Fanconi Anemia Core Complex Research Articles

FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response.

Fanconi anemia (FA) is an inherited cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA proteins act in a linear hierarchy: following ICL detection on chromatin, the FA core complex monoubiquitinates and recruits the central FANCI and FANCD2 proteins that subsequently coordinate ICL removal and repair of the ensuing DNA double-stranded break by homology-dependent repair (HDR). FANCD2 also functions during the replication stress response by mediating the restart of temporarily stalled replication forks thereby suppressing the firing of new replication origins. To address if FANCI is also involved in these FANCD2-dependent mechanisms, we generated isogenic FANCI-, FANCD2- and FANCI:FANCD2 double-null cells. We show that FANCI and FANCD2 are partially independent regarding their protein stability, nuclear localization and chromatin recruitment and contribute independently to cellular proliferation. Simultaneously, FANCD2—but not FANCI—plays a major role in HDR-mediated replication restart and in suppressing new origin firing. Consistent with this observation, deficiencies in HDR-mediated DNA DSB repair can be overcome by stabilizing RAD51 filament formation in cells lacking functional FANCD2. We propose that FANCI and FANCD2 have partially non-overlapping and possibly even opposing roles during the replication stress response.

Abstract LB-170: A novel, nontoxic RXR agonist, UAB30, targets Fanconi anemia DNA repair pathway to act as potent cancer chemopreventive agent

Abstract UAB30, a synthetic analog of 9-cis-RA is a potent retinoid X receptors (RXR) agonist. It was found to have significantly low retinoic acid receptor (RAR)-binding activity. This unique property of UAB30 is responsible for its low lipid toxicity. Thus, it does not cause elevation in serum triglycerides both in humans and in experimental animals. These properties with favorable pharmacokinetics make it a suitable candidate for chronic use as chemopreventive agent. Here, we show that UAB30 is highly effective in inhibiting UVB-induced pathogenesis of skin cancers in Ptch1+/-/SKH-1 hairless mice. Similar to humans, this unique genetically engineered murine model develops both basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) of the skin following chronic UVB irradiation. Compared to controls, the UAB30-treated group showed a 48% reduction in tumor number and a 78% reduction in tumor volume. The number of papillomas, BCCs, and SCCs were reduced by 33%, 74%, and 76%, respectively. UAB30 also diminished UVB-induced skin inflammation, hyperplasia, and enhanced differentiation of suprabasal epidermal cells. The expression of cyclins A, D1, D2, and D3, and cdc25A were decreased in UAB30 treated SCCs, while pro-apoptotic proteins, Bax and cleaved caspase 3 were increased. UAB30 when topically applied 30 min after each UVB irradiation for 30 weeks, it showed significant reduction in cancer development. A 57% reduction in tumor number and 72% reduction in tumor volume were observed. BCCs and SCCs were reduced by 55% and 60%, respectively. We also demonstrate that UAB30 acts by a unique molecular mechanism. Induction of lethal DNA double strand breaks (DSBs) characterized by the presence of p-γH2AX was observed following UAB30 treatment. These effects were more prominent in cancer cells than normal keratinocytes. Induction of DSBs was associated with the activation of DNA damage response (DDR) signaling leading to increased phosphorylation of ATM, Rad50, and Chk2. These effects paralleled with the reduction in cell cycle proteins and cell cycle arrest in G2/M phase followed by apoptosis of arrested cells. UAB30-induced DSBs and DDR were not dependent on ROS as we did not observe significant induction of ROS in UAB30-treated cells and did not notice any protective effect of N-acetyl cysteine. These effects were similar to DDR and changes in proliferation/apoptosis response observed following treatment with cisplatin which is known inducer of DSBs. Moreover, other RXR agonists were significantly less potent in inducing DSBs and DDR responses. The underlying mechanism for the accumulation of DSBs was the diminished ability of cancer cells to repair these DNA lesions. Reduction in FANCD2 and FANCC, the Fanconi anemia core complex proteins which are known to be involved in DSBs repair appear to be the cause of extensive DNA damage associated tumor cell death. Citation Format: Sandeep C. Chaudhary, Craig A. Elmets, Mohammad Athar. A novel, nontoxic RXR agonist, UAB30, targets Fanconi anemia DNA repair pathway to act as potent cancer chemopreventive agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-170. doi:10.1158/1538-7445.AM2017-LB-170

Fanconi anemia protein FANCD2 is activated by AICAR, a modulator of AMPK and cellular energy metabolism.

