Title: FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway
Abstract: Article29 March 2007free access FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway Chen Ling Chen Ling Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Masamichi Ishiai Masamichi Ishiai Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki, Okayama, Japan Search for more papers by this author Abdullah Mahmood Ali Abdullah Mahmood Ali Division of Experimental Hematology, Cincinnati Children's Hospital Research Foundation and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Annette L Medhurst Annette L Medhurst Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands Search for more papers by this author Kornelia Neveling Kornelia Neveling Department of Human Genetics, University of Wurzburg, Wurzburg, Germany Search for more papers by this author Reinhard Kalb Reinhard Kalb Department of Human Genetics, University of Wurzburg, Wurzburg, Germany Search for more papers by this author Zhijiang Yan Zhijiang Yan Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Yutong Xue Yutong Xue Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Anneke B Oostra Anneke B Oostra Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands Search for more papers by this author Arleen D Auerbach Arleen D Auerbach Laboratory of Human Genetics and Hematology, The Rockefeller University, New York, NY, USA Search for more papers by this author Maureen E Hoatlin Maureen E Hoatlin Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Detlev Schindler Detlev Schindler Department of Human Genetics, University of Wurzburg, Wurzburg, Germany Search for more papers by this author Hans Joenje Hans Joenje Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands Search for more papers by this author Johan P de Winter Johan P de Winter Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands Search for more papers by this author Minoru Takata Minoru Takata Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki, Okayama, Japan Department of Human Genetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan Search for more papers by this author Amom Ruhikanta Meetei Corresponding Author Amom Ruhikanta Meetei Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Division of Experimental Hematology, Cincinnati Children's Hospital Research Foundation and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Weidong Wang Corresponding Author Weidong Wang Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Chen Ling Chen Ling Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Masamichi Ishiai Masamichi Ishiai Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki, Okayama, Japan Search for more papers by this author Abdullah Mahmood Ali Abdullah Mahmood Ali Division of Experimental Hematology, Cincinnati Children's Hospital Research Foundation and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Annette L Medhurst Annette L Medhurst Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands Search for more papers by this author Kornelia Neveling Kornelia Neveling Department of Human Genetics, University of Wurzburg, Wurzburg, Germany Search for more papers by this author Reinhard Kalb Reinhard Kalb Department of Human Genetics, University of Wurzburg, Wurzburg, Germany Search for more papers by this author Zhijiang Yan Zhijiang Yan Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Yutong Xue Yutong Xue Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Anneke B Oostra Anneke B Oostra Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands Search for more papers by this author Arleen D Auerbach Arleen D Auerbach Laboratory of Human Genetics and Hematology, The Rockefeller University, New York, NY, USA Search for more papers by this author Maureen E Hoatlin Maureen E Hoatlin Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Detlev Schindler Detlev Schindler Department of Human Genetics, University of Wurzburg, Wurzburg, Germany Search for more papers by this author Hans Joenje Hans Joenje Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands Search for more papers by this author Johan P de Winter Johan P de Winter Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands Search for more papers by this author Minoru Takata Minoru Takata Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki, Okayama, Japan Department of Human Genetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan Search for more papers by this author Amom Ruhikanta Meetei Corresponding Author Amom Ruhikanta Meetei Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Division of Experimental Hematology, Cincinnati Children's Hospital Research Foundation