Journal: Nature Communications

Article Title: XPC is an RNA polymerase II cofactor recruiting ATAC to promoters by interacting with E2F1

doi: 10.1038/s41467-018-05010-0

Figure Lengend Snippet: Association of XPC-bound promoters with KAT2A. a Overlapping of peaks for Pol II and XPC with KAT2A peaks in XP-C WT cells after ATRA induction. These peaks correspond to recurrent peaks found in three independent ChIP-seq experiments. b Diagrams representing the fragment depth of KAT2A ChIP-seq experiment for XPC-negatively regulated (left panels) and XPC-positively regulated (right panels) genes, as they were previously determined in XP-C WT and XP-C DEL cells. c UCSC genome browser for XPC and KAT2A at CCND1 and DAPK1 promoters as XPC-negatively regulated and XPC-positively regulated genes, respectively. d Occupancy of KAT2B and KAT2A monitored by ChIP at CCND1 and DAPK1 promoters as XPC-negatively regulated and XPC-positively regulated genes. Error bars represent the standard deviation of three independent experiments

Article Snippet: Antibodies directed toward KAT2A (A4013) were obtained from Epigentek while those against Spt7L (A302–803A) and hSET1 (A300–803A) from Bethyl.

Techniques: ChIP-sequencing, Standard Deviation

Journal: Nature Communications

Article Title: XPC is an RNA polymerase II cofactor recruiting ATAC to promoters by interacting with E2F1

doi: 10.1038/s41467-018-05010-0

Figure Lengend Snippet: XPC interacts directly with KAT2A. a Immunoprecipitation performed on nuclear extract of untreated XP-C WT or XP-C DEL fibroblasts with antibody against GFP, KAT2A, or IgG. b Blue staining of recombinant Flag-KAT2A and duplex strep-XPC/His-HR23B (left panel). In vitro co-immunoprecipitation assay performed by antibodies against Flag tag and IgG with the recombinant protein Flag-KAT2A and the duplex strep-XPC/His-HR23B. c Immunoprecipitation performed on nuclear extract of untreated XP-C WT fibroblasts with antibody against TRRAP, ZZZ3, GFP, or IgG. Western blot was revealed with antibodies directed against GFP, KAT2A, KAT2B, ZZZ3, WDR5 (ATAC), TRRAP, and SPT7L (SAGA). d ChIP experiment monitoring the recruitment of KAT2B, KAT2A, ZZZ3, and TRRAP at CCND1 and DAPK1 promoters, before and after ATRA treatment. Error bars represent the standard deviation of three independent experiments

Article Snippet: Antibodies directed toward KAT2A (A4013) were obtained from Epigentek while those against Spt7L (A302–803A) and hSET1 (A300–803A) from Bethyl.

Techniques: Immunoprecipitation, Staining, Recombinant, In Vitro, Co-Immunoprecipitation Assay, FLAG-tag, Western Blot, Standard Deviation

Journal: Nature Communications

Article Title: XPC is an RNA polymerase II cofactor recruiting ATAC to promoters by interacting with E2F1

doi: 10.1038/s41467-018-05010-0

Figure Lengend Snippet: Ménage-à-trois between XPC, KAT2A, and E2F1. a Diagrams representing the fragment depth of E2F1 from ENCODE ChIP-seq data for XPC-negatively regulated (green) and XPC-positively regulated (blue) genes, as they were previously determined in XP-C WT and XP-C DEL . b ChIP experiment investigating the occupancy of E2F1 at CCND1 and DAPK1 promoters, with or without ATRA treatment. Error bars represent the standard deviation of three independent experiments. c Immunoprecipitation performed on nuclear extracts from untreated XP-C WT fibroblasts with antibody against E2F1 or IgG. Western blot was revealed with antibodies directed against E2F1, XPC, and KAT2A. d Left panel; blue staining of recombinant Flag-KAT2A, duplex Strep-XPC/His-HR23B, and c-myc-E2F1. Right panel; in vitro co-immunoprecipitation assay performed by antibodies against Flag tag with the recombinant protein Flag-KAT2A, the duplex Strep-XPC/His-HR23B, and c-myc-E2F1

Article Snippet: Antibodies directed toward KAT2A (A4013) were obtained from Epigentek while those against Spt7L (A302–803A) and hSET1 (A300–803A) from Bethyl.

