FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4312853831> ?p ?o ?g. }

• W4312853831 abstract "Article Figures and data Abstract Editor's evaluation Introduction Materials and methods Results Discussion Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PALB2 binding to chromatin. In unperturbed cells, the 7K-patch is targeted for KAT2A/2B-mediated acetylation, which in turn enhances the direct association of PALB2 with nucleosomes. Importantly, DNA damage triggers a rapid deacetylation of ChAM and increases the overall mobility of PALB2. Distinct missense mutations of the 7K-patch render the mode of PALB2 chromatin binding, making it either unstably chromatin-bound (7Q) or randomly bound with a reduced capacity for mobilisation (7R). Significantly, both of these mutations confer a deficiency in RAD51 foci formation and increase DNA damage in S phase, leading to the reduction of overall cell survival. Thus, our study reveals that acetylation of the ChAM 7K-patch acts as a molecular switch to enable dynamic PALB2 shuttling for HR repair while protecting active genes during DNA replication. Editor's evaluation The manuscript provides fundamental insights into the role of acetylation of PALB2, a protein involved in Fanconi anemia and homologous recombination though its association with BRCA1 and BRCA2. The evidence that PALB2 acetylation regulates its nuclear mobility is multi-faceted and convincing and the major strength is the definition of the role of de-acetylation of the ChAM domain of PALB2 to mobilize the protein under genotoxic stress. Individuals with an interest in genome stability will be the audience for this important study. https://doi.org/10.7554/eLife.57736.sa0 Decision letter eLife's review process Introduction PALB2, the partner and localizer of the breast cancer susceptibility 2 protein (BRCA2) (Xia et al., 2006), plays essential roles in the maintenance of cellular homeostasis and disease prevention in humans. Biallelic mutations in PALB2 cause Fanconi anaemia (FA), a rare genetic disorder characterised by bone marrow failure, developmental abnormalities, and an increased incidence of childhood cancers (Reid et al., 2007; Xia et al., 2007). Hereditary monoallelic PALB2 mutations also increase the risk of breast and pancreatic cancer (Jones et al., 2009; Rahman et al., 2007), similarly to inherited BRCA1 and BRCA2 mutations (ODonovan and Livingston, 2010). The physiological importance of PALB2 is further highlighted by the recent large-scale functional analysis of PALB2 mutations in cancer patients (Boonen et al., 2019; Rodrigue et al., 2019; Wiltshire et al., 2020). Canonically, PALB2 works together with BRCA1 and BRCA2 to promote error-free repair of highly genotoxic double-strand DNA breaks (DSBs) by homologous recombination (HR) (Ducy et al., 2019). In this process, BRCA1 acts as a DNA damage sensor, which in turn recruits PALB2 and BRCA2 to sites of DNA damage. Subsequently, the essential RAD51 recombinase is recruited to form nucleoprotein filaments, which catalyse the strand invasion and homology search phases of HR repair (Sy et al., 2009b; Xia et al., 2006). Besides the role of PALB2 in promoting HR, our recent study revealed a repair-independent role of PALB2 in protecting transcriptionally active chromatin during DNA replication (Bleuyard et al., 2017b). This role of PALB2 is mediated through its high-affinity binding partner the MORF-related gene on chromosome 15 protein (MRG15), which recognises an epigenetic marker of active genes, histone H3 trimethylated at lysine 36 (H3K36me3), via its N-terminal chromodomain (Bleuyard et al., 2017b; Hayakawa et al., 2010; Sy et al., 2009a). Moreover, PALB2 intrinsic chromatin association is reinforced by its chromatin-association motif (ChAM), an evolutionarily conserved domain uniquely found in PALB2 orthologues, which directly binds to nucleosomes (Bleuyard et al., 2012). Notably, our genome-wide chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq) analyses revealed that PALB2 associates with a small fraction of actively transcribed genes. Notably, locus-specific analyses through ChIP followed by quantitative