Cancer-causing mutations in the tumor suppressor PALB2 reveal a novel cancer mechanism using a hidden nuclear export signal in the WD40 repeat motif

Pauty, Joris; Couturier, Anthony M.; Rodrigue, Amélie; Caron, Marie-Christine; Coulombe, Yan; Dellaire, Graham; Masson, Jean‐Yves

doi:10.1093/nar/gkx011

Cited by 35 publications

(31 citation statements)

References 36 publications

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“…We show that in vitro , the native human SAGA complex (affinity-purified through its specific SUPT7L subunit; Figure S3B) displays both H2BK120 deubiquitinase and acetyltransferase activities, making it a primary candidate for this switch (Figure S3C). Furthermore, depletion of SUPT7L as well as PCAF, one HAT paralog present in the SAGA complex (Figures S3D and S3E), triggered decreases in both HR at an endogenous locus ( LMNA ) following CRISPR/Cas9 breakage (Pauty et al., 2017) and NHEJ in a cell reporter system (Jacquet et al., 2016) (Figures S3F and S3G), indicating that SAGA indeed contributes to DSB repair in human cells.…”

Section: Resultsmentioning

confidence: 99%

“…Cell Line 4D-Nucleofector X KitLonzaCat# V4XC1012 Deposited Data Raw data (ChIP-seq and BLESS)This paperArrayExpress: E-MTAB-5817RAD51 and XRCC4 ChIP-seqAymard et al., 2014ArrayExpressE-MTAB-1241RNA polymerase II S2P ChIP-seqCohen et al., 2018ArrayExpressE-MTAB-6318MethylCap-seqDeplus et al., 2014GEO GSE26810 Experimental Models: Cell Lines DIvA cellIacovoni et al., 2010N/AU20S-ISceI GFP-RFP (NHEJ)Jacquet et al., 2016N/A3xFlag-Twin-strep-tagged SUPT7L K562Dalvai et al., 2015N/AU2OSN/AATCC HTB-96, RRID:CVCL_0042 Oligonucleotides HR-DSB1 for ChIP-qPCRFW GATTGGCTATGGGTGTGGACREV CATCCTTGCAAACCAGTCCTAymard et al., 2014N/AHR-DSB2 for ChIP-qPCRFW CCGCCAGAAAGTTTCCTAGAREV CTCACCCTTGCAGCACTTGAymard et al., 2014N/ANHEJ-DSB for ChIP-qPCRFW TGCCGGTCTCCTAGAAGTTGREV GCGCTTGATTTCCCTGAGTAymard et al., 2014N/AACTB for ChIP-qPCRFW AGCCGGGCTCTTGCCAATREV AGTTAGCGCCCAAAGGACCAThis paperN/ATAF12 for ChIP-qPCRFW GCTGAGACGAACGCTTCACTREV CCTTCGAACACTGACCCACTThis paperN/AsiRNA siSUPT7L-46 CUACUAGACCCAACAGAAA[dT] [dT] UUUCUGUUGGGUCUAGUAG[dT] [dT]This paperN/AsiRNA siSUPT7L-47 CUAUCACAGUUACAUGCUA[dT] [dT]UAGCAUGUAACUGUGAUAG[dT] [dT]This paperN/AsiRNA siKAT2B (PCAF) CUCUAAUCCUCACUCAUUU[dT] [dT]AAAUGAGUGAGGAUUAGAG[dT] [dT]This paperN/AsiRNA siKAT2A (GCN5) GCUACUACGUGACCCGGAA[dT] [dT]UUCCGGGUCACGUAGUAGC[dT] [dT]This paperN/A Recombinant DNA pX330-LMNAgRNA1Pauty et a...…”

Section: Methodsmentioning

confidence: 99%

“…The pX330-LMNAgRNA1 plasmid used in this study was modified from the original plasmid to remove the 3XFlag tag from the Cas9 endonuclease previously described (Pauty et al., 2017). U2OS cells were seeded and transfected with indicated siRNA using Lipofectamine RNAiMAX (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

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Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

et al. 2018

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SummaryDouble-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

et al. 2018

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“…On one hand, the trafficking of a specific protein can be altered by mutations that either interfere with the ac-tivity of its transport signals (NLSs or NESs) or that create a novel signal in the mutant protein. For example, aberrant localization of tumor suppressors BRCA2 [67] and PALB2 [68] to the cytoplasm can result from mutations that unmask normally hidden NESs, whereas cytoplasmic mislocalization of certain NPM1 mutants is the result of a frameshift mutation that creates a novel strong NES, not present in the wild-type protein [69] .…”

Section: Altered Nucleocytoplasmic Localization Of Proteins In Cancermentioning

confidence: 99%

Hitting a moving target: inhibition of the nuclear export receptor XPO1/CRM1 as a therapeutic approach in cancer

Sendino¹,

Omaetxebarria²,

Rodríguez³

2018

CDR

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Cellular homeostasis crucially relies on the correct nucleocytoplasmic distribution of a vast number of proteins and RNA molecules, which are shuttled in and out of the nucleus by specialized transport receptors. The nuclear export receptor XPO1, also called CRM1, mediates the translocation of hundreds of proteins and several classes of RNA to the cytoplasm, and thus regulates critical signaling pathways and cellular functions. The normal function of XPO1 appears to be often disrupted in malignant cells due to gene mutations or, most commonly, aberrant overexpression. Due to its important physiological roles and its frequent alteration in human tumors, XPO1 is a promising target for cancer therapy. XPO1 inhibitors have undergone extensive testing as therapeutic agents in preclinical models of cancer, with promising results. One of these inhibitors, Selinexor, is currently being evaluated in multiple clinical trials of different types of solid tumors and hematological malignancies. Here, we review several key aspects of XPO1 function, as well as the mechanisms that may lead to its alteration in cancer, and provide an update on the status of XPO1 inhibitors being developed as drugs for cancer therapy, including the definitive results of the first clinical trials with Selinexor that have been recently published. Figure 1. Receptor-mediated nucleocytoplasmic transport of proteins. A: Illustrative overview of the nuclear import of a cargo protein bearing a "classical" NLS mediated by the Importina/Importinb heterodimer (left) and the nuclear export of a cargo protein bearing a "leucine-rich" NES mediated by XPO1 (right); B: schematic depiction of the nuclear pore complex, illustrating its main structural features; C: examples of nucleocytoplasmic transport signals. The NLSs of SV40 large T antigen (monopartite) and nucleoplasmin (bipartite) are shown, with the basic residues that characterize "classical" NLSs highlighted in red. Below, the NES of PKI and a general consensus sequence of "leucine-rich" NESs are shown. The characteristic hydrophobic residues (represented by f in the consensus) are highlighted in colors and underlined. NLS: nuclear localization signal; NES: nuclear export signal Conception of the work, draft and revision of the work: Sendino M, Omaetxebarria MJ, Rodríguez JA

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“…The resulting siRNA-resistant construct was 510 then used as a template to generate the mutant construct YFP-PALB2 146AAAA with the primers 511 JYM3909/JYM3910. Flag-tagged PALB2 146AAAA mutant was also obtained via site-directed 512 mutagenesis on pcDNA3-Flag PALB2 (Pauty et al, 2017). 513…”

Section: Plasmids and Sirna 508mentioning

confidence: 99%

Novel strand exchange activity of the human PALB2 DNA Binding Domain and its critical role for DNA repair in cells

Deveryshetty

Ryzhikov

Brahiti³

et al. 2018

Preprint

Self Cite

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17Breast cancer associated proteins 1 and 2 (BRCA1, -2) and partner and localizer of BRCA2 (PALB2) 18 protein are tumor suppressors linked to a spectrum of malignancies, including breast cancer and 19Fanconi anemia. They stimulate RAD51 recombinase during homology-directed repair (HDR). Along 20 with being a hub for a protein interaction network, PALB2 interacts with DNA. The mechanism of 21 PALB2 DNA binding and its function are poorly understood. We identified a major DNA-binding site 22 4.0±1.3 nM to 316±59 nM in the case of T1 and from 4.8±0.4 nM to 167±50 nM in the case of PB2-129 573 (Fig. 2). DNA binding was moderately affected by mutations of two other clusters, including 130 K45A/K50A, for which the Kd was increased to 28±5.2 nM (Fig. S4), and the triple mutant 131 R170A/K174A/R175A with similar change in Kd. From these experiments, we concluded that the main 132 DNA binding site is formed by residues 146-149 with a potential minor contribution from other basic 133 amino acids of the N-DBD. 134 135 Figure 2. Mutation of DNA binding residues. Isotherm of fluorescence anisotropy of FAM-ss49 (5 136 nM) titrated by PALB2 T1 (blue, open circles) and PB2-573 (magenta, open triangles) fragments and 137 their mutants: T1 146-RK/AA (filled blue circles), T1 146-RRKK/AAAA (crossed open blue circles), 138and 573 146-RRKK/AAAA (filled magenta triangles) under conditions identical to those in Fig. 1. 139 Figure S4. DNA binding of T1 mutants. Equilibrium binding of 5 nm D) to PALB2 T1 (blue). In A) and B) isotherm for K45A is in green, for K50A in red, for K45A/K50A 141 in black. In C) and D) isotherm for R170A is in red, for K174A in green, for R170A/K174A in magenta, 142 for R170A/R175A in orange, and for R170A/K174A/R175A in black. The binding buffer is 20 mM Tris-143 acetate pH 7.0, 100 mM NaCl, 5% glycerol, 10% DMSO. 144

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Cancer-causing mutations in the tumor suppressor PALB2 reveal a novel cancer mechanism using a hidden nuclear export signal in the WD40 repeat motif

Cited by 35 publications

References 36 publications

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

Hitting a moving target: inhibition of the nuclear export receptor XPO1/CRM1 as a therapeutic approach in cancer

Novel strand exchange activity of the human PALB2 DNA Binding Domain and its critical role for DNA repair in cells

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