FANCD2 is a pivotal molecule in the pathogenesis of Fanconi anemia (FA), an autosomal recessive human syndrome with diverse clinical phenotypes, including cancer predisposition, short stature, and hematological abnormalities. In our previous study, we detected the functional association of FANC proteins, whose mutations are responsible for the onset of FA, with AMPK in response to DNA interstrand crosslinking lesions. Because AMPK is well known as a critical sensing molecule for cellular energy levels, we checked whether FANCD2 activation occurs after treatments affecting AMPK and/or cellular energy status. Among the treatments tested, AMPK‐activating 5‐aminoimidazole‐4‐carboxamide‐ribonucleoside (AICAR) induced monoubiquitination and nuclear foci formation of FANCD2, which are biomarkers of FANCD2 activation. FANCD2 activation was abolished by treatments with Compound C, an AMPK inhibitor, or after AMPKα1 knockdown, substantiating the involvement of AMPK in AICAR‐induced FANCD2 activation. Similarly, FANCA protein, which is a component of the FA core complex monoubiquitinating FANCD2, was required for this event. Furthermore, FANCD2 repression enhanced cell death upon AICAR treatments in transformed fibroblasts and cell cycle arrest in the renal cell carcinoma cell line Caki‐1. Overall, this study showed FANCD2 involvement in response to AICAR, a chemical modulating cellular energy metabolism.

Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway

Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substratereceptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal invitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis fortemporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia.

The FA Core Complex Contains a Homo-dimeric Catalytic Module for the Symmetric Mono-ubiquitination of FANCI-FANCD2

SummaryActivation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core complex. FANCI and FANCD2 form a stable complex; however, the molecular basis of their ubiquitination is ill defined. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism. How FANCI mono-ubiquitination is achieved remains unclear. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs.

Rare Variants Affecting the Fanconi Anaemia DNA Repair Genes Associate with Increased Risk for AML

While there have been extensive studies to define the roles of recurrent somatic mutations in AML, the contribution of germline variants to AML initiation and progression is less well established. DNA repair disorders often predispose patients to developing myeloid malignancies. In particular, biallelic mutations affecting FANC genes cause the recessive heritable bone marrow failure syndrome Fanconi Anemia (FA), which is associated with >800-fold increased risk of progression to AML. A recent explosion of cancer predisposition studies has also revealed the importance of germlineFANC variants in elevated cancer risk (Cancer Treat Rev 2012; 38:89). To investigate the role of FANC gene variants in AML we have performed a case-control study, analyzing rare, deleterious somatic and germline variants for the 19 FANC genes in adult AML and healthy controls cohorts.Whole exome sequencing was performed on diagnosis samples from 131 adult Caucasian AML patients from two major Australian centers, and a cohort of 329 healthy females. We identified rare Tier 1 variants using a minor allele frequency (MAF) < 0.001, as reported in common dbSNP137, 1000 Genome and NHLBI-ESP project databases. Combined Annotation Dependent Depletion algorithm (CADD, Nat Genet 2014; 46: 310) >10 was used to filter for FANC gene variants with high probability of pathogenicity. Sanger sequencing of matched tumour/non-tumour DNA showed the large majority of variants tested to be germline (90%), consistent with previous studies reporting that somatic FANC genes variants are extremely rare in AML (< 1%). Overall, we identified 52 FANC gene variants in 44 cases with 34% of AML cases carrying one or more variant.For independent validation we determined the presence of somatic and germline FANC variants in the TCGA AML cohort using an identical pipeline and filtering analysis. In line with our results, we found that 36% of TCGA AML patients carry at least one germline FANC variant. We investigated known disease-causing (D-C) variants in these two AML cohorts using the FA (FAMutdb) and breast cancer (kConFab and BIC) mutation databases. We found 8 D-C FANC variants in the Australian AML cohort and 5 in the TCGA cohort, with 1 variant present in both cohorts. Moreover, the frequency of D-C variants in our cohort of females with AML (n=51) is 13.7%, while the frequency in the healthy female cohort is 4.5%, comparable to that reported in the ESP database for female European-Americans (2.1%, Hum Mol Genet 2014; 23: 6815). Accordingly, we determined that deleterious FANC germline variants confer a significant increased risk of AML (P=0.018, OR=3.3 for the Australian AML cohort).Finally, we performed mutational burden analysis to investigate enrichment of variants associated with particular FANC genes across the AML cohort. This revealed a significant enrichment of FANCL variants in AML vs healthy controls (P=0.008, Figure 1). FANCL is the enzymatic component of the FA core complex that monoubiquitinates the FANCD2/I heterodimer initiating DNA repair, and its down-regulation has been linked to AML (Oncogene 2016; doi:10.1038). Several FANCL variants, found in our AML cohort, affect the catalytic RING domain and are of particular interest. These include a D-C null variant present in 2 patients, a frame shift variant in 2 patients who presented with AML at a very early age (27 and 46 years old), and a variant affecting a critical conserved residue required for monoubiquitination of FANCD2/I.In conclusion, we show enrichment of rare potentially deleterious FANC gene mutations in AML, associated with a 3-fold increased risk of developing the disease. We hypothesise that, in hematopoietic stem/progenitor cells, these variants confer a subtle defect in interstrand cross-link repair leading to an increased accumulation of mutations and subsequent development of AML. Consistent with this there have been several reports of defective DNA damage repair and increased sensitivity to DNA damaging agents in cells from FANC carriers compared to normal controls (Nat Commun 2014; 5:5496; Mutagenesis 2009; 24:67). Importantly, it is possible to target defects in several DNA repair pathways, and our finding identifies a group of AML patients who may benefit from approaches that target defective FA and homologous recombination pathways. [Display omitted] DisclosuresGill:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The PTEN phosphatase functions cooperatively with the Fanconi anemia proteins in DNA crosslink repair.

Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure and increased cancer risk. The FA proteins function primarily in DNA interstrand crosslink (ICL) repair. Here, we have examined the role of the PTEN phosphatase in this process. We have established that PTEN-deficient cells, like FA cells, exhibit increased cytotoxicity, chromosome structural aberrations, and error-prone mutagenic DNA repair following exposure to ICL-inducing agents. The increased ICL sensitivity of PTEN-deficient cells is caused, in part, by elevated PLK1 kinase-mediated phosphorylation of FANCM, constitutive FANCM polyubiquitination and degradation, and the consequent inefficient assembly of the FA core complex, FANCD2, and FANCI into DNA repair foci. We also establish that PTEN function in ICL repair is dependent on its protein phosphatase activity and ability to be SUMOylated, yet is independent of its lipid phosphatase activity. Finally, via epistasis analysis, we demonstrate that PTEN and FANCD2 function cooperatively in ICL repair.

The Fanconi anemia pathway controls oncogenic response in hematopoietic stem and progenitor cells by regulating PRMT5-mediated p53 arginine methylation.

The Fanconi anemia (FA) pathway is involved in DNA damage and other cellular stress responses. We have investigated the role of the FA pathway in oncogenic stress response by employing an in vivo stress-response model expressing the Gadd45β-luciferase transgene. Using two inducible models of oncogenic activation (LSL-K-rasG12D and MycER), we show that hematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA core complex components Fanca or Fancc exhibit aberrant short-lived response to oncogenic insults. Mechanistic studies reveal that FA deficiency in HSPCs impairs oncogenic stress-induced G1 cell-cycle checkpoint, resulting from a compromised K-rasG12D-induced arginine methylation of p53 mediated by the protein arginine methyltransferase 5 (PRMT5). Furthermore, forced expression of PRMT5 in HSPCs from LSL-K-rasG12D/CreER-Fanca−/− mice prolongs oncogenic response and delays leukemia development in recipient mice. Our study defines an arginine methylation-dependent FA-p53 interplay that controls oncogenic stress response.

Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice.

After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance) protein. Fanconi anemia (FA) proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2–3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line.

