Regualr articleYeast two-hybrid screens imply involvement of fanconi anemia proteins in transcription regulation, cell signaling, oxidative metabolism, and cellular transport
Introduction
Fanconi anemia (FA) is an autosomal recessive disorder with an occurrence of one in 360,000 live births. Typically the disorder is diagnosed in children at ages 5 to 10 years, presenting with pancytopenia or aplastic anemia. The main cause of death is bone marrow failure. Advances in treatment of anemia and the availability of bone marrow and cord blood transplants has increased the survival of these patients. Hematopoiesis is however not the only defect found in FA patients. The phenotype is characterized by a wide variety of clinical abnormalities, which occur in various combinations and at diverse frequencies. The main abnormalities are skeletal and urogenital anomalies, both of which are congenital, growth retardation, and hyperpigmentation of the skin. A further complication, especially if children survive to adulthood, is the increased risk of cancer, especially acute myeloid leukemia and squameous cell carcinomas. Cells from FA patients show increased chromosomal breakage, which is exacerbated by DNA crosslinking agents, such as mitomycin C. This observation has been used for the development of a clinical test, which is applied currently as diagnostic confirmation of the other clinical observations [1].
Cellular complementation analysis has revealed that at least eight complementation groups exist [2], [3] and there is evidence for two additional groups (manuscript in preparation, Joenje et al.) The cloning of seven genes within the past decade has shown that each complementation group contains defects in distinct disease genes. The functional hypotheses that arose from the clinical phenotype include involvement in transcription, apoptosis, cell cycle, DNA repair, oxidative metabolism, and cell signaling. The structure of the proteins FANCA, FANCC, FANCE, FANCF, and FANCG gave no clues to a functional mechanism or pathway; however, the recent identification of FANCD2 and FANCD1 has allowed the association of these proteins with the DNA repair mechanism through their interactions with repair proteins [3], [4]. FANCD2 was found to associate with the breast cancer protein BRCA1 in a DNA damage-dependent manner [5] and FANCD1 has now been identified as the breast cancer-associated protein BRCA2 [4]. These connections show an involvement of at least part of the FA pathway in DNA repair mechanisms. Nevertheless a wealth of previous research has shown that FA proteins are involved in pathways connected to oxidative metabolism, apoptosis, cell signaling, and transcription, functions which contribute to the pleiotropic cellular and clinical FA phenotype [2], [6], [7], [8].
The yeast two-hybrid system is a very powerful tool for the exploration of protein function [9], [10]. This technique has successfully been exploited to unravel a large number of protein interactions, determine new protein partners, and even describe larger protein complexes [11], [12], [13], [14], [15]. The yeast two-hybrid system allows screening for novel interacting proteins using a protein of interest as “bait.” This genetic screen is well suited to the search for functional partners of the FA proteins, particularly since patient-derived inactivating mutations are available to test candidates for biologically significant interactions. Six of 16 proteins so far described to interact with FA proteins have been identified by the yeast two-hybrid technique; they are GRP94, FAZF, SNX5, BRG1, CYP2E1, and IκB kinase [16], [17], [18], [19], [20], [21] (see also Table 1). Here we report 69 novel potential FANCA, FANCC, or FANCG interacting proteins. These candidate proteins were collected from the experiments performed in four different laboratories using five libraries derived from various tissues. All interactor candidates have undergone testing in the yeast two-hybrid system, and many were additionally evaluated by in vitro and in vivo coimmunoprecipitation and colocalization experiments. Since full characterization of the biological significance of these interactions is hampered by the absence of functional assays for the FA proteins, further confirmatory experiments will be required to fully validate their in vivo involvement in the FA pathway. We present these interactions here in order to disseminate the data that have been accumulated from several yeast two-hybrid screens. Our data provide new evidence for a connection of the FA pathway with a range of cellular functions linked to hematopoiesis, cancer, and detoxification.
Section snippets
Bacterial and yeast strains
Escherichia coli bacterial strains DH5α and HB101 were used for propagation of plasmid constructs. Saccharomyces cerevisiae yeast strains Y190, Y187, AH109 (Clontech), EGY48, and RFY206 were used as hosts in the two-hybrid assays. AH109 contains the two nutritional reporter genes ADE2 and HIS3, while Y190 contains the reporter gene HIS3. EGY48 contains LEU2 as the reporter gene and RFY206 contains URA3 as reporter gene. All five strains contain the LacZ reporter gene.
Plasmids and yeast two-hybrid library
The bait vectors used in
Results
We have used five FANCC, four FANCA, and one FANCG yeast two-hybrid bait constructs to screen five different cDNA libraries for interacting proteins by yeast two-hybrid analysis. The human cDNA libraries used were derived from lymphocytes, B-lymphocytes, HeLa cells, skeletal muscle, placenta, and brain (Table 2). In total we have screened 36.5 × 106 clones from these libraries. Table 1 lists all proteins that reproducibly interacted with the bait protein while lacking interaction with control
Discussion
Our collection of yeast two-hybrid interaction screens using 10 different bait constructs with whole or partial sequences of FANCA, FANCC, or FANCG has resulted in the identification of 69 novel FA protein interactors potentially involved in the FA pathway. Functional classification of these proteins shows that they belong mainly to four different functional groups, transcription, oxidative metabolism, signaling, and transport. The GAL4-based system employed by three laboratories seemed to
Conclusions
The use of yeast two-hybrid library screens for the identification of new functional interactions is a powerful tool. Nevertheless this report shows that this technique can lead to a quantity of data that is difficult to manage and to process toward meaningful physiological results. The laborious and time-consuming follow-up studies necessary for the yeast two-hybrid technique have hampered the communication of a wealth of data produced in such screens, a fact that this report seeks to correct.
Acknowledgements
This work was supported by the Schroeder–Kurth Fonds (Wuerzburg, Germany), MRC (UK), Cancer Research Campaign UK, Leukaemia Research Fund (UK), Dutch Cancer Society (NL), Fanconi Anemia Research Fund (USA), and NIH Grant HL56045 (USA)
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2016, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesCitation Excerpt :The overexpression of both proteins in a myeloid progenitor cell line prevents apoptosis. FANCC increases GSTP1 activity after the induction of apoptosis [26,27]. Although FANCC lacks homology with conventional disulfide reductases, it seems to have a role in preventing the formation of inactivating disulfide bonds within GSTP1 during apoptosis.
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2015, Journal of Molecular BiologyCitation Excerpt :We demonstrated by co-immunoprecipitation that the interaction of FANCEΔ4 with FANCC, FANCF and FANCD2 is maintained. Hairy and the enhancer of split 1 HES1 is known to interact directly with the core complex members (FANCA, FANCF, FANCG and FANCL), but not FANCE [36], while an interaction between STAT1 and FANCC was also demonstrated in the literature [64–66]. Hence, we determined that FANCE-HA and FANCEΔ4-HA co-immunoprecipitated with HES1-FLAG and STAT1-FLAG in HEK293T cells.
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These authors contributed equally to this work.