Techniques: ChIP-sequencing, Standard Deviation, Immunoprecipitation, Western Blot, Staining, Recombinant, In Vitro, Co-Immunoprecipitation Assay, FLAG-tag

Journal: Nature Communications

Article Title: XPC is an RNA polymerase II cofactor recruiting ATAC to promoters by interacting with E2F1

doi: 10.1038/s41467-018-05010-0

Figure Lengend Snippet: XPC and E2F1 are mutually necessary for their recruitment and co-operate to recruit KAT2A at promoters upon transcription. a Expression of KAT2A and E2F1 in XP-C WT cells treated with siRNA targeting KAT2A (si KAT2A), E2F1 (si E2F1), or scrambled siRNA (si Ctrl) monitored by western blot (left panel) and qPCR (middle and left panels). b Relative mRNA expression of CCND1 and DAPK1 , before and after ATRA treatment in si Ctrl, si KAT2A, and si E2F1 XP-C WT cells. c , d , e Corresponding recruitment of Pol II, XPC ( c ), KAT2A, and E2F1 ( d ) as well as histone H3K9ac/H3 ( e ) at CCND1 and DAPK1 promoters monitored by ChIP. All the error bars represent the standard deviation of three independent experiments

Article Snippet: Antibodies directed toward KAT2A (A4013) were obtained from Epigentek while those against Spt7L (A302–803A) and hSET1 (A300–803A) from Bethyl.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

doi: 10.1007/s00018-024-05469-9

Figure Lengend Snippet: GCN5 is recruited to DNA DSBs post IR and ensures efficient DSB repair kinetics. A) Representative cropped immunofluorescence images of 8 Gy IR treated U87MG and LN229 cells at the indicated time points. Cells were stained for GCN5 (green) and γH2AX (red). Nuclei were stained with DAPI (blue). B) Colocalization between GCN5 and ɣH2AX is shown by calculation of Pearson’s Correlation Coefficient after drawing nuclear ROIs using Coloc 2 Plugin of Fiji (ImageJ). Foci number of each nucleus is calculated using particle counter of Fiji (ImageJ). Significance of each time point post IR was calculated w.r.t no IR. Mean ± SD of Pearson’s R value was plotted (N = 3; ****P < 0.0001; *** P < 0.001; Unpaired Student’s t test). Mean ± SEM of average foci number/nucleus have been plotted (N = 3; ns: non-significant; *P < 0.05; **P < 0.01; ****P < 0.0001). C) Representative cropped immunofluorescence images of untreated and 2 h 4-OHT treated DIvA U2OS cells stained for GCN5 (green) and γH2AX (red). Bar graph represents Mean ± SD of Pearson’s R value (N = 3; P < 0.0001; Unpaired Student’s t test). D) Representative cropped immunofluorescence images of scrambled and GCN5 Knockdown U87MG and LN229 cells at indicated time points post IR stained for γH2AX (red). Images were acquired using Nikon AX confocal microscope. E) Line graphs depicting average nuclear intensities of scrambled (Scr) and GCN5 knockdown (shGCN5) U87MG and LN229 cells at the indicated time points. Mean ± SD were plotted (N = 3; *P < 0.05; *P < 0.01, ***P < 0.001; ****P < 0.0001; Multiple two-tailed t test). F) Neutral comet assays were performed in U87MG and LN229 at indicated time points post 8 Gy IR. Representative images were obtained using Zeiss Upright Microscope at 20X magnification. G) 100 comet tails from each group were taken for quantification of neutral comet assay. Bar graphs depict the average (N = 2) percent tail moment of scrambled (Scr) and GCN5 knockdown (shGCN5) U87MG and LN229 cells at the indicated time points. Mean ± SD were plotted. (**P < 0.001; *P < 0.05; ns: non-significant; Multiple T test)

Article Snippet: Briefly, 25 ng of recombinant GCN5 (Novus Biologicals, NBP1-72,426) and 0.5, 1, 1.5, 2, 2.5 units of purified DNA-PK enzyme (Promega,V5811) or 0.1 μM DNA-PK wild type, K3241A, K3260A, K3241A + K3260A mutant peptides (Supplementary Table S5) or 0.5, 1, 1.5, 2, 2.5 ng of extracted histones or FGF (Promega,PHG0021) were incubated with a mixture containing 2X HAT buffer, HAT Substrate I, HAT substrate II and NADH Generating Enzyme in 96 well Blackwell plate in triplicates at 37 oC for 2 h with shaking in plate reader (Biotek, Cytation5).

