KDM5A KO cells were treated ± 5 μM PARPi Olaparib for 24 h and immunostained for the DNA break marker γH2AX. γH2AX foci were quantified and plotted in B. >100 cells were quantified per condition. Error bars represents SEM. (D) Western blot analysis of KDM5 proteins in cells from B. (E) Endogenous KDM5A interacts with PAR chains. Untreated or IR treated U2OS cells were immunoprecipitated with KDM5A antibody and analyzed with indicated antibodies. (F) PAR IP identifies increased KDM5A interactions following DNA damage. Experiment was performed as in . (G) IPed KDM5A from U2OS cells binds PAR. Experiment was performed as in . (H) Analysis of MBP-KDM5A fragments binding to PAR. 10 pM of H2A, BSA, MBP, and MBP-KDM5A fragments F1 to F8 were spotted onto nitrocellulose and PAR binding analysis was performed as in G.

Article Snippet: HCT116 WT or KDM5A KO cells were treated with 5 μM DMSO or Olaparib for 24 h. After treatment, DNA breaks were analyzed using CometAssay Reagent Kit (Trevigen) according to the manufacturer’s instruction.

Techniques: Western Blot, Inhibition, Marker, Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: Poly(ADP-ribose)-binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions

doi: 10.1101/2020.07.27.223131

Figure Lengend Snippet: (A) Clonogenic survival assay of HCT116 WT, KDM5A KO and KDM5A KO + GFP-KDM5A complemented cells. Cells were treated with PARPi, Olaparib at the indicated doses for 24 h, and colonies were quantified after 2 weeks; Error bars represent SD; n=3. (B) Clonogenic survival assay of U2OS cells treated with DMSO, 25 μM CPI455 and Olaparib at indicated doses for 24 h. Colonies were analyzed as in A. Error bars represent SD; n=3. (C) KDM5A suppresses micronuclei formation. HCT116 WT and KDM5A KO cells were treated with 5 μM Olaparib for 24 h and immunostained with DAPI to detect micronuclei (ex. arrows). (D) Quantification of C. >100 cells quantified per condition; n=2. Scale bars, 10 μm. (E) Loss of KDM5A increases DSBs as detected by neutral comet assay. Cells were treated as in D. Scale bars = 100 μm (F) Quantification of E. Olive tail moment for >100 cells quantified per condition; n=2. Error bars represent SEM. P-values were calculated by Turkey’s multiple comparison test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant).

Article Snippet: HCT116 WT or KDM5A KO cells were treated with 5 μM DMSO or Olaparib for 24 h. After treatment, DNA breaks were analyzed using CometAssay Reagent Kit (Trevigen) according to the manufacturer’s instruction.

Techniques: Clonogenic Cell Survival Assay, Neutral Comet Assay, Comparison

Journal: bioRxiv

Article Title: Poly(ADP-ribose)-binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions

doi: 10.1101/2020.07.27.223131

Figure Lengend Snippet: (A) Endogenous KDM5A and PARP1 interact. KDM5A was immunoprecipitated from HEK293T cells ± 10 Gy IR. Samples were analyzed by Western blotting with the indicated antibodies. (B) Reciprocal IP with endogenous PARP1 was performed as in A. (C) KDM5A binds to PAR in cells in a PARP-dependent manner. HEK293T cells were treated with Olaparib (5 μM, 1 h) followed by 10 Gy IR or H 2 O 2 (1mM, 15 min) and examined by IP-Western with an anti-PAR antibody. (D) KDM5A binds PAR non-covalently. U2OS cells were treated with 10 Gy IR for 2 min, and cell lysates were incubated with KDM5A or IgG antibodies ± 1% SDS. Purified samples were either blotted on nitrocellulose membrane to detect PAR chains or resolved on SDS-PAGE and immunoblotted to detect KDM5A. (E) Purified KDM5A from U2OS cells binds PAR chains in vitro . Purified proteins were spotted onto nitrocellulose and probed with purified PAR chains with binding detected with an anti-PAR antibody. (F) Domain organization of KDM5A. Schematics for overlapping KDM5A fragments (F1 to F8) are indicated. (G) Expression of affinity purified MBP-tagged KDM5A fragments. Purified fragments were analyzed by SDS-PAGE and Coomassie staining. (H) Purified MBP and MBP-KDM5A fragments F1 to F8 were analyzed for PAR binding as in E. (I) Schematics for KDM5A F8 and derivatives. Blue box indicates PHD3 domain. (J) PAR binding resides in KDM5A-F8-2 and F8-3. PAR binding was performed as in E with indicated proteins. (K) GFP-KDM5A F8 sufficient for recruitment to laser damage. Laser micro-irradiation was performed in U2OS cells and damaged region is indicated by dotted white circle. (L) Quantification of K from one representative experiment. N ≥10 cells. Error bars represent SEM. AU, arbitrary units. Scale bars, 5 μm.

Article Snippet: HCT116 WT or KDM5A KO cells were treated with 5 μM DMSO or Olaparib for 24 h. After treatment, DNA breaks were analyzed using CometAssay Reagent Kit (Trevigen) according to the manufacturer’s instruction.

Techniques: Immunoprecipitation, Western Blot, Incubation, Purification, Membrane, SDS Page, In Vitro, Binding Assay, Expressing, Affinity Purification, Staining, Irradiation

Journal: bioRxiv

Article Title: Poly(ADP-ribose)-binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions

doi: 10.1101/2020.07.27.223131

Figure Lengend Snippet: (A) KDM5A interacts with macroH2A1. Inducible SFB-KDM5A expressing HEK293T cells were treated with ± 10 Gy IR. SFB-KDM5A was immunoprecipitated with streptavidin beads and interactions were detected by Western blotting. Input shows expression and loading of proteins. (B) Endogenous KDM5A interacts with macroH2A1.2. Co-IPs were performed using HepG2 cells depleted with shRNA control (shCtrl) or shRNA macroH2A (mH2A KD), and complemented shRNA-macroH2A with Flag-macroH2A1.2 or Flag-macroH2A2. (C) KDM5A and macroH2A1.2 interactions are PARP-dependent. SFB-KDM5A expressing HEK293T cells were treated with DMSO or Olaparib (5 μM, 1 h) followed by ± 10 Gy IR treatments. Co-IPs were performed as in A. (D) The PID of KDM5A is required for macroH2A1.2 and PARP1 interactions. HEK293T cells expressing GFP, GFP-KDM5A, or GFP-KDM5AΔPID were analyzed by Co-IP Western blot analysis with the indicated antibodies. (E) Comparison of domain structure of histone H2A and macroH2A variants with PAR and KDM5A binding summary. (F) KDM5A and macroH2A1.2 promote HDR. HR efficiency in WT and KDM5A KO cells ± siRNA depletion of macroH2A1.2 was performed as in ; (n=2). Error bars represent SEM. P-values were calculated using an unpaired Student’s t-test (*, P<0.05; **, P<0.01; ns, not significant).

Article Snippet: HCT116 WT or KDM5A KO cells were treated with 5 μM DMSO or Olaparib for 24 h. After treatment, DNA breaks were analyzed using CometAssay Reagent Kit (Trevigen) according to the manufacturer’s instruction.

Techniques: Expressing, Immunoprecipitation, Western Blot, shRNA, Control, Co-Immunoprecipitation Assay, Comparison, Binding Assay