PCR (ChIP-qPCR) showed a decrease in PALB2 association with these genes upon exposure to an inhibitor of DNA topoisomerase I (TOP1), camptothecin (CPT), suggesting that the mode of PALB2 chromatin association is actively regulated (Bleuyard et al., 2017b). Despite these observations, the regulatory mechanism by which PALB2 switches between different modes of chromatin association (i.e. damage-induced association to promote HR repair versus steady-state association to protect active genes during DNA replication) remains unclear. Numerous studies in recent decades have provided evidence that reversible post-translational modifications (PTMs), such as phosphorylation, ubiquitylation, SUMOylation, poly(ADP-ribosyl)ation, methylation, and acetylation, are orchestrated to promote genome stability, including the DNA damage response (DDR) (Dantuma and van Attikum, 2016). For example, the damage-responsive ATM and ATR kinases mediate phosphorylation of PALB2 at residues S59, S177, and S376, which in turn facilitates PALB2 interaction with BRCA1, RAD51 foci formation, and hence HR repair of DSBs (Ahlskog et al., 2016; Buisson et al., 2017; Guo et al., 2015). Conversely, in G1, the E3 ligase KEAP1-CUL3-RBX1 ubiquitylates PALB2 at K25, a key residue involved in BRCA1 interaction and, in this way, suppresses PALB2-BRCA1 interaction and HR activation (Orthwein et al., 2015). Furthermore, our recent work identified PALB2 as a key substrate of the lysine acetyltransferases 2A (KAT2A/GCN5) and 2B (KAT2B/PCAF) (Fournier et al., 2016), two well-known transcriptional regulators (reviewed in Nagy et al., 2010), in undamaged cells. However, the physiological role of these acetylation events is as yet unknown. Notably, KAT2A/2B use the metabolite acetyl coenzyme A (acetyl-CoA) as a cofactor (Tanner et al., 2000), and hence are proposed to fine-tune cellular processes in accordance with the metabolic status of the cell (Wellen et al., 2009). Therefore, an understanding of the functional significance of PALB2 acetylation would have important implications in the context of tumorigenesis, as cancer cells frequently exhibit reprogrammed metabolism and elevated genome instability (Fouad and Aanei, 2017). In this study, we investigated the role of KAT2A/2B-mediated lysine acetylation in regulating PALB2. We found that KAT2A/2B acetylate a cluster of seven lysine residues (the 7K-patch) within the PALB2 ChAM. ChAM acetylation enhanced its direct association with nucleosomes. Notably, DNA damage triggered rapid ChAM deacetylation and increased the mobility of PALB2. Importantly, lysine to glutamine (Q) or arginine (R) substitutions in the 7K-patch rendered PALB2 either constitutively unbound or non-specifically chromatin-bound, resulting in impaired RAD51 foci formation in S phase and reduced cell survival. On the basis of these observations, we propose that PALB2 chromatin association is dynamically regulated by KAT2A/2B in a context-dependent manner, which plays a significant role in the maintenance of genome stability. Materials and methods Cell culture and cell lines Request a detailed protocol All cells were grown at 37 °C in an incubator with a humidified atmosphere with 5% CO2. HEK293T cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. A U2OS Flp-In T-REx P2shRNA cell line (Bleuyard et al., 2017b), carrying a doxycycline-inducible shRNA targeting the endogenous PALB2 3’-UTR, referred to as U2OS-shPALB2, was used to generate stable isogenic cell lines with constitutive or inducible expression of N3xFLAG- or FLAG-EGFP-PALB2 variants, respectively. U2OS-shPALB2 cells were co-transfected with pOG44 and pcDNA5/FRT GW/N3×FLAG-PALB2 or pcDNA5/FRT/TO/FLAG-EGFP-PALB2 (7Q or 7R), and resultant stable cell lines were selected in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 10 µg/mL blasticidin, 200 μg/mL hygromycin B (100 µg/mL to maintain the cell lines), and 1 µg/mL puromycin. Stable U2OS-shPALB2 lines carrying the empty pcDNA5/FRT GW/N3×FLAG vector or the pcDNA5/FRT GW/N3×FLAG-PALB2 WT vector have been described previously (Bleuyard et al., 2017b). All cell lines tested negative for Mycoplasma contamination using