Techniques: Immunofluorescence, Staining, Knockdown, Microscopy, Two Tailed Test, Neutral Comet Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

doi: 10.1007/s00018-024-05469-9

Figure Lengend Snippet: PARP1 is required for GCN5 recruitment to DNA DSBs. A-F) Representative cropped immunofluorescence images of DMSO and 10 µm Mirin (A) , 10 µm NU7026 (B) treated, scrambled (Scr) and DNA-PKcs knockdown (siDNA-PKcs) (C), DMSO and 5 µm olaparib treated with IR (D) and 5 µM doxorubicin (E) , scrambled (Scr) and PARP1 knockdown (shPARP1) (F) U87MG with/without 8 Gy IR stained for GCN5 (green) and γ-H2AX (red). For A-F , images were acquired at 63X objective magnification and Pearson’s R value of each nucleus was calculated using Coloc2 plugin of Fiji (ImageJ). Minimum 50 cells for each group were taken for analysis. Data is represented as Mean ± SD (N = 3; ns: non-significant; *p < 0.05, **P < 0.01; Unpaired two-tailed t test with Welch’s correction)

Article Snippet: Briefly, 25 ng of recombinant GCN5 (Novus Biologicals, NBP1-72,426) and 0.5, 1, 1.5, 2, 2.5 units of purified DNA-PK enzyme (Promega,V5811) or 0.1 μM DNA-PK wild type, K3241A, K3260A, K3241A + K3260A mutant peptides (Supplementary Table S5) or 0.5, 1, 1.5, 2, 2.5 ng of extracted histones or FGF (Promega,PHG0021) were incubated with a mixture containing 2X HAT buffer, HAT Substrate I, HAT substrate II and NADH Generating Enzyme in 96 well Blackwell plate in triplicates at 37 oC for 2 h with shaking in plate reader (Biotek, Cytation5).

Techniques: Immunofluorescence, Knockdown, Staining, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

doi: 10.1007/s00018-024-05469-9

Figure Lengend Snippet: PARP1 PARylates GCN5 post genotoxic stress. A) Western blots showing GCN5 and PARP1 after immunoprecipitation with GCN5 and IgG in U87MG and LN229 control, 30 min post 8 Gy IR and 2 h post 5 µM doxorubicin treated cells. Vinculin was used as loading control. B) GCN5-PARP1 interaction foci are shown using Duolink insitu detection reagent red (DU092008) post proximity ligation assay probed with GCN5 (anti-mouse) and PARP1 (anti-rabbit) antibodies in U87MG and LN229 control, IR and doxorubicin treated conditions. Nuclei are stained with DAPI and images are acquired using 63X objective. Mean ± SEM of 60 cells (N = 2) are plotted and unpaired t -test with Welch’s correction has been performed****P < 0.0001. C, D) Western blots showing GCN5 and Poly-ADP Ribose (PAR) following immunoprecipitation with GCN5 and IgG (C) , PAR and IgG (D) in U87MG and LN229 control, 30 min post 8 Gy IR and 2 h post 5 µM doxorubicin treated cells. Actin and vinculin were used as control for U87MG and LN229. E) Western blots of GCN5 and Poly-ADP Ribose (PAR) following immunoprecipitation with GCN5 and IgG in U87MG control and 30 min post 8 Gy IR, in combination with 5 µM olaparib. Actin was used as loading control. F) Western blots of PARP1 and Poly-ADP Ribose (PAR) following immunoprecipitation with GCN5 and IgG in U87MG scrambled and PARP1 knockdown (shPARP1) cells. Actin was used as loading control. The smears obtained in PAR inputs (C-F) indicate all PARylated proteins within the mentioned molecular weight range

