Research Article: Fanconi Anemia Proteins and Their Interacting Partners: A Molecular Puzzle

Date Published: March 29, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Tagrid Kaddar, Madeleine Carreau.


In recent years, Fanconi anemia (FA) has been the subject of intense investigations, primarily in the DNA repair research field. Many discoveries have led to the notion of a canonical pathway, termed the FA pathway, where all FA proteins function sequentially in different protein complexes to repair DNA cross-link damages. Although a detailed architecture of this DNA cross-link repair pathway is emerging, the question of how a defective DNA cross-link repair process translates into the disease phenotype is unresolved. Other areas of research including oxidative metabolism, cell cycle progression, apoptosis, and transcriptional regulation have been studied in the context of FA, and some of these areas were investigated before the fervent enthusiasm in the DNA repair field. These other molecular mechanisms may also play an important role in the pathogenesis of this disease. In addition, several FA-interacting proteins have been identified with roles in these “other” nonrepair molecular functions. Thus, the goal of this paper is to revisit old ideas and to discuss protein-protein interactions related to other FA-related molecular functions to try to give the reader a wider perspective of the FA molecular puzzle.

Partial Text

Fanconi anemia (FA) is a complex disease that is considered a congenital form of aplastic anemia. The genetic mode of transmission is both autosomal and X-linked, and a growing number of identified genes are distributed among the various chromosomes. The common clinical manifestation in most patients with FA, which may occur in all FA patients eventually, is life-threatening bone marrow failure (BMF) [1, 2]. FA is also associated with diverse birth defects and a predisposition to malignancies. FA-associated congenital malformations can affect many organ systems including the central nervous system, the gastrointestinal system, and the skeletal system [3–8]. Other findings in patients with FA include short stature, skin pigmentation abnormalities, and small facial features. In addition, more than 70% of patients with FA show endocrine dysfunctions including deficiencies in growth hormone and thyroid hormone as well as diabetes [9, 10]. All of these disease manifestations suggest a role for FA genes in mechanisms that bear on hematopoiesis, development, and neoplasia.

Patients with FA are classified into complementation groups (to date 14 groups from A to P have been identified), and all of these groups correspond to one of the following cloned genes: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BRIP1/BACH1, FANCL/PHF9, FANCM/HEF, FANCN/PALB2, and FANCP/SLX4 [11–27]. Approximately 85% of FA patients have a defective FANCA, FANCC or FANCG gene, while the other genes account for less than 5% of the mutations found in FA patients. To date, some patients still remain unassigned indicating the possibility of novel FA genes [28]. Mutations in the RAD51C gene (provisionally termed FANCO) have been associated with a FA-like disorder, suggesting that this gene may represent yet another FA gene [29, 30]. Patients with mutations in one of the 15 FA and FA-like genes present clinical FA aspects to various degrees but show a common cellular phenotype: hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC), diepoxybutane, and cisplatin [28, 31]. When exposed to those agents, cells from FA patients show an abnormally prolonged cell cycle arrest in the G2/M phase, increased chromosomal aberrations, and reduced survival. These cellular features define FA, and presumably, all FA proteins cooperate in a pathway, termed the FA pathway, to maintain chromosome integrity.

One intriguing aspect of the FA molecular pathway is the cellular distribution of FA proteins. Although the well-characterized function of this pathway in DNA crosslink damage occurs in the nucleus, FA core complex proteins can be found in different cellular compartments in addition to the nucleus.

FA proteins undergo multiple posttranslational modifications, including monoubiquitination, phosphorylation and proteolytic processing. The most widely studied modification is the monoubiquitination of FANCD2 and FANCI. Although only two of the fifteen FA proteins are monoubiquitinated, several FA proteins, including FANCA, FANCE, FANCG, FANCD2, FANCI, and FANCM, are phosphorylated, and two FA proteins are regulated through a caspase-mediated proteolytic process. These data suggest that posttranslational modifications play an important role in FA proteins activity. In this section, we will only discuss FA protein modifications other than the ubiquitination of FANCD2 and FANCI because it has been extensively discussed [47, 76, 77].

The abnormal sensitivity of FA cells to reactive oxygen species (ROS) was first suggested in 1977 by Nordenson [96] who showed reduced chromosomal breaks in FA lymphocytes cultured in the presence of superoxide dismutase, catalase, or both enzymes. The role of oxygen on chromosomal instability in FA mutant cells was confirmed by Joenje et al. in 1981; they showed attenuated chromosomal aberrations at low oxygen tension (5%) but aggravated chromosomal aberrations at high concentrations of oxygen [97]. Subsequently, several reports indicated that FA cells were hypersensitive to oxygen radicals showing reduced growth and blockage in the G2 phase of the cell cycle [98–101]. Increased ROS in FA leucocytes has also been reported [99]. In addition, the overexpression of detoxifying enzymes, the inhibition of enzymes involved in oxidation or the use of antioxidants in FA cells reduced the rate of spontaneous chromosomal breakage and abolished the DNA damaging effects of MMC [98, 102–106]. Other studies have established a link between an altered redox state and reduced proliferation, reduced growth, and altered cytokine responses in FA cells including hematopoietic progenitors [107–110]. Studies from FancC/Sod1 double mutant mice exhibiting defects in hematopoiesis including bone marrow hypocellularity and cytopenia, which is reminiscent of phenotypes observed in patients with FA, suggest that an abnormal redox state contribute to BMF in FA [111].

It is well established that FA mutant cells are prone to apoptosis. The FA literature is rich in reports pertaining to FA mutant cells (human bone-marrow-derived cells, lymphocytes, fibroblasts, and mouse embryonic fibroblasts) that show increased apoptosis or reduced cell growth in response to various agents including ROS inducers, DNA damaging agents, growth factor withdrawal, and cytokines. It is clear from many studies of patient-derived cells and cells from FA mouse models that FA proteins are involved in pathways that regulate cell survival or cell death [116–121]. For instance, two FA proteins, FANCC and FANCD2, are caspase targets [94, 95], and FANCC overexpression or the inhibition of its caspase-mediated cleavage prevents or delays apoptosis, even in wildtype cells supporting the idea of a cell survival function of the FA proteins [94, 119, 122]. The role of FANCC in cell survival has been linked to oxidative metabolism as described above but it may also be linked to cytokine-mediated cellular responses because many cytokine-mediated signaling events lead to apoptosis. It has been suggested that abnormal cytokine regulation may account for the progressive BMF observed in patients with FA because TNF-α  overproduction and underproduction of Il-6 have been detected in the sera of patients with FA [123–125]. FA-C mutant cells and FancC−/− progenitor and stem cells are hypersensitive to the inhibitory cytokines including TNF-α  and IFN-γ, and show suppressed growth and increased apoptosis at doses that do not affect normal cells [116, 122, 126, 127]. In addition, the continuous injection of low IFN-γ  doses in vivo leads to BMF in FA mice [128, 129], whereas TNF-α  leads to clonal evolution and leukemia in this FA mouse model [130]. In support of these altered cytokine responses in FA cells, the cytokine-response genes myxovirus A (MxA), IFN response factor 1 (IRF1), p21CIP/WAF, and IFN-stimulated gene factor 3 (ISGF3γ) were highly expressed in FA mutant cells without exogenous cytokine stimulation, while corrected cells suppressed this overproduction and restored their MMC resistance [116, 131, 132]. These data suggest that FA proteins, or at least FANCC, function to modulate a cytokine-mediated signal. Indeed, FANCC was shown to directly interact with signal transducer and activator of transcription 1 (STAT1), which is an IFN signal transducer [133]. FANCC functions as a control factor for STAT1 docking at the IFN-γR complex and subsequent activation of the IFN type II signaling cascade [133]. Thus, the STAT1 activation defect observed in FA-C cells results in an altered nuclear STAT1-DNA complex, which diminishes the expression of IRF1. The STAT1-FANCC interaction is also induced by other cytokines, including IFN-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor, whereas mutant FANCC does not associate with STAT1 in cells stimulated with these factors. FANCC seems to regulate IFNγ-inducible genes (e.g., IRF1, p21WAF, and ISGF3γ) independently of STAT1 binding. An altered response to type I IFN was also observed in FANCC mutant and FancC−/− cells, as shown by the reduced phosphorylation of the Janus kinases, Jak1 and Tyk2, and the subsequently diminished phosphorylation of STAT1, STAT3, and STAT5 [134]. This altered Tyk2 response translates into reduced numbers of CD4-positive cells in FancC−/− mice. Because Tyk2 plays a role in the differentiation and maintenance of T helper cells, failure of FANCC to normally activate Jak/STAT signaling may result in impaired immune cell differentiation and immune defects, as reported in patients with FA [135–139].

Another FA protein role less considered is the regulation of transcription. Several FA proteins have interacting partners directly involved in transcriptional regulation. The first FA protein partner identified that acts in transcription is FAZF (FA Zinc Finger) [147]. FAZF, also known as RoG (for repressor of GATA) [148], PLZP (for PLZF-like zinc finger protein) [149] and TZFP (for testis zinc finger) [150], is a transcriptional repressor that belongs to the BTB/POZ family of proteins and is similar to the PLZF protein [147]. This family of transcriptional repressors was shown to be important for several developmental processes including tissue proliferation and differentiation and tumor formation. FAZF was identified in a yeast 2-hybrid screen with FANCC. FAZF was shown to be highly expressed in CD34-positive progenitor cells; it further increased during proliferation of these cells and decreased during their terminal differentiation [151]. FAZF acts as a negative regulator of transcription. Because a disease-causing mutation in FANCC interferes with FAZF binding [147], and FancC−/− hematopoietic stem/progenitor cells show increased cycling and aberrant cell cycle control [152], a plausible hypothesis is that the FANCC-FAZF interaction in hematopoietic stem/progenitor cells leads to the repression of critical target genes required for growth suppression.

Since the discovery of FANCC, the first identified FA gene in 1992 [15], there have been significant advances in the FA molecular biology field. These advances mostly include characterization of the canonical FA pathway, which is activated in response to DNA crosslink damage. It is clear that FA proteins are required for DNA crosslink repair; however, the question of how a defective FA protein leads to BMF, and developmental abnormalities remains elusive. It is obvious that absence of a functional FA protein affects many cellular and molecular functions and leads to an array of cellular phenotypes (see Figure 1). A perplexing question is whether FA proteins interactions with their nonrepair partners act only as modifiers of the clinical manifestation of FA. Once we reconcile all the notions related to FA proteins role in these various cellular and molecular activities, we may then obtain a clearer picture of the complexities of this molecular puzzle.




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