the MycoAlert detection kit (Lonza). Antibodies The primary antibodies used for western blot (WB: with their respective working dilutions) were: anti-FLAG (Sigma F1804, mouse, WB: 1/1000), anti-pan-acetyl lysine (AcK) (Cell Signaling Technology 9441 S, rabbit, WB: 1/1000), anti-PALB2 (Bethyl A301-246A, rabbit, WB: 1/500; in-house antibody raised in rabbit (Rodrigue et al., 2019), WB: 1/5000), anti-BRCA2 (Millipore OP95, mouse, WB: 1/1000), anti-RAD51 (Yata et al., 2014) (7946, rabbit, WB: 1/5000), anti-lamin A (Sigma L1293, rabbit, WB: 1/2000), anti-γ-H2A.X (Millipore 05–636, mouse, WB: 1/1000), anti-GFP (Sigma G1544, mouse, WB: 1/1000), anti-histone H3 (Bethyl A300-823A, rabbit, WB: 1/1000), anti-MRG15 (Cell Signaling Technology D2Y4J, rabbit, WB: 1/1000), anti-BRCA1 (Sigma OP107, mouse, WB: 1/1000), anti-GST (Santa Cruz Biotechnology sc-138, mouse, WB: 1/1000), biotin-HRP conjugated (Sigma A0185, mouse, WB: 1/1000), anti-KAT2A/GCN5 (Cell Signaling Technology 3305, rabbit, WB: 1/1000), anti-α-tubulin (Cell Signaling Technology 3873, mouse, WB: 1/2000) and anti-vinculin (Sigma V9131, mouse, WB: 1/200,000). Secondary antibodies coupled with horseradish peroxidase (HRP): goat anti-mouse (Dako P0447, 1/1000; Jackson ImmunoResearch 515-035-062, 1/20,000), goat anti-rabbit (Dako P0448, 1/1000; Jackson ImmunoResearch 111-035-144, 1/20,000). Antibodies used for immunofluorescence (IF) were: anti-γ-H2A.X (Millipore 05–636, mouse, IF: 1/2000) and anti-RAD51 (BioAcademia 70–001, rabbit, IF: 1/1000). Alexa Fluor conjugated secondary antibodies: goat anti-mouse (Invitrogen A-11001, IF: 1/1000; or Invitrogen A-11017, IF: 1/400) and goat anti-rabbit (Invitrogen A-11011, IF: 1/1000). For ChIP, control IgG (Jackson Immunoresearch 015-000-003, mouse) and anti-FLAG (Sigma F1804, mouse) were used. Plasmids For bacterial expression, full-length PALB2 and fragments 1–4 were PCR amplified using primer pairs, numbered 1 and 8 (full length), 1 and 2 (Fr. 1), 3 and 4 (Fr. 2), 5 and 6 (Fr. 3), or 7 and 8 (Fr. 4) listed in Table 1, from pCMV-SPORT6-PALB2 (IMAGE clone 6045564, Source BioSciences) and cloned into the BamHI/NotI sites of the pGEX-6P-1 vector (GE Healthcare). For mammalian expression of ChAM fragments of varying lengths, PALB2 cDNA was first PCR amplified using primer pairs, numbered 9 and 11 (#1), 9 and 12 (#2), 10 and 12 (#3), 10 and 11 (#4), or 10 and 13 (#5) listed in Table 1, cloned into the BamHI/XhoI sites of the pENTR3C Gateway entry vector (Thermo Fisher Scientific), and subsequently transferred to pcDNA-DEST53 (Invitrogen) using Gateway cloning. PALB2 Q and R missense mutations were introduced by inverse PCR, where 5’-phosphorylated oligonucleotides containing the desired mutations were used to create blunt-ended products, which were then recircularised by intramolecular ligation. For bacteria expression of ChAM missense variants, pGEX4T3-ChAM (Bleuyard et al., 2017b) was modified using primer pairs numbered 18 and 20 (7Q), 18 and 15 (3Q4K), 14 and 20 (3K4Q), or 15 and 16 (3R4K) listed in Table 1. For PALB2 7Q and 7R full-length missense variants, pENTR3C-PALB2 was modified using primer pairs numbered 18 and 19 (7Q), or 16 and 17 (7R) listed in Table 1. To generate N3xFLAG-fusion or FLAG-EGFP-fusion mammalian expression vectors, PALB2 variants were subsequently transferred to pcDNA5/FRT-GW/N3×FLAG using Gateway cloning (Bleuyard et al., 2017b), or cloned into the NotI/XhoI sites of pcDNA5/FRT/TO/FLAG-EGFP (Bleuyard et al., 2017b). Table 1 List of oligonucleotides used in this study. NameSequenceNo.PALB2-F1_fo15’-atggatccatggacgagcctccc-3’1PALB2-F1_re15’-atgcggccgcattagaacttgtgggcag-3’2PALB2-F2_fo15’-atggatccgcacaaggcaaaaaaatg-3’3PALB2-F2_re15’-atgcggccgctgtgatactgagaaaagac-3’4PALB2-F3_fo15’-atggatccttatccttggatgatgatg-3’5PALB2-F3_re15’-atgcggccgcagctttccaaagagaaac-3’6PALB2-F4_fo15’-atggatcctgttccgtagatgtgag-3’7PALB2-F4_re15’-atgcggccgcttatgaatagtggtatacaaat-3’8PALB2_395_Fo5’- actggatcctcttgcacagtgcctg-3’9PALB2_353_Fo5’- actggatccaaatctttaaaatctcccagtg-3’10PALB2_450_Re5’- tatctcgagttaatttttacttgcatccttattttta-3’11PALB2_433_Re5’- tatctcgagttacaaatgactctgaatgacagc-3’12PALB2_499_Re5’- tatctcgagttacaagtcattatcttcagtggg-3’13Patch 