Article Snippet: Briefly, 25 ng of recombinant GCN5 (Novus Biologicals, NBP1-72,426) and 0.5, 1, 1.5, 2, 2.5 units of purified DNA-PK enzyme (Promega,V5811) or 0.1 μM DNA-PK wild type, K3241A, K3260A, K3241A + K3260A mutant peptides (Supplementary Table S5) or 0.5, 1, 1.5, 2, 2.5 ng of extracted histones or FGF (Promega,PHG0021) were incubated with a mixture containing 2X HAT buffer, HAT Substrate I, HAT substrate II and NADH Generating Enzyme in 96 well Blackwell plate in triplicates at 37 oC for 2 h with shaking in plate reader (Biotek, Cytation5).

Techniques: Western Blot, Immunoprecipitation, Control, Proximity Ligation Assay, Staining, Knockdown, Molecular Weight

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

doi: 10.1007/s00018-024-05469-9

Figure Lengend Snippet: GCN5 interacts with and acetylates DNA-PKcs required for its phosphorylation. A) Table showing the total combined number of peptides corresponding to each interactor of GCN5 following mass spectrometry post IR and doxorubicin treatment. B, C) Representative western blots of DNA-PKcs, GCN5 and Ku80 following immunoprecipitation with GCN5 and IgG in control, 30 min post 8 Gy IR and 2 h post 5 µM doxorubicin treated U87MG (B) and LN229 (C) cells. Vinculin was used as loading control. D) In vitro acetyltransferase assay was performed using 25 ng purified GCN5 and DNA-PKcs concentrations as indicated. Each line represents the % Histone acetyltransferase (HAT) activity with different DNA-PKcs concentrations at the indicated time points. E) Table shows the amino acid sequences of DNA-PKcs peptides used for acetyltransferase assay. The amino acids highlighted in yellow are the ones that are mutated. In vitro acetyltransferase assay was performed using 25 ng purified GCN5 and 0.1 mM of indicated DNA-PKcs peptides. Each line represents the % Histone acetyltransferase (HAT) activity of each mentioned DNA-PKcs peptide at the indicated time points. F) Western blots showing expression of pDNA-PKcs, DNA-PKcs, GCN5, H3K27Ac in U87MG and LN229 cells transfected with empty (Control) and GCN5 acetyltransferase mutant (Y621A/P622A) vectors, with or without IR. Tubulin and total H3 were used as loading control. Quantification of H3K27Ac w.r.t total H3 has been shown, calculated using ImageJ. Images are representative of 2 biological replicates. G) Representative cropped immunofluorescence images of YFPDNA-PKcs expressing U2OS cells transfected with scrambled (Scr) and 2 shRNAs (shGCN5-1 and shGCN5-2) targeting GCN5 post 4 Gy IR. pDNA-PKcs (red) was stained with Alexa-Fluor (594) and YFP DNA-PKcs imaging was done at 488 nm excitation channel using Nikon AX Confocal microscope at 63X magnification. Fluorescence intensities of mentioned proteins were calculated by drawing nuclear ROIs for minimum 100 cells using Fiji (ImageJ). Data is represented as Mean ± SD (N = 3; Unpaired t test with Welch’s correction was performed; ***P < 0.0001)

Article Snippet: Briefly, 25 ng of recombinant GCN5 (Novus Biologicals, NBP1-72,426) and 0.5, 1, 1.5, 2, 2.5 units of purified DNA-PK enzyme (Promega,V5811) or 0.1 μM DNA-PK wild type, K3241A, K3260A, K3241A + K3260A mutant peptides (Supplementary Table S5) or 0.5, 1, 1.5, 2, 2.5 ng of extracted histones or FGF (Promega,PHG0021) were incubated with a mixture containing 2X HAT buffer, HAT Substrate I, HAT substrate II and NADH Generating Enzyme in 96 well Blackwell plate in triplicates at 37 oC for 2 h with shaking in plate reader (Biotek, Cytation5).

