ALBERT

Description: Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome. The phenotype includes developmental defects, bone marrow failure, and cell cycle abnormalities. At least eight complementation groups (A-H) exist, and although three of the corresponding complementation group genes have been cloned, they lack recognizable motifs, and their functions are unknown. We have isolated a binding partner for the Fanconi anemia group C protein (FANCC) by yeast two-hybrid screening. We show that the novel gene, FAZF, encodes a 486 amino acid protein containing a conserved amino terminal BTB/POZ protein interaction domain and three C-terminal Krüppel-like zinc fingers. FAZF is homologous to the promyelocytic leukemia zinc finger (PLZF) protein, which has been shown to act as a transcriptional repressor by recruitment of nuclear corepressors (N-CoR, Sin3, and HDAC1 complex). Consistent with a role in FA, BTB/POZ-containing proteins have been implicated in oncogenesis, limb morphogenesis, hematopoiesis, and proliferation. We show that FAZF is a transcriptional repressor that is able to bind to the same DNA target sequences as PLZF. Our data suggest that the FAZF/FANCC interaction maps to a region of FANCC deleted in FA patients with a severe disease phenotype. We also show that FAZF and wild-type FANCC can colocalize in nuclear foci, whereas a patient-derived mutant FANCC that is compromised for nuclear localization cannot. These results suggest that the function of FANCC may be linked to a transcriptional repression pathway involved in chromatin remodeling.

Description: The Fanconi Anemia (FA) Group C complementation group gene (FANCC) encodes a protein, FANCC, with a predicted Mr of 63000 daltons. FANCC is found in both the cytoplasmic and the nuclear compartments and interacts with certain other FA complementation group proteins as well as with non-FA proteins. Despite intensive investigation, the biologic roles of FANCC and of the other cloned FA gene products (FANCA and FANCG) remain unknown. As an approach to understanding FANCC function, we have studied the molecular regulation of FANCC expression. We found that although FANCCmRNA levels are constant throughout the cell cycle, FANCC is expressed in a cell cycle-dependent manner, with the lowest levels seen in cells synchronized at the G1/S boundary and the highest levels in the M-phase. Cell cycle–dependent regulation occurred despite deletion of the 5′ and 3′ FANCC untranslated regions, indicating that information in the FANCC coding sequence is sufficient to mediate cell cycle–dependent regulation. Moreover, inhibitors of proteasome function blocked the observed regulation. We conclude that FANCC expression is controlled by posttranscriptional mechanisms that are proteasome dependent. Recent work has demonstrated that the functional activity of FA proteins requires the physical interaction of at least FANCA, FANCC, and FANCG, and possibly of other FA and non-FA proteins. Our observation of dynamic control of FANCC expression by the proteasome has important implications for understanding the molecular regulation of the multiprotein complex.