1-K-Rev5’-tcagagtcatttggatgtcaagaaaaaaggttt-3’14Patch 2-WT-Fwd5’-aaaaataaaaataaggatgcaagtaaaaat-315Patch 1-R-Rev5’-tcagagtcatttggatgtcaggagaagagggttt-3’16Patch 2-R-Fwd_FL5’-agaaatagaaatagggatgcaagtagaaatttaaacctttccaat-3’17Patch 1-Q-Rev5’-tcagagtcatttggatgtccagcaacaaggttt-3’18Patch 2-Q-Fwd_FL5’-caaaatcaaaatcaggatgcaagtcaaaatttaaacctttccaat-3’19Patch 2-Q-Fwd_ChAM5’-caaaatcaaaatcaggatgcaagtcaaaattgagcggccgcact-3’20Beta-Actin_in3-fo5’-taacactggctcgtgtgacaa-3’21Beta-Actin_in3-re5’-aagtgcaaagaacacggctaa-3’22Chr5_TCOF1_peak2_fo5’-ctacccgatccctcaggtca-3’23Chr5_TCOF1_peak2_re5’-tcagggctctatgaggggac-3’24Chr11_WEE1_mid_fo5’-ggccgaggcttgaggtatatt-3’25Chr11_WEE1_mid_re5’-ataaccccaaagaacacaggtca-3’26 DNA damage and drug treatment Request a detailed protocol For ionising radiation-induced DNA damage, cells were exposed to 4 Gy γ-rays using a 137Cs source delivering a dose rate of 1.68 Gy/min (Gravatom) or a CellRad X-ray irradiator (Precision X-Ray Inc). KDAC inhibition was performed by treating cells with a cocktail of 5 mM sodium butyrate (NaB, Sigma 303410), 5 μM trichostatin (TSA, Sigma T8552) and 0.5 mM nicotinamide (NaM) for 2 hr at 37 °C. DMSO was used as negative vehicle control. siRNA treatment Request a detailed protocol For KAT2A/GCN5 and KAT2B/PCAF knockdowns, U2OS cells at 30% confluence were transfected using DharmaFECT 1 (Dharmacon) according to the manufacturer’s instructions, with 50 pmol each of ON-Targetplus SMARTpools siRNAs targeting KAT2A (Dharmacon L-009722-02-0005) and KAT2B (Dharmacon L-005055-00-0005) in serum-free DMEM. As a negative control, 100 pmol of ON-TARGETplus non-targeting pool siRNAs (Dharmacon D001810-10-05) were used. The serum-free medium was replaced with DMEM supplemented with 10% FBS at 24 hr after transfection, and after further 48 hr incubation, the cells were collected by trypsinisation (total of 72 hr siRNA exposure). Fluorescence recovery after photobleaching (FRAP) Request a detailed protocol Cells were plated into CELLview cell culture dishes (Greiner Bio-One) and analysed in phenol red-free Leibovitz’s L15 medium (Gibco). FRAP experiments were performed on a spinning-disk confocal microscope (Ultra-View Vox, Perkin Elmer) mounted on an IX81 Olympus microscope with an Olympus 60x1.4 oil PlanApo objective, in a controlled chamber at 37 °C and 5% CO2 (TOKAI HIT stage top incubator). The fluorescence signal was detected using an EMCCD camera (ImagEM, Hamamatsu C9100-13). Cells were bleached in the GFP channel at maximum laser power with a single pulse for 20 ms, within a square region of interest of 5 µm2. After bleaching, GFP fluorescence recovery was monitored within the bleached area every second for 40 s. FRAP parameters were controlled using Volocity software 6.0 (Quorum Technologies). FRAP data were fitted and normalised for overall bleaching of the entire cell (whole-cell) using the FRAP plugins in ImageJ/Fiji (https://imagej.net/mbf/intensity_vs_time_ana.htm#FRAP) (Schindelin et al., 2012). From the FRAP curve fitting, half-time recovery time values after photobleaching (t1/2) were extracted and plotted in GraphPad Prism 7.02 (GraphPad Software), in which statistical analyses were performed. Protein purification Request a detailed protocol FLAG-KAT2A, FLAG-KAT2A catalytic mutant, and FLAG-KAT2B proteins were purified as described previously (Fournier et al., 2016). GST-PALB2 full-length and fragments were purified from 1 L of ArcticExpress cells (Agilent Technologies), grown at 37 °C in LB broth medium containing 50 µg/mL ampicillin and 25 µg/mL gentamycin. Protein expression was induced by 0.1 mM IPTG exposure for 24 hr at 13 °C. Cells were collected by centrifugation for 15 min at 1,400 × g at 4 °C and washed with ice-cold phosphate-buffered saline (PBS). Cells lysis was performed by 30 min incubation on ice in 15 mL of ice-cold extraction buffer (50 mM Tris-HCl pH 8.0, 150 mM KCl, 1 mM EDTA, 2 mM DTT, 10% glycerol, and protease inhibitor cocktail (PIC, Sigma P2714)) supplemented with 2 mg/mL Lysozyme (Sigma) and 0.2% Triton X-100, followed by sonication. Cell lysates were collected after 45 min centrifugation at 35,000 × g, 4 °C. GST-fusion proteins were pulled down with glutathione Sepharose 4B beads (GE Healthcare), pre-washed with ice-cold PBS. After overnight incubation at 4 °C on a rotating wheel, beads were washed three times with ice-cold extraction buffer, three times with 5 mL of ice-cold ATP-Mg buffer (50 mM Tris-HCl pH 7.5, 500 mM KCl, 2 mM DTT, 20 mM MgCl2, 5 mM ATP, and 10% glycerol) to release chaperone binding from the recombinant protein PALB2 and three times with ice-cold equilibration buffer (50 mM Tris-HCl pH 8.8, 150 mM KCl, 2 mM DTT, and 10% glycerol). Proteins were eluted from beads in ice-cold elution buffer (50 mM Tris-HCl pH 8.8, 150 mM KCl, 2 mM DTT, 0.1% Triton X-100, 25 mM L-glutathione, and 10% glycerol). For GFP-ChAM purification for mass spectrometry analysis, HEK293T cells (3×107 cells) transiently expressing GFP-ChAM were collected by centrifugation for 5 min at 500 × g, 4 °C and washed once with ice-cold PBS. Cells were further resuspended in 5 mL ice-cold sucrose buffer (10 mM Tris-HCl pH 8.0, 20 mM KCl, 250 mM sucrose, 2.5 mM MgCl2, 10 mM benzamidine hydrochloride (Benz-HCl) and PIC). After addition of Triton X-100 (Sigma) to a final concentration of 0.3% w/v, the cell suspension was vortexed four times for 10 s at 1 min intervals. The intact nuclei were collected by centrifugation for 5 min at 500 × g, 4 °C, and the supernatant was discarded. The nuclear pellet was washed once with ice-cold sucrose buffer and resuspended in ice-cold NETN250 buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 2 mM EDTA, 0.5% NP-40, 10 mM Benz-HCl and PIC). After 30 min incubation on ice, the chromatin fraction was collected by centrifugation for 5 min at 500 × g, 4 °C, washed once with 5 mL ice-cold NETN250 buffer and lysed for 15 min at room temperature (RT) in ice-cold NETN250 buffer supplemented 5 mM MgCl2 and 125 U/mL benzonase (Novagen 71206–3). After addition of EDTA and EGTA to respective final concentrations of 5 mM and 2 mM to inactivate the benzonase and centrifugation for 30 min at 16,100 × g, 4 °C, the supernatant was collected as the chromatin-enriched fraction. GFP-ChAM was pulled down using 15 µL GFP-Trap Agarose (Chromotek), pre-washed three times with ice-cold NETN250 buffer and blocked for 3 hr at 4 °C on a rotating wheel with 500 µL ice-cold NETN250 buffer supplemented with 2 mg/mL bovine serum albumin (BSA, Sigma). After 3 hr protein binding at 4 °C on a rotating wheel, the GFP-Trap beads were collected by centrifugation for 5 min at 1,000 × g, 4 °C and washed four times with ice-cold NETN250 buffer. For the analysis of ChAM acetylation upon DNA damage, a GFP-ChAM fusion was transiently expressed from pDEST53-GFP-ChAM for 24 hr in HEK293T cells. Whole-cell extracts (WCEs) were prepared from ∼1.5×107 cells resuspended in NETN150 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA and 0.5% NP-40 alternative [NP-40 hereafter] [Millipore 492018]) supplemented with 1 mM DTT, PIC, lysine deacetylase inhibitor (5 mM NaB), 1 mM MgCl2 and 125 U/mL benzonase. After 30 min incubation on ice, cell debris was removed by 30 min centrifugation at 4 °C, and the supernatant was collected as WCE. WCE was then incubated with 15 µl of GFP-Trap Agarose for GFP-pull down. After 1 hr protein binding at 4 °C on a rotating wheel, GFP-Trap beads were collected by 5 min centrifugation at 500 × g at 4 °C and washed three times with NET150 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl and 2 mM EDTA) supplemented with 0.1% NP-40, 1 mM DTT, PIC, 5 mM NaB and 1 mM MgCl2. Proteins were eluted from beads by heating at 85 °C for 10 min in Laemmli buffer supplemented with 10 mM DTT. The proteins were separated by SDS-PAGE and analysed by western blot. For the nucleosome pull-down assays, GFP-ChAM variants were affinity-purified from HEK293T cells (3×107 cells) following transient expression. After collecting cells by centrifugation for 5 min at 500 × g, 4 °C, the cell pellet was washed twice with ice-cold PBS and resuspended in ice-cold NETN150 buffer supplemented with 10 mM Benz-HCl and PIC. After 30 min incubation on ice, the chromatin was pelleted by centrifugation for 5 min at 500 × g, 4 °C, and