and University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Weidong Wang Corresponding Author Weidong Wang Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Author Information Chen Ling1,‡, Masamichi Ishiai2,‡, Abdullah Mahmood Ali3, Annette L Medhurst4, Kornelia Neveling5, Reinhard Kalb5, Zhijiang Yan1, Yutong Xue1, Anneke B Oostra4, Arleen D Auerbach6, Maureen E Hoatlin7, Detlev Schindler5, Hans Joenje4, Johan P de Winter4, Minoru Takata2,8, Amom Ruhikanta Meetei 1,3 and Weidong Wang 1 1Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA 2Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki, Okayama, Japan 3Division of Experimental Hematology, Cincinnati Children's Hospital Research Foundation and University of Cincinnati College of Medicine, Cincinnati, OH, USA 4Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands 5Department of Human Genetics, University of Wurzburg, Wurzburg, Germany 6Laboratory of Human Genetics and Hematology, The Rockefeller University, New York, NY, USA 7Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR, USA 8Department of Human Genetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan ‡These authors contributed equally to this work *Corresponding authors: Laboratory of Genetics, National Institute on Aging, NIH, 333 Cassell Drive, TRIAD Center Room 3000, Baltimore, MD 21224, USA. Tel.: +1 410 558 8334; Fax: +1 410 558 8331; E-mail: [email protected] Division of Experimental Hematology, Cincinnati Children's Hospital Research Foundation and University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. Tel.: +1 513 636 1768; Fax: +1 513 636 3768; E-mail: [email protected] The EMBO Journal (2007)26:2104-2114https://doi.org/10.1038/sj.emboj.7601666 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Fanconi anemia (FA) core complex plays a central role in the DNA damage response network involving breast cancer susceptibility gene products, BRCA1 and BRCA2. The complex consists of eight FA proteins, including a ubiquitin ligase (FANCL) and a DNA translocase (FANCM), and is essential for monoubiquitination of FANCD2 in response to DNA damage. Here, we report a novel component of this complex, termed FAAP100, which is essential for the stability of the core complex and directly interacts with FANCB and FANCL to form a stable subcomplex. Formation of this subcomplex protects each component from proteolytic degradation and also allows their coregulation by FANCA and FANCM during nuclear localization. Using siRNA depletion and gene knockout techniques, we show that FAAP100-deficient cells display hallmark features of FA cells, including defective FANCD2 monoubiquitination, hypersensitivity to DNA crosslinking agents, and genomic instability. Our study identifies FAAP100 as a new critical component of the FA-BRCA DNA damage response network. Introduction Fanconi anemia (FA) is a rare genetic disease characterized by congenital defects, bone marrow failure, chromosomal instability, and cancer susceptibility (Joenje and Patel, 2001; Kennedy and D'Andrea, 2005). A hallmark feature of this disease is that cells derived from FA patients are highly sensitive to DNA crosslinking agents such as mitomycin C (MMC) and cisplatin. For this reason, the disease has been considered as a useful model for studying the repair mechanism of DNA interstrand crosslinks (ICLs). To date, 13 FA complementation groups (FANC-A, B, C, D1, D2, E, F, G, I, J, L, M, and N) have been described, and 12 of the corresponding disease genes have been identified (see reviews Niedernhofer et al, 2005; Thompson, 2005; Patel, 2007). The only group whose corresponding gene has not been identified is FA-I. The FA proteins participate in a DNA damage response pathway, termed the FA pathway. A key step in this pathway is the monoubiquitination of FANCD2, an event that occurs either during S-phase of the cell cycle or following a variety of DNA damages, including ICLs, double-stranded DNA breaks, and blocked replication forks (Garcia-Higuera et al, 2001; Taniguchi and D'Andrea, 2002). At least nine FA gene products, FANC-A, B, C, E, F, G, L, M, and I, act upstream of this reaction, because cells deficient in any one of these proteins are defective in FANCD2 monoubiquitination (Garcia-Higuera et al, 2001; Taniguchi and D'Andrea, 2002; Meetei et al, 2003a, 2004, 2005; Levitus et al, 2004). Conversely, three FA proteins, FANCD1, FANCN, and FANCJ, are dispensable for FANCD2 monoubiquitination and are thought to function either downstream or in parallel pathways. Recent evidence suggests that the FA proteins may not act in a simple linear pathway, but rather, form an