Techniques: Phospho-proteomics, Mass Spectrometry, Western Blot, Immunoprecipitation, Control, In Vitro, Purification, Activity Assay, Expressing, Transfection, Mutagenesis, Immunofluorescence, Staining, Imaging, Microscopy, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

doi: 10.1007/s00018-024-05469-9

Figure Lengend Snippet: GCN5 is responsible for activation of p-ATM and p-BRCA1 at DSBs in U87MG and LN229 cells . A-F) Representative cropped immunofluorescence images of scrambled (Scr) and GCN5 knockdown (siGCN5) U87MG and LN229 cells at 30 min post 8 Gy IR treatment stained for Mre11 (A) , PARP1 (B) , Ku80 (C) , pATM (D) , pBRCA1 (E) , pATR (F) in green. Nuclei were stained with DAPI (blue). 100 cells for each group were taken for analysis. Average foci/nucleus were calculated using particle counter of Fiji (ImageJ). Data is represented as Mean ± SEM (N = 2; Unpaired T test with Welch’s correction; * P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Briefly, 25 ng of recombinant GCN5 (Novus Biologicals, NBP1-72,426) and 0.5, 1, 1.5, 2, 2.5 units of purified DNA-PK enzyme (Promega,V5811) or 0.1 μM DNA-PK wild type, K3241A, K3260A, K3241A + K3260A mutant peptides (Supplementary Table S5) or 0.5, 1, 1.5, 2, 2.5 ng of extracted histones or FGF (Promega,PHG0021) were incubated with a mixture containing 2X HAT buffer, HAT Substrate I, HAT substrate II and NADH Generating Enzyme in 96 well Blackwell plate in triplicates at 37 oC for 2 h with shaking in plate reader (Biotek, Cytation5).

Techniques: Activation Assay, Immunofluorescence, Knockdown, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

doi: 10.1007/s00018-024-05469-9

Figure Lengend Snippet: GCN5 regulates PRKDC expression by acetylating H3K27 in its promoter . A, B) Bar graphs show qPCR analysis of HR and NHEJ pathway associated genes in GCN5 knockdown and scrambled U87MG (A) and LN229 (B) cells at 30 min post IR. Each bar represents the average fold change in transcripts of the respective genes post GCN5 knockdown w.r.t. scrambled. Data is represented as mean ± SEM (N = 3; Multiple two-tailed t-test ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns- not significant). C) Schematic of PRKDC gene regulatory region coordinates that were amplified by qPCR from immunoprecipitated DNA following ChIP by GCN5, H3K27ac and IgG in LN229 cells at 30 min post IR. D-F) ChIP with GCN5 + IgG (D) , H3K27Ac + IgG (E) , GCN5 followed by H3K27Ac + IgG (F) were performed in LN229 cells at 30 min post IR treatment followed by qPCR using the primer sets(S1-S8) corresponding to PRKDC promoter sequences. The bar plots represent the Mean ± SEM of % input enrichment (n = 2; Multiple t test; *P < 0.05). G) Luciferase reporter-based promoter assay result depicted by bar plots representing the fold change of PRKDC promoter (-1273 bp to + 52 w.r.t TSS) w.r.t. luciferase control vector in LN229 cells transfected with GCN5 overexpression construct, GCN5 Acetyltransferase mutant construct (GCN5 Acmutant), GCN5 shRNA. Fold change of shGCN5 has been calculated w.r.t pLKO scrambled and both GCN5 wt and Acmutant w.r.t pcDNA3-EGFP empty vector. Mean ± SEM from 3 replicates have been plotted (Multiple t test; ****P < 0.0001; *P < 0.05)

Article Snippet: Briefly, 25 ng of recombinant GCN5 (Novus Biologicals, NBP1-72,426) and 0.5, 1, 1.5, 2, 2.5 units of purified DNA-PK enzyme (Promega,V5811) or 0.1 μM DNA-PK wild type, K3241A, K3260A, K3241A + K3260A mutant peptides (Supplementary Table S5) or 0.5, 1, 1.5, 2, 2.5 ng of extracted histones or FGF (Promega,PHG0021) were incubated with a mixture containing 2X HAT buffer, HAT Substrate I, HAT substrate II and NADH Generating Enzyme in 96 well Blackwell plate in triplicates at 37 oC for 2 h with shaking in plate reader (Biotek, Cytation5).