Description: Fanconi anemia (FA) is an inherited hematological disorder characterized by bone marrow failure, birth defects, and cancer susceptibility, typically leading to squamous cell carcinomas and acute myelogenous leukemia. Twelve FA genes have been described, eight of which function together in a multiprotein, upstream “FA core complex” to mediate the S-phase and DNA damage-induced monoubiquitylation of two downstream proteins, FANCD2 and FANCI. Despite this knowledge the precise function of the FA proteins is not well understood because they function as part of a network of proteins that have not been completely defined. Recently we developed a new animal model for FA research using extracts from the eggs of Xenopus laevis. Xenopus extracts are cell cycle synchronized and contain nuclear proteins that are stockpiled for DNA replication. We showed that FA gene orthologs (xFA), like their human counterparts, form complexes that are required for the monoubiquitylation of xFANCD2 in response to DNA damage. Xenopus laevis extracts are thus a powerful system to analyze the endogenous state of xFA protein complexes and their components in an S phase, replication-competent context. The objective of this study was to isolate protein complexes containing the xFA core complex protein, xFANCM and xFANCM-interacting proteins. Using a co-immunoprecipitation approach followed by mass spectrometry, we identified a novel protein-binding partner of xFANCM (termed xMIP-1, for xFANCM Interacting Protein 1). The interaction was confirmed by reciprocal coimmunoprecipitation in both Xenopus extracts and human cells. Surprisingly, co-fractionation demonstrated that xFANCM was present in two protein complexes during S phase; one containing FA core complex members (900 kDa) containing xMIP-1. Because xMIP-1 is a partner protein of xFANCM we wanted to determine if xMIP-1, like xFANCM, was required for the monoubiquitylation of xFANCD2. This was done using a DNA stimulation assay, where upon immunodepletion of xMIP-1 from egg extracts, we were able to observe the monoubiquitylation of xFANCD2 in response to DNA structures as a size shift via immunoblot. The absence of xMIP-1 had no detectable effect on the monoubiquitylation of xFANCD2 suggesting that xMIP-1, unlike xFANCM, was not required for xFANCD2 monoubiquitylation. To explore a functional link between xFANCM and xMIP-1 we used egg extracts to show that xMIP-1, like xFANCM, was recruited to replicating chromatin and exhibited a size shift during the replication process. Furthermore immunodepletion of xFANCM from egg extracts reduced recruitment of xMIP-1 to replicating chromatin, suggesting that xMIP-1 chromatin binding was dependent on xFANCM. In contrast, xMIP-1 recruitment to replicating chromatin was not affected by the immunodepletion of other FA core complex proteins tested, suggesting that xMIP-1 chromatin binding is independent of the FA core complex. To further characterize the observed DNA binding activity of xMIP-1 we used the DNA stimulation assay and several defined DNA structures. Surprisingly xMIP-1 showed a double-stranded DNA stimulated mobility shift similar to those reported previously for xFANCD2 (Sobeck et al., 2007) and xMRE11 (Costanzo et al., 2001) suggesting xMIP-1 may play a role in the DNA damage response. Our data suggests xFANCM is a member of an S phase complex that has not been previously described with a “non-FA” partner protein that may function with xFANCM during the DNA damage response to maintain genomic stability.

Description: Objective: The Fanconi anemia (FA) pathway is a DNA damage response network involved in the cellular resistance against DNA interstrand crosslinks (ICLs). A recent study showed that the FA pathway is synthetic lethal with several other DNA repair genes (Kennedy, 2007) such as ATM, NBS1, RAD54B and TP53BP1. Defects in those genes have been linked to a wide range of inherited and sporadic hematological malignancies including B-CLL, ALL, AML, CML, non-Hodgkin lymphoma, mantle cell lymphoma and multiple myeloma. FA pathway inhibitors may therefore selectively kill malignant cells bearing these defects. Curcumin, a natural product, was the first identified FA pathway inhibitor with activity in the micromolar range in cells (Chirnomas, 2006). However, the poor bioavailability of curcumin hinders its clinical efficacy. Identification of a curcumin analog with better activity, bioavailability and low toxicity could overcome this obstacle. We recently developed a cell-free assay for FA pathway function using Xenopus egg extracts to test the activity of curcumin analogs. As a pilot study we evaluated how well the assay identified inhibitors of the FA pathway in human cells. Methods: Fourteen curcumin analogs previously assayed in the NCI anticancer cell line screen (Adams, 2004) were tested for their activity on the FA pathway. Xenopus egg extracts were used to measure the relative inhibitory activity of the analogs on FANCD2 monoubiquitylation (FANCD2-L) and phosphorylation of other DNA damage response proteins. The underlying mechanism of inhibition was explored by testing the integrity of the core complex, the recruitment of the core complex to DNA and chromatin, and analyzing DNA replication and proteasome activity. Activity of several analogs was confirmed in HeLa cells by evaluation of the inhibition of hydroxyurea (HU)-induced FANCD2-L and FANCD2 foci. Results: EF24 (Adams, 2005) and three structurally similar analogs were 10 times more active than curcumin for FANCD2-L inhibition in Xenopus extracts. These analogs inhibited Mre11 phosphorylation at similar concentrations but had no effect on RPA32 and H2AX phosphorylation. In contrast to curcumin, EF24 did not display significant proteasome inhibition activity and did not affect integrity of the core complex or its recruitment to DNA and chromatin, ruling out these mechanisms to explain inhibition of the FA pathway. In HU-treated HeLa cells, EF24 strongly inhibited FANCD2-L and FANCD2 foci with an IC50 of 350 nM, confirming the results observed in Xenopus extracts. Conclusions: EF24 is a more potent FA pathway inhibitor than curcumin both in Xenopus extracts and in human cells, and as such may be effective as a single agent in targeted therapies against hematological malignancies deficient in ATM, NBS1, RAD54B or TP53BP1. In addition, this study demonstrates that Xenopus extracts are a powerful tool to identify and evaluate small molecules that modulate the FA and other DNA damage response pathways.

