Elsevier

Experimental Cell Research

Volume 289, Issue 2, 1 October 2003, Pages 211-221
Experimental Cell Research

Regualr article
Yeast two-hybrid screens imply involvement of fanconi anemia proteins in transcription regulation, cell signaling, oxidative metabolism, and cellular transport

https://doi.org/10.1016/S0014-4827(03)00261-1Get rights and content

Abstract

Mutations in one of at least eight different genes cause bone marrow failure, chromosome instability, and predisposition to cancer associated with the rare genetic syndrome Fanconi anemia (FA). The cloning of seven genes has provided the tools to study the molecular pathway disrupted in Fanconi anemia patients. The structure of the genes and their gene products provided few clues to their functional role. We report here the use of 3 FA proteins, FANCA, FANCC, and FANCG, as “baits” in the hunt for interactors to obtain clues for FA protein functions. Using five different human cDNA libraries we screened 36.5 × 106 clones with the technique of the yeast two-hybrid system. We identified 69 proteins which have not previously been linked to the FA pathway as direct interactors of FANCA, FANCC, or FANCG. Most of these proteins are associated with four functional classes including transcription regulation (21 proteins), signaling (13 proteins), oxidative metabolism (10 proteins), and intracellular transport (11 proteins). Interaction with 6 proteins, DAXX, Ran, IκBγ, USP14, and the previously reported SNX5 and FAZF, was additionally confirmed by coimmunoprecipitation and/or colocalization studies. Taken together, our data strongly support the hypothesis that FA proteins are functionally involved in several complex cellular pathways including 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)

References (57)

  • R Gongora et al.

    An essential role for Daxx in the inhibition of B lymphopoiesis by type I interferons

    Immunity

    (2001)
  • M Dasso

    Running on Rannuclear transport and the mitotic spindle

    Cell

    (2001)
  • P Kalab et al.

    The ran GTPase regulates mitotic spindle assembly

    Curr. Biol.

    (1999)
  • M Hetzer et al.

    GTP hydrolysis by Ran is required for nuclear envelope assembly

    Mol. Cell

    (2000)
  • M Kieran et al.

    The DNA binding subunit of NF-kappa B is identical to factor KBF1 and homologous to the reloncogene product

    Cell

    (1990)
  • C.M Pickart

    Ubiquitin enters the new millennium

    Mol. Cell

    (2001)
  • M.S Dai et al.

    The effects of the Fanconi anemia zinc finger (FAZF) on cell cycle, apoptosis, and proliferation are differentiation stage-specific

    J. Biol. Chem.

    (2002)
  • I Garcia-Higuera et al.

    The Fanconi anemia proteins FANCA and FANCG stabilize each other and promote the nuclear accumulation of the Fanconi anemia complex

    Blood

    (2000)
  • F.A.E Kruyt et al.

    Abnormal microsomal detoxification implicated in Fanconi anemia group C by interaction of the FAC protein with NADPH cytochrome P450 reductase

    Blood

    (1998)
  • W Ruppitsch et al.

    Overexpression of thioredoxin in Fanconi anemia fibroblasts prevents the cytotoxic and DNA damaging effect of mitomycin C and diepoxybutane

    FEBS Lett.

    (1998)
  • S Fagerlie et al.

    The Fanconi anemia group C gene product. Signaling functions in hematopoietic cells

    Exp. Hematol.

    (2001)
  • G.M Kupfer et al.

    The Fanconi anemia polypeptide, FAC, binds to the cyclin-dependent kinase, cdc2

    Blood

    (1997)
  • T Taniguchi et al.

    Convergence of the Fanconi anemia and ataxia telangiectasia signaling pathways

    Cell

    (2002)
  • T Otsuki et al.

    Phosphorylation of Fanconi anemia protein, FANCA, is regulated by Akt kinase

    Biochem. Biophys. Res. Commun.

    (2002)
  • L.W McMahon et al.

    Human alpha Spectrin II and the Fanconi anemia proteins FANCA and FANCC interact to form a nuclear complex

    J. Biol. Chem.

    (1999)
  • A.D Auerbach et al.

    Prenatal and postnatal diagnosis and carrier detection of Fanconi anemia by a cytogenetic method

    Pediatrics

    (1981)
  • H Joenje et al.

    The emerging genetic and molecular basis of Fanconi anaemia

    Nat. Rev. Genet.

    (2001)
  • N.G Howlett et al.

    Biallelic inactivation of BRCA2 in Fanconi anemia

    Science

    (2002)
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