interactive network (Venkitaraman, 2004). Importantly, this network includes the breast cancer susceptibility gene products, BRCA1 and BRCA2. One FA gene, FANCD1, is identical to BRCA2 (Howlett et al, 2002), whereas another FA gene, FANCJ, is identical to BRIP1 (also called BACH1) (Levitus et al, 2005; Levran et al, 2005; Litman et al, 2005), a DNA helicase that interacts with BRCA1(Cantor et al, 2004). Most recently, a third FA gene, FANCN, is found to be PALB2 (Reid et al, 2007; Xia et al, 2007), which interacts with and is essential for the function of BRCA2 (Xia et al, 2006). The connection between the FA and BRCA proteins argues that defects in FA proteins not only play a role in the pathophysiology of FA, but also in genome instability and tumorigenesis in non-FA patients. Indeed, inactivation of FA genes, by either mutations or epigenetic silencing, has been reported in cancers derived from non-FA patients (Taniguchi et al, 2003; van der Heijden et al, 2003). Thus, investigation of the FA-associated protein function may provide novel insight into the general mechanism of genome maintenance and cancer prevention. We have previously purified the FA core complex with an FANCA antibody (Meetei et al, 2003a, 2003b). This complex includes not only the five known FA proteins (FANC-A, C, E, F, and G), but also four new polypeptides, which are named FAAPs for FANCA-associated polypeptides. Three of the four FAAPs, FAAP43, FAAP95, and FAAP250, have been shown to be integral components of the FA core complex and essential for FANCD2 monoubiquitination. Moreover, the genes encoding these proteins are defective in FA complementation groups L, B, and M, respectively (Meetei et al, 2003a, 2004, 2005). Thus, eight of the nine components of the FA core complex are FA proteins (FANC-A, B, C, E, F, G, L, and M). Furthermore, two of the newly discovered FA proteins have enzymatic activities: FANCL is a ubiquitin ligase essential for FANCD2 monoubiquitination in vivo (Meetei et al, 2003a), whereas FANCM is an ortholog of archeal DNA repair protein Hef (Meetei et al, 2005; Mosedale et al, 2005), and has a DNA-stimulated ATPase and DNA translocase activity (Meetei et al, 2005). It was proposed that the entire FA core complex functions as an E3 ubiquitin ligase that monoubiquitinates FANCD2 in response to DNA damage (Meetei et al, 2005). Moreover, the core complex appears to have at least two additional functions that are also essential in repair of crosslinked DNA damage: one is to recruit monoubiquitinated FANCD2 to chromatin and the other is an uncharacterized step after FANCD2 binding to chromatin (Matsushita et al, 2005). How FA core complex accomplishes its multiple functions remains unclear. Here, we describe a novel component of the FA core complex, termed FAAP100, which is essential for the stability and function of the complex. We demonstrate that cells depleted of FAAP100 by siRNA or cells in which the FAAP100 gene is deleted display hallmark features of FA cells. Our data suggest that FAAP100 plays an essential role in the FA DNA damage response network, and its gene could be mutated in FA patients of a novel complementation group. Results FAAP100 is an integral component of the FA core complex FAAP100 is the 100 kDa polypeptide in the FA core complex purified with an FANCA antibody (Figure 1A) (Meetei et al, 2003a, 2003b). We identified this polypeptide by mass spectrometry as LOC80233, a hypothetical protein with unknown function (gene name: C17orf70; accession number: NP_079437). The LOC80233 sequence in the GenBank is incomplete. We identified a full-length cDNA for this gene, which encodes a protein of 881 amino-acid residues (accession number: DQ989324). An antibody against LOC80233 recognized the 100 kDa polypeptide in the FA complex purified by the FANCA antibody (Figure 1B), indicating that LOC80233 is FAAP100. Figure 1.FAAP100 is a new intrinsic component of the FA core complex. (A) A silver-stained SDS gel showing the FA core complex purified by an FANCA antibody from HeLa nuclear extract (Meetei et al, 2003a). (B) Immunoblotting shows the presence of FAAP100 in the FANCA and FANCB immunoprecipitates. (C) Immunoblotting shows that the antibody against FAAP100 co-immunoprecipitated several FA core complex components. (D) Immunoblotting shows that the Superose 6 gel filtration profile of FAAP100 is coincidental with that of FANCL, and is somewhat different from that of FANCA and FANCG. Download figure Download PowerPoint The following lines of evidence suggest that FAAP100 is an integral component of the FA core complex. First, FAAP100 was co-immunoprecipitated by antibodies against multiple core complex components, including FANCA and FANCB (Figure 1B). Second, reciprocal immunoprecipitation with the FAAP100 antibody obtained every FA core complex component that has been tested, including FANCA, FANCB, FANCL, and FANCM (Figure 1C). We noticed that the amounts of FANCB and FANCL isolated by the FAAP100 antibody are much higher than that of FANCM and FANCA, hinting that the interactions between FAAP100, FANCB, and/or FANCL could be more direct and thus stronger. Third, the gel filtration profile of FAAP100 significantly overlaps with that of several other FA proteins, indicating that these proteins may associate in one or more complexes (Figure 1D). Specifically, the profile of FAAP100 is coincidental with that of FANCL, suggesting that these two proteins are predominantly present in the same subcomplex(es) in vivo. However, the profiles of FANCA and FANCG are almost identical to each other, suggesting that the majority of these latter two proteins are present in the same complexes. FAAP100 interacts with FANCB and FANCL We investigated whether there is direct binding of FAAP100 to any of the other FA core complex members, using the mammalian two-hybrid assay (Medhurst et al, 2006). FAAP100 fused to the GAL4 DNA-binding domain (BD) was cotransfected with various FA proteins fused to the VP16 activation domain (AD). Only when BD-FAAP100 was coexpressed with AD-FANCB, was an induction of reporter gene activation over controls detected, indicating a direct interaction between this protein pair (Figure 2A). Figure 2.FAAP100 interacts with the FANCB and FANCL. (A) Mammalian two-hybrid assay shows direct interaction between FAAP100 and FANCB. BD-FAAP100 was cotransfected with AD-FA proteins and a luciferase reporter construct to test for direct interactions in HEK 293 cells. Fold induction of luciferase expression is relative to FAAP100 alone. Each experimental data set was obtained in triplicate. (B) Mammalian three-hybrid assay shows that the interaction between FAAP100 and FANCL depends on the presence of FANCB. HEK 293 cells stably expressing FLAG-FANCB were cotransfected with the indicated protein pairs and assayed for luciferase activation. Fold induction relative to reporter gene activation when full-length FAAP100 is expressed alone. Each experimental data set was obtained in triplicate. (C, D) Silver-stained SDS gels showing HF-FANCL-associated polypeptides purified from either cytosol (C), or nuclear extract (D), of HeLa cells by tandem-affinity purification. The major polypeptides identified by mass spectrometry are indicated by arrows. The 'MOCK' lanes have control samples that were obtained by purification using HeLa cells that do not express HF-FANCL. The salt concentration of the washing buffer (250 or 500 mM) used in affinity purification was indicated. Download figure Download PowerPoint We also performed three-hydrid analysis in human embryonic kidney (HEK) 293 cells that stably express FANCB. The rationale is that the interactions between FAAP100 and some other core complex components may depend on the presence of FANCB. Coexpression of BD-FAAP100 with AD-FANCA or AD-FANCG in these cells did not activate the reporter gene over background levels (Figure 2B). However, when BD-FAAP100 and AD-FANCL were coexpressed, there was a strong activation of the reporter gene (26-fold higher than controls). These data are consistent with the co-immunoprecipitation and fractionation data presented above (Figure 1), and suggest that FAAP100 may form a subcomplex with FANCB and FANCL through direct interaction. A stable subcomplex containing FANCL, FANCB, and FAAP100 can be biochemically purified The importance of FANCL as the crucial catalytic subunit of the FA core complex prompted us to purify its associated proteins with an unbiased biochemical approach. This should also test more directly the issue whether FANCL indeed forms a stable subcomplex with FAAP100 and FANCB. We generated a stable HeLa cell line expressing FANCL linked to two tags, a FLAG epitope and a 6-histidine tag, to allow its tandem-affinity purification with a FLAG antibody and a metal affinity column, respectively. This fusion protein, termed HF-FANCL, can complement an FA-L patient cell line (Supplementary Figure 1A and B), indicating that the tags do not interfere with the function of the protein. The majority of FAAP100 co-immunoprecipitated with HF-FANCL, as evidenced by significant depletion of FAAP100 after HF-FANCL precipitation (Supplementary Figure 1C), which is consistent with the notion that the majority of these two proteins coexist in vivo. HF-FANCL-associated