the supernatant was collected as NETN150 soluble fraction and centrifuged for 30 min at 16,100 × g, 4 °C to remove cell debris and insoluble material. For each sample, 10 µL of GFP-Trap Agarose were washed three times with 500 µL ice-cold NETN150 buffer. NETN150 soluble proteins (2.5 mg) in a total volume of 1 mL ice-cold NETN150 buffer were incubated with the GFP-Trap beads to perform a GFP pull-down. After 2 hr incubation at 4 °C on a rotating wheel, the GFP-Trap beads were collected by centrifugation for 5 min at 500 × g, 4 °C and washed four times with ice-cold NETN150 buffer. Human nucleosomes were partially purified from HEK293T cells (4×107 cells), collected by centrifugation for 5 min at 500 × g, 4 °C, washed twice with ice-cold PBS and lysed in ice-cold NETN150 buffer supplemented with 10 mM Benz-HCl and PIC. After 30 min of incubation on ice, the chromatin was pelleted by centrifugation for 5 min at 500 × g, 4 °C, washed once with ice-cold NETN150 buffer and digested for 12 min at 37 °C with 50 gel units of micrococcal nuclease (NEB) per milligram of DNA in NETN150 buffer containing 5 mM CaCl2, using 200 µL buffer per mg of DNA. The reaction was stopped with 5 mM EGTA and the nucleosome suspension cleared by centrifugation for 30 min at 16,100 × g, 4 °C. Acetyltransferase assays Request a detailed protocol Radioactive 14C-acetyltransferase assays on recombinant proteins were performed by incubating purified GST-PALB2 (full-length and fragments) or purified RAD51 with purified FLAG-KAT2B in the presence of 14C-labeled acetyl-CoA in the reaction buffer (50 mM Tris-HCl pH 8.0, 10% glycerol, 100 mM EDTA, 50 mM KCl, 0.1 M NaB, PIC, and 5 mM DTT) for 1 hr at 30 °C. The reactions were stopped by addition of Laemmli buffer containing 10% beta-mercaptoethanol, boiled for 5 min, resolved by SDS-PAGE, and stained using Coomassie blue to reveal overall protein distribution. The acrylamide gel was then dried and exposed to phosphorimager to reveal 14C-labeled proteins. Non-radioactive acetyltransferase assays were performed as described above using cold acetyl-CoA instead. After 1 hr incubation at 30 °C, the reactions were stopped by addition of Laemmli buffer containing 10 mM DTT, boiled for 5 min, resolved by SDS-PAGE, and after Ponceau S staining of the membrane to reveal overall protein distribution, analysed by western blot using anti-acetyl lysine antibody. Acetyltransferase assays performed for mass spectrometry analyses were performed as previously described (Fournier et al., 2016). Nucleosome pull-down assay Request a detailed protocol Nucleosome pull-down assays were performed by mixing 250 µg of partially purified nucleosomes and GFP-ChAM variants immobilised on GFP-Trap beads in NETN150 buffer supplemented with 2 mg/mL BSA, followed by 30 min incubation at RT, then 1.5 h incubation at 4 °C, on a rotating wheel. GFP-Trap beads were further washed four times with NETN150 buffer, and samples were analysed by SDS-PAGE and western blot. Chemical cell fractionation and whole-cell extract Request a detailed protocol HEK293T cells transiently expressing GFP-ChAM variants were collected using TrypLE Express reagent (Gibco), washed twice with ice-cold PBS and resuspended in sucrose buffer (10 mM Tris-HCl pH 7.5, 20 mM KCl, 250 mM sucrose, 2.5 mM MgCl2, 10 mM Benz-HCl and PIC), using 1 mL buffer per 100 mg of weighed cell pellet. After addition of Triton X-100 (Sigma) to a final concentration of 0.3% w/v, the cell suspensions were vortexed three times for 10 s at 1 min intervals. The intact nuclei were collected by centrifugation for 5 min at 500 x g, 4 °C, and the supernatant collected as the cytoplasmic fraction. The nuclei pellet was washed once with ice-cold sucrose buffer and resuspended in ice-cold NETN150 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, 10 mM Benz-HCl and PIC), using 400 µL buffer per 100 mg of initially weighed cell pellet. After 30 min incubation on ice, the chromatin fraction was collected by centrifugation for 5 min at 500 x g, 4 °C and the supernatant collected as nuclear soluble fraction. The chromatin pellet was washed once with ice-cold NETN150 buffer and finally solubilised for 1 hr on ice in NETN150 buffer containing 2 mM MgCl2 and 125 U/mL Benzonase nuclease (Merck Millipore), using 250 µL buffer per 100 mg of initial weighed cell pellet. Cytoplasmic, nuclear soluble and chromatin-enriched fractions were centrifuged for 30 min at 16,100 x g, 4 °C to remove cell debris and insoluble material. For whole-cell extract, cells were directly lysed in NETN150 buffer containing 2 mM MgCl2 and 125 U/mL Benzonase for 1 hr on ice and centrifuged for 30 min at 16,100 x g, 4 °C to remove cell debris and insoluble material. Cell survival Request a detailed protocol U2OS-shPALB2 cells complemented with FLAG-PALB2 WT or its variants were seeded in 96-wells plates and grown in the presence or absence of 2 μg/mL doxycycline for 4 days at 37 °C. Cell survival was then measured using WST-1 reagent (Roche Applied Science) following manufacturer’s protocol. Protein structure prediction with AlphaFold2 Request a detailed protocol The predictions of full-length PALB2 (amino acid 1–1186) and the ChAM variants (amino acid 395–450) were conducted via the ColabFold: AlphaFold2 using MMseqs2 (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb) (Mirdita et al., 2022; Steinegger and Söding, 2017). The resultant structures were visualised using UCSF Chimera (https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004). Immunofluorescence microscopy Request a detailed protocol For γ-H2A.X foci analysis, cells were grown on coverslips and washed with PBS before pre-extraction with 0.1% Triton in PBS for 30 s at RT. Cells were then fixed twice with 4% PFA in PBS, first for 10 min on ice and then for 10 min at RT. After permeabilisation in 0.5% Triton X-100 in PBS for 10 min at RT, cells were blocked with 5% BSA in PBS supplemented with 0.1% Tween 20 solution (PBS-T-0.1) and incubated with anti-γ-H2A.X antibody for 3 hr at RT. After washing with PBS-T-0.1 for 5 min at RT, cells were incubated with secondary antibodies coupled with a fluorophore, washed with PBS-T-0.1 for 5 min at RT, and mounted on slides using a DAPI-containing solution. Cells were analysed on a spinning-disk confocal microscope (Ultra-View Vox, Perkin Elmer) mounted on an IX81 Olympus microscope, with a 40x1.3 oil UPlan FL objective. The fluorescence signal was detected using an EMCCD camera (ImagEM, Hamamatsu C9100-13). Images were processed in Image J (https://imagej.nih.gov/ij/) (Schneider et al., 2012). Click-iT fluorescent EdU labelling and immunofluorescence microscopy Request a detailed protocol For γ-H2A.X and RAD51 foci analysis, U2OS-shPALB2 stably expressing FLAG (EV) or FLAG-PALB2 variants were grown on coverslips in the presence of 2 μg/mL doxycycline for 4 or 5 days. When applicable, cells were exposed to irradiation at 4 Gy using a CellRad X-ray irradiator (Precision X-Ray Inc) and returned at 37 °C for 2 hr 30 min. Then, cells were incubated with 10 μM EdU in media for 30 min at 37 °C before washing with PBS and fixation in 4% PFA in PBS for 10 min. After permeabilisation in 0.5% Triton X-100 in PBS for 5 min, cells were blocked with 1% BSA, 10% goat serum in PBS for 30 min. EdU staining was performed with the Click-iT Alexa Fluor 647 Imaging Kit for 30 min (Invitrogen C10340), using 1/50 the recommended volume of Alexa Fluor azide, and samples were protected from light from this point on. Primary antibody incubation was performed for 1 hr with anti- γ-H2A.X (Millipore 05–636) and anti-RAD51 (BioAcademia 70–001) diluted in PBS-1% BSA at 1:2000 and 1:1000, respectively. Secondary antibodies Alexa Fluor 488 goat anti-mouse (Invitrogen A-11001) and Alexa Fluor 568 goat anti-rabbit (Invitrogen A-11011) were diluted 1:1000 in PBS-1% BSA and applied for 1 hr. Nuclei were stained for 10 min with 4, 6-diamidino-2-phenylindole (DAPI) prior to mounting on slides with ProLong Gold antifade solution (Invitrogen). All immunofluorescence steps were performed at RT with 3 intervening PBS washes. Slide images were acquired on a CellDiscoverer 7 widefield imaging system (Carl Zeiss Microscopy) using a 50 x/1.2 water immersion objective with a ×0.5 magnification changer. Acquired images were pr" @default.
• W4312853831 created "2023-01-05" @default.
• W4312853831 creator A5003311334 @default.
• W4312853831 creator A5014821752 @default.
• W4312853831 creator A5017404099 @default.
• W4312853831 creator A5022291235 @default.
• W4312853831 creator A5033399441 @default.
• W4312853831 creator A5034434657 @default.
• W4312853831 creator A5050294216 @default.
• W4312853831 creator A5055093602 @default.
• W4312853831 creator A5074760080 @default.
• W4312853831 creator A5076716605 @default.
• W4312853831 creator A5077153564 @default.
• W4312853831 creator A5088056326 @default.
• W4312853831 date "2021-08-11" @default.
• W4312853831 modified "2023-10-18" @default.
• W4312853831 title "Author response: KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity" @default.
• W4312853831 doi "https://doi.org/10.7554/elife.57736.sa2" @default.
• W4312853831 hasPublicationYear "2021" @default.
• W4312853831 type Work @default.
• W4312853831 citedByCount "0" @default.
• W4312853831 crossrefType "peer-review" @default.
• W4312853831 hasAuthorship W4312853831A5003311334 @default.
• W4312853831 hasAuthorship W4312853831A5014821752 @default.
• W4312853831 hasAuthorship W4312853831A5017404099 @default.
• W4312853831 hasAuthorship W4312853831A5022291235 @default.
• W4312853831 hasAuthorship W4312853831A5033399441 @default.
• W4312853831 hasAuthorship W4312853831A5034434657 @default.
• W4312853831 hasAuthorship W4312853831A5050294216 @default.
• W4312853831 hasAuthorship W4312853831A5055093602 @default.
• W4312853831 hasAuthorship W4312853831A5074760080 @default.
• W4312853831 hasAuthorship W4312853831A5076716605 @default.
• W4312853831 hasAuthorship W4312853831A5077153564 @default.
• W4312853831 hasAuthorship W4312853831A5088056326 @default.
• W4312853831 hasBestOaLocation W43128538311 @default.
• W4312853831 hasConcept C104317684 @default.
• W4312853831 hasConcept C119157956 @default.
• W4312853831 hasConcept C141231307 @default.
• W4312853831 hasConcept C144133560 @default.
• W4312853831 hasConcept C155202549 @default.
• W4312853831 hasConcept C185592680 @default.
• W4312853831 hasConcept C2780771206 @default.
• W4312853831 hasConcept C54355233 @default.
• W4312853831 hasConcept C70721500 @default.
• W4312853831 hasConcept C83640560 @default.
• W4312853831 hasConcept C86803240 @default.
• W4312853831 hasConceptScore W4312853831C104317684 @default.
• W4312853831 hasConceptScore W4312853831C119157956 @default.
• W4312853831 hasConceptScore W4312853831C141231307 @default.
• W4312853831 hasConceptScore W4312853831C144133560 @default.
• W4312853831 hasConceptScore W4312853831C155202549 @default.
• W4312853831 hasConceptScore W4312853831C185592680 @default.
• W4312853831 hasConceptScore W4312853831C2780771206 @default.
• W4312853831 hasConceptScore W4312853831C54355233 @default.
• W4312853831 hasConceptScore W4312853831C70721500 @default.
• W4312853831 hasConceptScore W4312853831C83640560 @default.
• W4312853831 hasConceptScore W4312853831C86803240 @default.
• W4312853831 hasLocation W43128538311 @default.
• W4312853831 hasOpenAccess W4312853831 @default.
• W4312853831 hasPrimaryLocation W43128538311 @default.
• W4312853831 hasRelatedWork W1986653602 @default.
• W4312853831 hasRelatedWork W2009936943 @default.
• W4312853831 hasRelatedWork W2159081745 @default.
• W4312853831 hasRelatedWork W2383803137 @default.
• W4312853831 hasRelatedWork W2411851904 @default.
• W4312853831 hasRelatedWork W2594022603 @default.
• W4312853831 hasRelatedWork W2884361791 @default.
• W4312853831 hasRelatedWork W2887293316 @default.
• W4312853831 hasRelatedWork W4303449006 @default.
• W4312853831 hasRelatedWork W4360611266 @default.
• W4312853831 isParatext "false" @default.
• W4312853831 isRetracted "false" @default.
• W4312853831 workType "peer-review" @default.