Techniques: Expressing, Knockdown, Two Tailed Test, Amplification, Immunoprecipitation, Luciferase, Promoter Assay, Control, Plasmid Preparation, Transfection, Over Expression, Construct, Mutagenesis, shRNA

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

doi: 10.1007/s00018-024-05469-9

Figure Lengend Snippet: Pharmacological and genetic perturbation of PARP1 inhibits GCN5 repair activity in U87MG . A-D) Representative cropped immunofluorescence images of DMSO and Olaparib treated U87MG, with or without 8 Gy IR stained for H3K9Ac (A) , H3K27Ac (B) , pDNA-PKcs (C) , pATM (D) (green) and γ-H2AX (red). E–H) Representative cropped immunofluorescence images of scrambled (Scr) and PARP1 knockdown (shPARP1) U87MG cells, with or without 8 Gy IR stained for H3K9Ac (E) , H3K27Ac (F) , pDNA-PKcs (G) , pATM (H) (green) and γ-H2AX (red). All the images of A-H were acquired at 63X objective magnification and mean fluorescence intensities of mentioned proteins were calculated for individual cells after drawing nuclear ROIs in FIJI (ImageJ). For each group, minimum 60 cells were taken for analysis. Data is represented as Mean ± SD (N = 3; ***p < 0.001, ****p < 0.0001, ns: non-significant, Unpaired two-tailed t test with Welch’s correction)

Article Snippet: Briefly, 25 ng of recombinant GCN5 (Novus Biologicals, NBP1-72,426) and 0.5, 1, 1.5, 2, 2.5 units of purified DNA-PK enzyme (Promega,V5811) or 0.1 μM DNA-PK wild type, K3241A, K3260A, K3241A + K3260A mutant peptides (Supplementary Table S5) or 0.5, 1, 1.5, 2, 2.5 ng of extracted histones or FGF (Promega,PHG0021) were incubated with a mixture containing 2X HAT buffer, HAT Substrate I, HAT substrate II and NADH Generating Enzyme in 96 well Blackwell plate in triplicates at 37 oC for 2 h with shaking in plate reader (Biotek, Cytation5).

Techniques: Activity Assay, Immunofluorescence, Staining, Knockdown, Fluorescence, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

doi: 10.1007/s00018-024-05469-9

Figure Lengend Snippet: PARP1 and GCN5 inhibition radio-sensitizes GBM cells due to impaired HR and NHEJ . A) Illustration of HR and NHEJ vectors, obtained from Dr. Vera Gorbunova, Department of Biology, University of Rochester, NY. B, C) HR and NHEJ vector reactivation assay was performed in scrambled (Scr) and PARP1 knockdown (B) or GCN5 knockdown (C) U87MG cells transfected with TdRed and HR or NHEJ vectors. % Repair efficiency was calculated by counting the number of GFP + /TdRed + cells*100 obtained by flow cytometry at 72 h post transfection. Bar graphs represent percent repair efficiency in each set as indicated. Data is represented as Mean ± SD. (N = 2; *P < 0.05; Unpaired two-tailed t test with Welch’s correction). D, E) Line graph shows the % surviving fraction of U87MG and LN229 cells transfected with either scrambled (Scr) or GCN5 shRNA (shGCN5) construct at the indicated doses of radiation. Data is represented as Mean ± SD (N = 3; **P < 0.01; ***p < 0.001, Two-tailed Multiple t test). F) Line graph shows the % surviving fraction of LN229 cells transfected with either vector control (pLKO 3.1 empty vector) or GCN5 overexpression construct at the indicated doses of radiation. Data is represented as Mean ± SD (N = 3; *P < 0.05, **P < 0.01, Two-tailed Multiple t test). G, H) Graph shows the number of cells counted by Trypan blue viability assay of scrambled (Scr) and GCN5 knockdown (shGCN5) U87MG (G) and LN229 (H) cells with or without 8 Gy IR, at the indicated days post radiation treatment. Data is represented as Mean ± SD (N = 3). The significance of shGCN5 was calculated w.r.t Scr, IR + shGCN5 w.r.t IR + scr for the mentioned time points (**P < 0.01, ***P < 0.001, ****P < 0.0001; Two-tailed Multiple t test). I) Trypan blue viability count of vector control and GCN5 acetyltransferase mutant (GCN5 Acmutant) transfected LN229 cells after exposure to 8 Gy IR, at the indicated days post radiation treatment. Data is represented as Mean ± SD (N = 3). The significance of GCN5 Acmutant was calculated w.r.t vector control, IR + Acmutant w.r.t IR + vector control for the mentioned time points (**P < 0.01, ****P < 0.0001, Two-tailed Multiple t test). J) Working model highlighting the detailed molecular mechanism of DSB repair regulation by GCN5

