The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.

Fanconi anemia (FA) is a chromosomal instability syndrome associated with a strong predisposition to cancer, particularly acute myeloid leukemia and squamous cell carcinoma. At the cellular level, FA is characterized by spontaneous chromosomal breakage and a unique hypersensitivity to DNA cross-linking agents. Complementation analysis has indicated that at least seven distinct genes are involved in the pathogenesis of FA. Despite the identification of four of these genes (FANCA, FANCC, FANCF and FANCG), the nature of the 'FA pathway' has remained enigmatic, as the FA proteins lack sequence homologies or motifs that could point to a molecular function. To further define this pathway, we studied the subcellular localizations and mutual interactions of the FA proteins, including the recently identified FANCF protein, in human lymphoblasts. FANCF was found predominantly in the nucleus, where it complexes with FANCA, FANCC and FANCG. These interactions were detected in wild-type and FA-D lymphoblasts, but not in lymphoblasts of other FA complementation groups. This implies that each of the FA proteins, except FANCD, is required for these complexes to form. Similarly, we show that the interaction between FANCA and FANCC is restricted to wild-type and FA-D cells. Furthermore, we document the subcellular localization of FANCA and the FANCA/FANCG complex in all FA complementation groups. Our results, along with published data, culminate in a model in which a multi-protein FA complex serves a nuclear function to maintain genomic integrity.

Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.

Fanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive bone marrow failure and cancer predisposition. Eight FA complementation groups have been identified. The FANCA, FANCC, FANCE, FANCF, and FANCG proteins form a nuclear complex required for the monoubiquination of the FANCD2 protein. To investigate the architecture of the FA protein complex, the yeast 2-hybrid system was used to map contact points of the FANCA/FANCG, FANCC/FANCE, and FANCF/FANCG interactions. FANCG was shown to interact with both the amino-terminus of FANCA and the carboxyl-terminal region of FANCF. A FANCG mutant truncated at the carboxyl-terminus retained the ability to interact with FANCA. The interaction between FANCG and FANCF was ablated by a Leu71Pro mutant of FANCG. A central region of FANCE was sufficient for FANCC binding. A Leu554Pro mutant of FANCC failed to interact with FANCE. To further examine complex assembly, the yeast 3-hybrid system was used to investigate the ability of FANCG to act as a molecular bridge in mediating interaction between other FA proteins. FANCG was able to mediate interaction between FANCA and FANCF, as well as between monomers of FANCA. Direct interaction between FANCE and FANCD2 was also demonstrated in the yeast 2-hybrid system. This interaction involving an amino-terminal region of FANCD2 may provide a link between the FA protein complex and its downstream targets.

The events responsible for repair of DNA interstrand cross-links in mammalian cells, the proteins involved and their interactions with each other are poorly understood. The present study demonstrates that the structural protein nonerythroid alpha spectrin (alphaSpIISigma*), present in normal human cell nuclei, plays an important role in repair of DNA interstrand cross-links. These results show that alphaSpIISigma* relocalizes to nuclear foci after damage of normal human cells with the DNA interstrand cross-linking agent 8-methoxypsoralen plus ultraviolet A (UVA) light and that FANCA and the known DNA repair protein XPF localize to the same nuclear foci. That alphaSpIISigma* is essential for this re-localization is demonstrated by the finding that in cells from patients with Fanconi anemia complementation group A (FA-A), which have decreased ability to repair DNA interstrand cross-links and decreased levels of alphaSpIISigma*, there is a significant reduction in formation of damage-induced XPF as well as alphaSpIISigma* nuclear foci, even though levels of XPF are normal in these cells. In corrected FA-A cells, in which levels of alphaSpIISigma* are restored to normal, numbers of damage-induced nuclear foci are also returned to normal. Co-immunoprecipitation studies show that alphaSpIISigma*, FANCA and XPF co-immunoprecipitate with each other from normal human nuclear proteins. These results demonstrate that alphaSpIISigma*, FANCA and XPF interact with each other in the nucleus and indicate that there is a close functional relationship between these proteins. These studies suggest that an important role for alphaSpIISigma* in the nucleus is to act as a scaffold, aiding in recruitment and alignment of repair proteins at sites of damage.

Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63-linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage-induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance.

Fanconi anemia (FA) is a genetic disorder that predisposes to hematopoietic failure, birth defects and cancer. We identified an interaction between the FA protein, FANCA and brm-related gene 1 (BRG1) product. BRG1 is a subunit of the SWI/SNF complex, which remodels chromatin structure through a DNA-dependent ATPase activity. FANCA was demonstrated to associate with the endogenous SWI/SNF complex. We also found a significant increase in the molecular chaperone, glucose-regulated protein 94 (GRP94) among BRG1-associated factors isolated from a FANCA-mutant cell line, which was not seen in either a normal control cell line or the mutant line complemented by wild-type FANCA. Despite this specific difference, FANCA did not appear to be absolutely required for in vitro chromatin remodeling. Finally, we demonstrated co-localization in the nucleus between transfected FANCA and BRG1. The physiological action of FANCA on the SWI/SNF complex remains to be clarified, but our work suggests that FANCA may recruit the SWI/SNF complex to target genes, thereby enabling coupled nuclear functions such as transcription and DNA repair.

Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

Fanconi anaemia (FA) is an autosomal-recessive disorder characterized by genomic instability, developmental defects, DNA crosslinking agent hypersensitivity and cancer susceptibility. Somatic-cell hybrid studies have revealed five FA complementation groups (A-E; refs 4-6) displaying similar phenotypes, suggesting that FA genes are functionally related. The two cloned FA genes, FAA and FAC, encode proteins that are unrelated to each other or to other proteins in GenBank. In the current study, we demonstrate the FAA and FAC bind each other and form a complex. Protein binding correlates with the functional activity of FAA and FAC, as patient-derived mutant FAC (L554P) fails to bind FAA. Although unbound FAA and FAC localize predominantly to the cytoplasm, the FAA-FAC complex is found in similar abundance in both cytoplasm and nucleus. Our results confirm the interrelatedness of the FA genes in a pathway, suggesting the cooperation of FAA and FAC in a nuclear function.