Abstract 2758: FANCF, a Fanconi anemia core complex protein involved in monoubiquitination of FANCD2, also has a role with nuclear alpha spectrin in DNA interstrand crosslink repair

Abstract Nonerythroid nuclear A spectrin (ASpII) is critical for repair of DNA interstrand crosslinks (ICLs) and for genomic stability. We have previously shown that there is a deficiency in ASpII in the inherited chromosomal instability disorder, Fanconi anemia (FA), which has a defect in ability to repair DNA ICLs and a predisposition to cancer. Eight FA proteins, FANC -A, B, C, E, F, G, L, and M, form a core complex essential for monoubiquitination of FANCD2 (FANCD2-Ub), a process which is critical for ICL repair. However, whether any of these FA core proteins play additional roles in ICL repair is not clearly known. The present study was undertaken to address this question and to examine whether one of these proteins, FANCF, is involved in steps in the ICL repair process in which ASpII also plays a role. Immunofluorescence microscopy was used to determine whether FANCF co-localizes with ASpII in nuclear foci in normal human cells after they are damaged with an ICL agent, 8-methoxypsoralen plus UVA light (8-MOP). Time course measurements showed that FANCF co-localized in nuclear foci with ASpII and followed a similar time course for formation. This time course was similar to that of the ICL repair protein, XPF, which produces incisions at sites of ICLs and acts downstream of FANCD2-Ub. FANCF foci, like ASpII and XPF foci, were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. This association of FANCF with ASpII was corroborated by co-immunoprecipitation studies which demonstrated that FANCF has enhanced binding to ASpII after ICL damage. These studies indicate that FANCF associates with ASpII and is involved with ASpII in the repair process. Since we have demonstrated that ASpII, like XPF, acts downstream of FANCD2-Ub and that FANCF co-localizes with ASpII after ICL damage, this suggests that FANCF interacts with ASpII in repair events downstream of FANCD2-Ub. In FA-A cells, FANCF is present as in normal cells, but does not form nuclear foci after ICL damage. Transfection of FA-A cells with a cDNA expressing FANCA, however, led to restoration of ASpII levels to normal and to formation of FANCF nuclear foci, which co-localized with ASpII foci. This indicates that ASpII is needed in localization of FANCF to sites of damage. These studies support a model we have proposed in which ASpII acts as a scaffold in the recruitment of proteins involved in the repair process to sites of ICL damage. They also show that FANCF has an additional function in ICL repair besides monoubiquitination of FANCD2. We propose that after DNA damage and monoubiquitination of FANCD2, ASpII and FANCF act downstream of FANCD2, along with XPF, and that the interaction between these proteins at sites of damage is critical for the repair process and maintenance of genomic stability. Citation Format: Muriel W. Lambert, Deepa Sridharan, Pan Zhang. FANCF, a Fanconi anemia core complex protein involved in monoubiquitination of FANCD2, also has a role with nuclear alpha spectrin in DNA interstrand crosslink repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2758.

FBW7 regulates DNA interstrand cross-link repair by modulating FAAP20 degradation.

Mutations that deregulate protein degradation lead to human malignancies. The SCF ubiquitin E3 ligase complex degrades key oncogenic regulators, thereby limiting their oncogenic potential. FBW7 is a substrate recognition subunit of SCFFBW7 and is among the most commonly mutated ubiquitin-proteasome system proteins in cancer. FBW7-mutated cancer cells display increased genome instability, but the molecular mechanism by which FBW7 preserves genome integrity remains elusive. Here, we demonstrate that SCFFBW7 regulates the stability of FAAP20, a critical component of the Fanconi anemia (FA) DNA interstrand cross-link (ICL) repair pathway. Phosphorylation of the FAAP20 degron motif by GSK3β provides a platform for recognition and polyubiquitination of FAAP20 by FBW7, and its subsequent degradation by the proteasome. Accordingly, enhanced GSK3β-FBW7 signaling disrupts the FA pathway. In cells expressing non-phosphorylatable FAAP20 mutant, the turnover of its binding partner, FANCA, is deregulated in the chromatin during DNA ICL repair, and the FA pathway is compromised. We propose that FAAP20 degradation, which is prompted by its phosphorylation, controls the dynamics of the FA core complex required for completing DNA ICL repair. Together, this study provides insights into how FBW7-mediated proteolysis regulates genome stability and how its deregulation is associated with tumorigenesis.

Modularized Functions of the Fanconi Anemia Core Complex

Modularized Functions of the Fanconi Anemia Core Complex

Hyper-active non-homologous end joining selects for synthetic lethality resistant and pathological Fanconi anemia hematopoietic stem and progenitor cells

The prominent role of Fanconi anemia (FA) proteins involves homologous recombination (HR) repair. Poly[ADP-ribose] polymerase1 (PARP1) functions in multiple cellular processes including DNA repair and PARP inhibition is an emerging targeted therapy for cancer patients deficient in HR. Here we show that PARP1 activation in hematopoietic stem and progenitor cells (HSPCs) in response to genotoxic or oxidative stress attenuates HSPC exhaustion. Mechanistically, PARP1 controls the balance between HR and non-homologous end joining (NHEJ) in double strand break (DSB) repair by preventing excessive NHEJ. Disruption of the FA core complex skews PARP1 function in DSB repair and led to hyper-active NHEJ in Fanca−/− or Fancc−/− HSPCs. Re-expression of PARP1 rescues the hyper-active NHEJ phenotype in Brca1−/−Parp1−/− but less effective in Fanca−/−Parp1−/− cells. Inhibition of NHEJ prevents myeloid/erythroid pathologies associated with synthetic lethality. Our results suggest that hyper-active NHEJ may select for “synthetic lethality” resistant and pathological HSPCs.

Leukemia and chromosomal instability in aged Fancc-/- mice.

Fanconi anemia (FA) is an inherited disorder of genomic instability associated with high risk of myelodysplasia and acute myeloid leukemia (AML). Young mice deficient in FA core complex genes do not naturally develop cancer, hampering preclinical studies on malignant hematopoiesis in FA. Here we describe that aging Fancc(-/-) mice are prone to genomically unstable AML and other hematologic neoplasms. We report that aneuploidy precedes malignant transformation during Fancc(-/-) hematopoiesis. Our observations reveal that Fancc(-/-) mice develop hematopoietic chromosomal instability followed by leukemia in an age-dependent manner, recapitulating the clinical phenotype of human FA and providing a proof of concept for future development of preclinical models of FA-associated leukemogenesis.

Hyper-Active Non-Homologous End Joining Selects for Synthetic Lethality Resistant and Pathological Hematopoietic Stem Cells

The prominent role of Fanconi anemia (FA) proteins involves homologous recombination (HR) repair. Poly[ADP-ribose] polymerase1 (PARP1) functions in multiple cellular processes including DNA repair and PARP inhibition is an emerging targeted therapy for cancer patients deficient in HR. Here we show that PARP1 activation in hematopoietic stem and progenitor cells (HSPCs) in response to genotoxic or oxidative stress attenuates HSPC exhaustion. Mechanistically, PARP1 controls the balance between HR and non-homologous end joining (NHEJ) in double strand break (DSB) repair by preventing excessive NHEJ. Disruption of the FA core complex skews PARP1 function in DSB repair and led to hyper-active NHEJ in Fanca-/- or Fancc-/- HSPCs. Re-expression of PARP1 rescues the hyper-active NHEJ phenotype in Brca1-/-Parp1-/- but less effective in Fanca-/-Parp1-/- cells. Inhibition of NHEJ prevents myeloid/erythroid pathologies associated with "synthetic lethality" and reduces human engraftment. Our results suggest that hyper-active NHEJ may select for "synthetic lethality" resistant and pathological HSPCs. DisclosuresNo relevant conflicts of interest to declare.

Forkhead transcription factor FoxF1 interacts with Fanconi anemia protein complexes to promote DNA damage response.

Forkhead box F1 (Foxf1) transcription factor is an important regulator of embryonic development but its role in tumor cells remains incompletely understood. While 16 proteins were characterized in Fanconi anemia (FA) core complex, its interactions with cellular transcriptional machinery remain poorly characterized. Here, we identified FoxF1 protein as a novel interacting partner of the FA complex proteins. Using multiple human and mouse tumor cell lines and Foxf1+/- mice we demonstrated that FoxF1 physically binds to and increases stability of FA proteins. FoxF1 co-localizes with FANCD2 in DNA repair foci in cultured cells and tumor tissues obtained from cisplatin-treated mice. In response to DNA damage, FoxF1-deficient tumor cells showed significantly reduced FANCD2 monoubiquitination and FANCM phosphorylation, resulting in impaired formation of DNA repair foci. FoxF1 knockdown caused chromosomal instability, nuclear abnormalities, and increased tumor cell death in response to DNA-damaging agents. Overexpression of FoxF1 in DNA-damaged cells improved stability of FA proteins, decreased chromosomal and nuclear aberrations, restored formation of DNA repair foci and prevented cell death after DNA damage. These findings demonstrate that FoxF1 is a key component of FA complexes and a critical mediator of DNA damage response in tumor cells.

FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

Cytokine overproduction and crosslinker hypersensitivity are unlinked in Fanconi anemia macrophages.

The Fanconi anemia proteins participate in a canonical pathway that repairs cross-linking agent-induced DNA damage. Cells with inactivated Fanconi anemia genes are universally hypersensitive to such agents. Fanconi anemia-deficient hematopoietic stem cells are also hypersensitive to inflammatory cytokines, and, as importantly, Fanconi anemia macrophages overproduce such cytokines in response to TLR4 and TLR7/8 agonists. We questioned whether TLR-induced DNA damage is the primary cause of aberrantly regulated cytokine production in Fanconi anemia macrophages by quantifying TLR agonist-induced TNF-α production, DNA strand breaks, crosslinker-induced chromosomal breakage, and Fanconi anemia core complex function in Fanconi anemia complementation group C-deficient human and murine macrophages. Although both M1 and M2 polarized Fanconi anemia cells were predictably hypersensitive to mitomycin C, only M1 macrophages overproduced TNF-α in response to TLR-activating signals. DNA damaging agents alone did not induce TNF-α production in the absence of TLR agonists in wild-type or Fanconi anemia macrophages, and mitomycin C did not enhance TLR responses in either normal or Fanconi anemia cells. TLR4 and TLR7/8 activation induced cytokine overproduction in Fanconi anemia macrophages. Also, although TLR4 activation was associated with induced double strand breaks, TLR7/8 activation was not. That DNA strand breaks and chromosome breaks are neither necessary nor sufficient to account for the overproduction of inflammatory cytokines by Fanconi anemia cells suggests that noncanonical anti-inflammatory functions of Fanconi anemia complementation group C contribute to the aberrant macrophage phenotype and suggests that suppression of macrophage/TLR hyperreactivity might prevent cytokine-induced stem cell attrition in Fanconi anemia.

Conserved Overlapping Gene Arrangement, Restricted Expression, and Biochemical Activities of DNA Polymerase ν (POLN)

DNA polymerase ν (POLN) is one of 16 DNA polymerases encoded in vertebrate genomes. It is important to determine its gene expression patterns, biological roles, and biochemical activities. By quantitative analysis of mRNA expression, we found that POLN from the zebrafish Danio rerio is expressed predominantly in testis. POLN is not detectably expressed in zebrafish embryos or in mouse embryonic stem cells. Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development. Analysis of transcripts revealed that vertebrate POLN has an unusual gene expression arrangement, sharing a first exon with HAUS3, the gene encoding augmin-like complex subunit 3. HAUS3 is broadly expressed in embryonic and adult tissues, in contrast to POLN. Differential expression of POLN and HAUS3 appears to arise by alternate splicing of transcripts in mammalian cells and zebrafish. When POLN was ectopically overexpressed in human cells, it specifically coimmunoprecipitated with the homologous recombination factors BRCA1 and FANCJ, but not with previously suggested interaction partners (HELQ and members of the Fanconi anemia core complex). Purified zebrafish POLN protein is capable of thymine glycol bypass and strand displacement, with activity dependent on a basic amino acid residue known to stabilize the primer-template. These properties are conserved with the human enzyme. Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase.

Biophysical characterization of the interaction between FAAP20-UBZ4 domain and Rev1-BRCT domain

FAAP20 (Fanconi anemia-associated protein 20) is a subunit of the Fanconi anemia (FA) core complex that repairs interstrand cross-links. To understand the molecular basis for the FA core complex-mediated recruitment of Rev1 to the DNA lesion, we characterized the interactions among FAAP20-UBZ4, Rev1-BRCT, and ubiquitin using NMR. We found that FAAP20-UBZ4 binds not only ubiquitin but also Rev1-BRCT. Mapping the protein–protein interactions showed that FAAP20-UBZ4 has distinct binding surfaces for ubiquitin and Rev1-BRCT. In addition, the chemical exchange patterns indicated that the interaction between FAAP20-UBZ4 and ubiquitin might enhance the binding affinity between FAAP20-UBZ4 and Rev1-BRCT. These results provide new insight into the Rev1 recognition mechanism by FAAP20.