Article Snippet: Briefly, 25 ng of recombinant GCN5 (Novus Biologicals, NBP1-72,426) and 0.5, 1, 1.5, 2, 2.5 units of purified DNA-PK enzyme (Promega,V5811) or 0.1 μM DNA-PK wild type, K3241A, K3260A, K3241A + K3260A mutant peptides (Supplementary Table S5) or 0.5, 1, 1.5, 2, 2.5 ng of extracted histones or FGF (Promega,PHG0021) were incubated with a mixture containing 2X HAT buffer, HAT Substrate I, HAT substrate II and NADH Generating Enzyme in 96 well Blackwell plate in triplicates at 37 oC for 2 h with shaking in plate reader (Biotek, Cytation5).

Techniques: Inhibition, Plasmid Preparation, Knockdown, Transfection, Flow Cytometry, Two Tailed Test, shRNA, Construct, Control, Over Expression, Viability Assay, Mutagenesis

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: Identification of KAT2A as a host factor promoting cccDNA transcription. (A) The reported succinyltransferases and desuccinylases were summarised in the table. (B) After 4 days of siRNA treatment, total RNA was extracted by TRNzol Universal reagent. The silencing efficiency of related genes in Huh-7 cells was analysed by real-time PCR with specific primers. β-actin was used as the internal control. (C) The indicated siRNA (50 pmol) and monomeric linearised HBV DNA (1 μg) were co-transfected into huh-7 cells by using Lipofectamine™ 3000 Transfection Reagent. The supernatant was collected and HBeAg and HBsAg levels were detected by ELISA. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A knockdown suppresses cccDNA transcription in the HBV infection cell model. HepG2-NTCP cells were infected HBV particles at 1-day post shRNA transduction. At 4 days after infection. (A) The expression of KAT2A was detected by western blot. (B) Northern blotting and real-time PCR were used to analyse the effect of KAT2A knockdown on HBV RNAs. (C) HBV cccDNA was extracted by a modified Hirt DNA extraction protocol and then Southern blotting and Taq-man probe qPCR were used to detect the HBV cccDNA level. The mitochondrial gene Cox1 was hybridised as the loading control for HBV cccDNA. (D) The level of HBV 3.5-kb RNA, total HBV RNAs, and cccDNA were used to calculate the ratio of HBV 3.5-kb RNA to cccDNA and total HBV RNAs to cccDNA. (E) HBV core DNA was quantified by Southern blotting and absolute quantification PCR. The level of β-actin was used as a loading control for HBV core DNA. (F) HBV core protein (HBc) was produced by HBV infected HepG2-NTCP cells. Intracellular HBc was visualised by immunofluorescence staining. The scale bar is 100 μm. (G) Western blot was used to detect the level of HBsAg in cells (left). Cell culture supernatant was collected for HBsAg and HBeAg analysis via ELISA (right). Cox1, cyclooxygenase 1; rRNA, ribosomal RNA. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Infection, shRNA, Transduction, Expressing, Western Blot, Northern Blot, Real-time Polymerase Chain Reaction, Modification, DNA Extraction, Southern Blot, Produced, Immunofluorescence, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A overexpression promotes HBV transcription in the HBV infection cell model. HepG2-NTCP cells were infected HBV particles at 1-day post lentivirus expressing KAT2A plasmids transduction. At 4 days after infection. (A) The expression of KAT2A was detected by western blot. (B) The effect of KAT2A overexpression on HBV RNAs was analysed by Northern blotting and real-time PCR. (C) Taq-man probe qPCR was used to detect the HBV cccDNA level. (D) The level of HBV 3.5-kb RNA, total HBV RNAs, and cccDNA were used to calculate the ratio of HBV 3.5-kb RNA to cccDNA and total HBV RNAs to cccDNA. (E) HBV core DNA was quantified by Southern blotting and absolute quantification PCR. The level of β-actin was used as a loading control for HBV core DNA. (F) HBc was produced by HBV-infected HepG2-NTCP cells. Intracellular HBc was visualised by immunofluorescence staining. The scale bar is 100 μm. (G) Cell culture supernatant was collected for HBsAg and HBeAg analysis via ELISA. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Over Expression, Infection, Expressing, Transduction, Western Blot, Northern Blot, Real-time Polymerase Chain Reaction, Southern Blot, Produced, Immunofluorescence, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A bound to cccDNA and regulated H3K79 succinylation on the cccDNA minichromosome. (A) HepG2-NTCP cells were infected with HBV particles for 4 days, HBV core protein and endogenous KAT2A were observed by immunofluorescence assay using the specific antibodies. The scale bar is 10 μm. (B) Cross-linked chromatin from the HBV-infected and non-infected HepG2-NTCP nucleus was immunoprecipitated with a specific antibody or the control lgG. Taq-man probe qPCR was used to detect the HBV cccDNA level. ChIP results are expressed as% of input. (C–E) HepG2-NTCP cells were infected with HBV particles at 1-day post shKAT2A transduction. On 4 days post-infection. (C) The levels of H3ac, H3K9ac, and H3K14ac associated with cccDNA, GAPDH, or MYH6 promoter were analysed by ChIP assay with anti-H3ac, anti-H3K9ac, anti-H3K14ac, and the corresponding IgG, respectively. (D) ChIP assay was performed with anti-Pan-succ antibody. (E) The levels of H3K79succ and H3K122succ associated with cccDNA, GAPDH, or MYH6 promoter were analysed by ChIP assay with anti-H3K79succ, anti-H3K122succ, and the corresponding lgG, respectively. (F) ChIP-Seq analysis of the H3K79succ modification on the cccDNA minichromosome. The HBV-specific reads were quantified and normalised by the reads mapped to the human genome. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Infection, Immunofluorescence, Immunoprecipitation, Transduction, ChIP-sequencing, Modification