proteins from both cytosol and nuclear extracts of HeLa cells were isolated and analyzed by mass spectrometry. In the cytosol preparation, three major polypeptides were obtained, which were identified as HF-FANCL, FAAP100, and FANCB (Figure 2C). The levels of these three polypeptides are roughly stoichiometric, consistent with the three-hybrid data that suggested an FAAP100/FANCB/FANCL subcomplex. Below, we abbreviate this complex as L-B-P100. FANCA was the only other FA protein identified by mass spectrometry, but its level was considerably lower than that in L-B-P100. These data suggest that L-B-P100 could represent the most abundant form of FANCL in the cytosol, with only a small fraction in association with FANCA. The fact that FAAP100 can be purified independently by antibodies against two different FA core complex subunits (FANCA and FANCL) provides further evidence that FAAP100 is an integral component of the same complex. In the nuclear preparation of HF-FANCL-associated proteins, three major polypeptides were also identified as HF-FANCL, FANCB, and FAAP100 (Figure 2D, lane 4). The stoichiometry of the three is indistinguishable from that of the cytosolic L-B-P100 complex, suggesting that they may have remained together as a unit when imported into the nucleus. Two other HF-FANCL-associated proteins were found to be FANCA and FANCM. The level of FANCA is roughly stoichiometric to proteins in L-B-P100, whereas that of FANCM appears to be substoichiometric. Other FA proteins may also be present but at levels too low to be detected by mass spectrometry (we detected the presence of FANCG by immunoblotting; data not shown). When HF-FANCL-associated proteins were purified from nuclear extract under high-salt washing conditions, only FAAP100 and FANCB remained in stoichiometric association with HF-FANCL, whereas FANCA and FANCM were largely removed (Figure 2D, lane 2). These data provide biochemical evidence for existence of an L-B-P100 subcomplex module within the FA core complex, and suggest that the interactions within this module are stronger than those between this module and the other components of the core complex. FAAP100 is conserved in vertebrates and contains a putative coiled-coil domain Searching the genomic sequence databases with the BLAST algorithm revealed that FAAP100 protein is conserved only in vertebrates including mouse, Xenopus, and fish Tetraodon nigroviridis (Supplementary Figure 2), but not in invertebrates and yeast. This feature of FAAP100 is shared by several other components of the FA core complex, including FANC-A, B, C, E, F, and G, suggesting that the genes encoding these proteins may have arisen at about the same time during evolution. Examination of FAAP100 sequence revealed the presence of a putative coiled-coil domain (also called leucine-zipper) (Supplementary Figure 2). This domain could mediate protein–protein interactions by either homodimerization (interacting with the same domain) or heterodimerization (interacting with a different coiled-coil domain). Potential coiled-coil domains are present in FANCA and FANCG (Lo Ten Foe et al, 1996; Demuth et al, 2000), which directly interact with each other (Garcia-Higuera et al, 1999; Waisfisz et al, 1999). We found that FANCB, the protein that directly interacts with FAAP100 in the two-hybrid assay (Figure 2A), may also contain a coiled-coil domain (Supplementary Figure 3). It remains to be determined whether the interaction between FAAP100 and FANCB is mediated through these domains. FAAP100 is essential for the monoubiquitination of FANCD2 and the stability of the FA core complex We depleted FAAP100 in HeLa cells by two different siRNA oligos and found that these cells not only have reduced levels of FAAP100 but also of other components of the FA core complex, including FANC-B, L, A, and G (Figure 3A). This feature mimics that of FANCB-depleted HeLa cells, which showed a reduced level of FANCL (Meetei et al, 2004). The data suggest that FAAP100, like FANCB, is required for stability of the FA core complex. Importantly, FAAP100-depleted cells display reduced levels of monoubiquitinated FANCD2 both in the absence and presence of DNA-damaging agents such as MMC, cisplatin, and hydroxyurea (Figure 3B and C). This feature is shared by cells depleted of other FA core complex components (Meetei et al, 2003a, 2004, 2005), indicating that FAAP100 is an essential part of the FA core complex required for FANCD2 monoubiquitination. Figure 3.FAAP100 is essential for FANCD2 monoubiquitination and the stability of the FA core complex. (A) Immunoblotting shows that HeLa cells depleted of FAAP100 by two different siRNA oligos not only have reduced levels of FAAP100, but also of several other FA core complex components, including FANCB, FANCL, FANCA, and FANCG. HeLa cells transfected with a scrambled siRNA oligo are shown as control. The image of FAAP100 depletion (top panel) was reproduced from the lanes 1–3 of the top panel in (B), to allow easy comparison of the reduction of FAAP100 protein level with those of other FA proteins. (B, C) Immunoblotting shows that HeLa cells depleted of FAAP100 display reduced level of monoubiquitinated FANCD2 in cells that are either untreated (−), or treated with mitomycin C (MMC), cisplatin, or hydroxyurea (+). The monoubiquitinated and non-ubiquitinated FANCD2 proteins are indicated by FANCD2-L and FANCD2-S, respectively. (D) Immunoblotting shows that HeLa cells depleted of FAAP100 by On-Targetplus SMARTpool siRNAs display a level of monoubiquitinated FANCD2 comparable to cells depleted of FANCM or FANCL (compare the relative ratio between FANCD2-L and FANCD2-S in lane 6 with those in lanes 3 and 4). In addition, cells depleted by On-Target siRNAs have lower levels of FAAP100 and monoubiquitinated FANCD2 (compare lane 6 with lane 2). A nonspecific polypeptide was marked with an asterisk, which was resolved from FAAP100 using a 6% SDS gel. (E) Immunoblotting shows that HeLa cells depleted of FAAP100 by On-Targetplus SMARTpool siRNAs (siFAAP100) reduced monoubiquitination of FANCD2 in the presence of several DNA-damaging drugs. Note that the FAAP100 level in depleted cells is similar that of a nonspecific polypeptide (marked by an asterisk), indicating that its level is very low and close to the detection limit of the antibody. The cell lysis buffer in (A), (B) and (C) includes 8 M urea, which allows more efficient extraction of FANCD2-L and FANCB. The cell lysis buffer in (D) and (E), and those used in previous studies of FANCL, FANCB, and FANCM, do not include urea. Thus, the results in (D) and (E) should be more suitable for comparison with the previous studies. Download figure Download PowerPoint We noticed that the level of monoubiquitinated FANCD2 in FAAP100-depleted cells is similar to that of cells depleted of FANCB (Meetei et al, 2004), but higher than those depleted of FANCL or FANCM (Meetei et al, 2003a, 2005). One possible explanation is that FAAP100 and FANCB are non-enzymatic components of the core complex, and are therefore less important for FANCD2 monoubiquitination than the enzymatic components, FANCL and FANCM. Another possibility is that both non-enzymatic and enzymatic subunits are of equal importance, but the former proteins are depleted less efficiently than the latter. The fact that cells from FA patients with mutations in the FANCB, FANCL, and FANCM genes all have complete absence of monoubiquitinated FANCD2 supports the second possibility (Meetei et al, 2003a, 2004, 2005). To distinguish further between these two possibilities, we utilized an improved siRNA technique, On-Targetplus SMARTpool siRNAs, to deplete FAAP100. These siRNAs are designed by better bioinformatics tools and chemically modified to reduce significantly the off-target effects of siRNA. The On-Targetplus siRNAs reduced FAAP100 protein level by about 90% (data not shown), which is more efficient than by regular siRNAs (Figure 3D, compare lane 6 to lane 2; and Figure 3E). Importantly, the new siRNAs decreased monoubiquitinated FANCD2 to levels comparable to cells depleted of FANCL or FANCM (Figure 3D, compare lane 6 with lanes 2 and 3; and E), which supports the hypothesis that FAAP100 is as important for FANCD2 monoubiquitination as FANCL and FANCM. Consistent with results obtained by using regular siRNAs (Figure 3A), the On-Targetplus siRNAs also reduced the levels of several subunits of the FA core complex, in particular FANCL and FANCB (Supplementary Figure 4A and B). The findings support the notion that FAAP100 is essential for stability of the FA core complex. In contrast to cells depleted of FANCL or FANCM (Meetei et al, 2003a, 2005), HeLa cells depleted of FAAP100 by either regular or On-Targetplus siRNA have no significant difference in MMC-induced chromosome breakage levels and MMC-sensitivity compared with cells treated with control siRNAs (data not shown). This could be due to the fact that the siRNA depletion is incomplete, so that the FAAP100 protein level fails to reach the critical threshold to disrupt its normal function in the latter two processes. In support of this possibility, we noticed that there was still a residual level of FAAP100 in FAAP100-silenced cells that co-immunoprecipitated with FANCB (Supplemen