Description: The Fanconi Anemia (FA) Group C complementation group gene (FANCC) encodes a protein, FANCC, with a predicted Mr of 63000 daltons. FANCC is found in both the cytoplasmic and the nuclear compartments and interacts with certain other FA complementation group proteins as well as with non-FA proteins. Despite intensive investigation, the biologic roles of FANCC and of the other cloned FA gene products (FANCA and FANCG) remain unknown. As an approach to understanding FANCC function, we have studied the molecular regulation of FANCC expression. We found that although FANCCmRNA levels are constant throughout the cell cycle, FANCC is expressed in a cell cycle-dependent manner, with the lowest levels seen in cells synchronized at the G1/S boundary and the highest levels in the M-phase. Cell cycle–dependent regulation occurred despite deletion of the 5′ and 3′ FANCC untranslated regions, indicating that information in the FANCC coding sequence is sufficient to mediate cell cycle–dependent regulation. Moreover, inhibitors of proteasome function blocked the observed regulation. We conclude that FANCC expression is controlled by posttranscriptional mechanisms that are proteasome dependent. Recent work has demonstrated that the functional activity of FA proteins requires the physical interaction of at least FANCA, FANCC, and FANCG, and possibly of other FA and non-FA proteins. Our observation of dynamic control of FANCC expression by the proteasome has important implications for understanding the molecular regulation of the multiprotein complex.

Description: Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

Description: Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome. The phenotype includes developmental defects, bone marrow failure, and cell cycle abnormalities. At least eight complementation groups (A-H) exist, and although three of the corresponding complementation group genes have been cloned, they lack recognizable motifs, and their functions are unknown. We have isolated a binding partner for the Fanconi anemia group C protein (FANCC) by yeast two-hybrid screening. We show that the novel gene, FAZF, encodes a 486 amino acid protein containing a conserved amino terminal BTB/POZ protein interaction domain and three C-terminal Krüppel-like zinc fingers. FAZF is homologous to the promyelocytic leukemia zinc finger (PLZF) protein, which has been shown to act as a transcriptional repressor by recruitment of nuclear corepressors (N-CoR, Sin3, and HDAC1 complex). Consistent with a role in FA, BTB/POZ-containing proteins have been implicated in oncogenesis, limb morphogenesis, hematopoiesis, and proliferation. We show that FAZF is a transcriptional repressor that is able to bind to the same DNA target sequences as PLZF. Our data suggest that the FAZF/FANCC interaction maps to a region of FANCC deleted in FA patients with a severe disease phenotype. We also show that FAZF and wild-type FANCC can colocalize in nuclear foci, whereas a patient-derived mutant FANCC that is compromised for nuclear localization cannot. These results suggest that the function of FANCC may be linked to a transcriptional repression pathway involved in chromatin remodeling.