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A binds to the cccDNA minichromosome through interaction with HBc. (A) HepG2-NTCP cells were co-transfected with plasmids encoding KAT2A and Flag-HBc for 3 days, the cells were subjected to Co-IP assay with the indicated antibody. The expression of the indicated proteins was analysed by western blot. (B,C) HepG2-NTCP cells were transduced with lentivirus expressing KAT2A or shKAT2A for 24 h, then infected with HBV wild type virus (HBV WT virus) and HBc-deficient virus (HBV-ΔHBc virus) for 4 days. Total HBV RNAs and cccDNA were extracted and quantified by real-time PCR and Taq-man probe qPCR for calculating the ratio of total HBV RNAs to cccDNA. The HBc proteins were detected by immunoblotting analysis. (D) HepG2-NTCP cells in 100 mm dishes were infected by HBV WT virus and HBV-ΔHBc virus. The cells were used for ChIP assays with the anti-KAT2A antibody. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Transfection, Co-Immunoprecipitation Assay, Expressing, Western Blot, Transduction, Infection, Real-time Polymerase Chain Reaction

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: Antiviral activity of KAT2A knockdown in vivo . (A) Schematic depiction of experiments in C57BL/6 mice. (B) Serum HBV DNA was detected by absolute quantification PCR. (C) Serum HBsAg was measured via ELISA. (D) HBV 3.5-kb RNA and total HBV RNAs in liver tissue were measured by real-time PCR. (E) HBV cccDNA in liver tissue was detected by Taq-man probe qPCR. (F) The level of HBV DNA in liver tissue was detected by absolute quantification PCR. (G) HBc in liver tissues was analysed by IHC. The scale bar is 50 μm. (H,I) The levels of H3K9ac, H3K14ac associated with cccDNA were analysed by ChIP assay. (J) The level of H3K79succ associated with cccDNA was analysed by ChIP assay. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Activity Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction