Regulated interaction of the Fanconi anemia protein, FANCD2, with chromatin (original) (raw)
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Identification of the FANCI Protein, a Monoubiquitinated FANCD2 Paralog Required for DNA Repair
Cell, 2007
Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.
Functional Interaction of Monoubiquitinated FANCD2 and BRCA2/FANCD1 in Chromatin
Molecular and Cellular Biology, 2004
Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, and L), and eight FA genes have been cloned. The FANCD1 gene is identical to the breast cancer susceptibility gene, BRCA2. The FA proteins cooperate in a common pathway, but the function of BRCA2/FANCD1 in this pathway remains unknown. Here we show that monoubiquitination of FANCD2, which is activated by DNA damage, is required for targeting of FANCD2 to chromatin, where it interacts with BRCA2. FANCD2-Ub then promotes BRCA2 loading into a chromatin complex. FANCD2 ؊/؊ cells are deficient in the assembly of DNA damage-inducible BRCA2 foci and in chromatin loading of BRCA2. Functional complementation with the FANCD2 cDNA restores BRCA2 foci and its chromatin loading following DNA damage. BRCA2 ؊/؊ cells expressing a carboxy-terminal truncated BRCA2 protein form IR-inducible BRCA2 and FANCD2 foci, but these foci fail to colocalize. Functional complementation of these cells with wild-type BRCA2 restores the interaction of BRCA2 and FANCD2. The C terminus of BRCA2 is therefore required for the functional interaction of BRCA2 and FANCD2 in chromatin. Taken together, our results demonstrate that monoubiquitination of FANCD2, which is regulated by the FA pathway, promotes BRCA2 loading into chromatin complexes. These complexes appear to be required for normal homology-directed DNA repair.
PLoS ONE, 2013
Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.
… and cellular biology, 2007
The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.
Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways
Human Molecular Genetics, 2004
Fanconi Anaemia [FA] is a chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinking [ICL] agents and a high risk of cancer. Six of the eight proteins encoded by the known FA genes form a nuclear complex which is required for the mono-ubiquitination of the FANCD2 protein. FANCD2 complexes and co-localises with BRCA1, but its presumptive role in DNA repair has not yet been clearly defined. We used yeast two hybrid analysis to test for interaction between FANCD2 and 10 proteins involved in homologous recombination repair. FANCD2 did not interact with RAD51, the five RAD51 paralogs, RAD52, RAD54 or DMC1. However, it bound to a highly conserved C-terminal site in BRCA2 that also binds FANCG/XRCC9. FANCD2 and BRCA2 can be co-immunoprecipitated from cell extracts of both human and Chinese hamster wild-type cells, thus confirming that the interaction occurs in vivo . Formation of nuclear foci of FANCD2 was normal in the BRCA2 mutant CAPAN-1 cells, which indicates that the recruitment of FANCD2 to sites of DNA-repair is independent of wildtype BRCA2 function. FANCD2 co-localised with RAD51 in foci following treatment with mitomycin C or hydroxyurea, and co-localised very tightly with PCNA after treatment with hydroxyurea. These findings suggest that FANCD2 may have a role in the cellular response to stalled replication forks or in the repair of replication-associated double-strand breaks, irrespective of the type of primary DNA lesion. by guest on November 1, 2015 http://hmg.oxfordjournals.org/ Downloaded from Homologous recombination repair [HRR] is thought to involve a number of proteins which include BRCA2, RAD51 and its five paralogs [XRCC2, XRCC3, RAD51B, RAD51C and RAD51D], RAD52 and RAD54 (24). We have investigated the by guest on November 1, 2015 http://hmg.oxfordjournals.org/ Downloaded from
Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein
2012
The Fanconi anemia (FA)-BRCA pathway is critical for the repair of DNA interstrand crosslinks (ICLs) and the maintenance of chromosome stability. A key step in FA-BRCA pathway activation is the covalent attachment of monoubiquitin to FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI localize in chromatin-associated nuclear foci where they interact with several well-characterized DNA repair proteins. Importantly, very little is known about the structure, function, and regulation of FANCD2. Herein, we describe the identification and characterization of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain (UBD) in FANCD2, and demonstrate that the CUE domain mediates noncovalent binding to ubiquitin in vitro. We show that while mutation of the CUE domain destabilizes FANCD2, the protein remains competent for DNA damage-inducible monoubiquitination and phosphorylation. Importantly, we demonstrate that the CUE domain is required for interaction with FANCI, retention of monoubiquitinated FANCD2 and FANCI in chromatin, and for efficient ICL repair. Our results suggest a model by which heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently attached to FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation. For personal use only. on October 4, 2017. by guest www.bloodjournal.org From FANCN/PALB2, FANCO/RAD51C and FANCP/SLX4. The FA proteins together with BRCA1 function cooperatively in the FA-BRCA pathway to repair damaged DNA and prevent cellular transformation. 2 Disruption of the FA-BRCA pathway leads to cellular hypersensitivity to the cytotoxic and clastogenic effects of DNA interstrand crosslinking agents. 3 The FA-BRCA pathway is activated following exposure to DNA damaging agents and during S-phase of the cell cycle. 4,5 Activation of the pathway occurs when the core FA complex comprised of FANCA,-B,-C,-E,-F,-G,-L, and-M, and other proteins, assembles in the nucleus and monoubiquitinates the paralogous proteins FANCD2 and FANCI. 4,6,7 FANCL, a RING domain-containing protein is the catalytic E3 ubiquitin ligase subunit of the FA core complex, while UBE2T is the E2 conjugating enzyme. 8-10 Monoubiquitination of FANCD2 and FANCI targets these proteins to discrete chromatin-associated nuclear foci, where they interact with several key DNA repair proteins, including BRCA1, FANCD1/BRCA2, and RAD51. 4,5,11 Following DNA repair, FANCD2 and FANCI are deubiquitinated by the USP1/UAF1 complex facilitating the release of these proteins from chromatin. 12,13 Recent studies indicate that FANCD2 monoubiquitination is necessary for the recruitment of the FAN1 and SLX4/FANCP endonucleases to sites of DNA damage. 14-19 Despite the critical functions of FANCD2 and FANCI in ICL repair, very little is known about their structure, function, and regulation. For personal use only. on October 4, 2017. by guest www.bloodjournal.org From fail to correct the MMC hypersensitivity of FA-D2 (FANCD2-/-) patient cells. Taken together, our results suggest that the heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently linked to K523 of FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation. Methods Cell culture and antibodies PD20 (FA-D2 (FANCD2-/-)), HeLa and COS-7 cells were grown in DMEM media supplemented with 12% v/v FBS, L-glutamine and penicillin/streptomycin. Stable FA-D2 cells were generated by infection with pMMP Moloney murine leukemia or pLenti6.2/V5-DEST (Invitrogen) virus harboring wild type or mutant FANCD2 cDNAs. 4,25 Stable cell lines were grown in DMEM media supplemented with either 1 μg/ml puromycin or 2 μg/ml blasticidin. The following antibodies were used: rabbit polyclonal antisera against FANCD2 (NB100-182; Novus Biologicals), FANCI (Dr. Patrick Sung, Yale University and A300-212A; Bethyl Laboratories), H2A (07-146; Millipore), and mouse monoclonal antisera against α-tubulin (MS-581-PO; Lab Vision) and V5 (R96025; Invitrogen). Immunofluorescence microscopy For immunofluorescence microscopy (IF) freely soluble cellular proteins were pre-extracted with 0.3% v/v Triton X-100 and cells fixed in 4% w/v paraformaldehyde and 2% w/v sucrose at 4°C followed by permeabilization in 0.3% v/v Triton X-100 in PBS. Fixed cells were blocked for 30 minutes in antibody dilution buffer (5% v/v goat serum, 0.1% v/v NP-40, in PBS) and incubated For personal use only. on October 4, 2017. by guest www.bloodjournal.org From 6 with primary antibody for 1 h. Cells were washed three times in PBS and incubated for 30 minutes at room temperature with an Alexa fluor 488-conjugated secondary antibody. Nuclear foci were analyzed using a Zeiss AxioImager.A1 upright epifluorescent microscope with AxioVision LE 4.6 image acquisition software. Immunoprecipitation Cells were lysed in NETN100 (20 mM Tris-HCl pH 7.4, 0.1% v/v NP-40, 100 mM NaCl, 1 mM EDTA, 1 mM Na 3 O 4 V, 1 mM NaF, supplemented with protease inhibitors), incubated on ice and sonicated briefly. 800 µg whole-cell lysates (WCL) were incubated with 3 µg of antibodies against FANCD2 (FI-17; Santa Cruz), V5 (R96025; Invitrogen) or mouse IgG (12-371B; Millipore). Plasmids and site-directed mutagenesis The FANCD2-P204A,-LP215AA, and-LL234AA cDNAs were generated by site-directed mutagenesis of the wild type FANCD2 cDNA using the Quikchange Site-directed Mutagenesis Kit (Stratagene). The forward and reverse oligonucleotide sequences used are as follows: P204A
Interaction of FANCD2 and NBS1 in the DNA damage response
Nature Cell Biology, 2002
Fanconi anaemia (FA) and Nijmegen breakage syndrome (NBS) are autosomal recessive chromosome instability syndromes with distinct clinical phenotypes. Cells from individuals affected with FA are hypersensitive to mitomycin C (MMC), and cells from those with NBS are hypersensitive to ionizing radiation. Here we report that both NBS cell lines and individuals with NBS are hypersensitive to MMC, indicating that there may be functional linkage between FA and NBS. In wild-type cells, MMC activates the colocalization of the FA subtype D2 protein (FANCD2) and NBS1 protein in subnuclear foci. Ionizing radiation activates the ataxia telangiectasia kinase (ATM)-dependent and NBS1-dependent phosphorylation of FANCD2, resulting in an S-phase checkpoint. NBS1 and FANCD2 therefore cooperate in two distinct cellular functions, one involved in the DNA crosslink response and one involved in the S-phase checkpoint response.
Molecular Cell, 2010
FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCMassociated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in (1) destabilization of FANCM and MHF2, (2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, (3) defective chromatin localization of FA nuclear core complex proteins, (4) elevated MMC-induced chromosome aberrations, and (5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.
ATR couples FANCD2 monoubiquitination to the DNA-damage response
Genes & …, 2004
Fanconi anemia (FA) is a multigenic autosomal recessive cancer susceptibility syndrome. The FA pathway regulates the monoubiquitination of FANCD2 and the assembly of damage-associated FANCD2 nuclear foci. How FANCD2 monoubiquitination is coupled to the DNA-damage response has remained undetermined. Here, we demonstrate that the ATR checkpoint kinase and RPA1 are required for efficient FANCD2 monoubiquitination. Deficiency of ATR function, either in Seckel syndrome, which clinically resembles Fanconi anemia, or by siRNA silencing, results in the formation of radial chromosomes in response to the DNA cross-linker, mitomycin C (MMC), thus mimicking the chromosome instability of FA cells.