Gamma interferon (IFN-␥) induces transcription of a distinct set of genes by activating STAT1, one member of a family of latent cytoplasmic transcription factors that are activated via phosphorylation on tyrosine residues (6). The IFN-␥ receptor (IFN-␥R) lacks intrinsic tyrosine kinase activity, but on ligand binding, receptor multimerization results in reciprocal activation of JAK1 and JAK2, two Janus tyrosine kinases noncovalently attached to the IFN-␥R ␣ and ␤ chains (2, 20, 22). Phosphorylation of the tyrosine residue on IFN-␥R␣ by JAK molecules creates a docking site for the Src homology 2 domain of STAT1, which is then phosphorylated on tyrosine and ultimately forms a homodimeric transcription factor that translocates to the nucleus (6, 21). The factors that govern the traffic of cytoplasmic STAT molecules to the docking site on the IFN-␥R are unknown. We have recently found that the activation of STAT1 in response to IFN-␥ is suppressed in hematopoietic cells from children with Fanconi anemia of type C (FA-C) and in mice nullizygous at the FA-C locus. However, in the ground state (uninduced by IFN), IFN response factor 1 (IRF-1) is expressed at high levels in mutant FA-C cells (35), suggesting that a non-STAT1 pathway is involved in constitutive activation of IRF-1 in FA cells. In addition, complementation of the defect by retrovirus mediated transfer of normal FANCC cDNA reconstitutes the normal STAT1 response (10,38).Linkage of FANCC function with that of STAT1 provided us with an opportunity to test whether the relationship of these two molecules was direct or indirect. We report herein results of experiments in which the assembly of the fully activated IFN-␥R complex, including STAT1, JAK1, and JAK2, was examined in isogenic murine and human FA-C cells. We report that in IFN-␥-stimulated FA-C cells, phosphorylation of JAK1, JAK2, and IFN-␥R␣ occurs normally, but STAT1 does not dock at the receptor ␣ chain. In FA-C cells nuclear STAT1 is reduced, and IFN fails to induce STAT1-specific DNA-binding complexes and expression of IRF-1. Expression of the normal FANCC cDNA in mutant cells results in normal STAT1 docking and phosphorylation as well as normal induction of nuclear STAT1-DNA complex and normal induction of IRF-1. We also find that a variety of cytokines and hematopoietic growth factors stimulate the association of STAT1 with glutathione S-transferase (GST) fusion proteins encoding the normal FANCC but not a naturally occurring inactivating mutant FANCC (L554P) and that the association occurs rapidly and prior to STAT1 phosphorylation on Y 701 . Coimmunoprecipitation experiments confirmed the IFN-inducible association of

Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2 ؊/؊ mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit ؉ Sca-1 ؉ Lineage ؊ (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2 ؊/؊ KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2 ؊/؊ mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2 ؊/؊ KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2 ؊/؊ bone marrow cells. We conclude that Fancd2 ؊/؊ mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies. IntroductionFanconi anemia (FA) is a rare, autosomal, recessive genetic disorder associated with severe birth defects, cancer predisposition, and bone marrow failure. Thirteen causative genes (FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, FANCI, FANCL/PHF9/Pog, FANCJ/BRIP1/BACH1, FANCM/Hef, and FANCN/ PALB2) have been identified and cloned to date, and the encoded proteins are believed to work together in a common DNA damageresponse pathway to maintain genomic integrity and protect the genome from DNA damage induced by cross-linking agents. 1,2 Although deficiency in DNA cross-link repair renders all FA cells susceptible to cross-linking agents, bone marrow is the most affected organ system. Mutations in any of the different FA genes almost universally lead to bone marrow failure, which is the primary cause of mortality in FA. 3 The pathogenesis of bone marrow failure in FA remains elusive. Mutations in several genes involved in DNA damage repair, including Atr, XPD, and Ercc1, caused either hematopoietic stem cell (HSC) loss or impaired HSC function under conditions of stress. [4][5][6] These studies suggest that the maintenance of genome integrity is critical for HSC survival and function. However, the extent to which genotoxicity, resulting from impaired DNA damage repair, contributes to bone marrow failure in FA is unclear. 7 Other pathways associated with hematopoietic failure, such as altered cytokine signaling, may also contribute to FA pathogenesis. 8,9 For example, levels of proapoptotic cytokines tumor necrosis factor-␣ (TNF-␣) and interferon-␥ (IFN-␥) are elevated in FA lymphocytes, bone marrow cells, and FA patient serum samples. 10-12 FA bone marrow cells (at least of the C complementation group) are also hypersensitive to these cytokines and undergo apoptosis when exposed to even low levels of them. [13][14][15] To better understand FA, multiple murine knockout models, includingand Fancl Ϫ/Ϫ m...

Tumor necrosis factor alpha (TNF-␣) production is abnormally high in IntroductionThe Fanconi anemia (FA) proteins play an important role in regulating genome stability, 1 but there is little evidence that the loss of the genoprotection per se in FA cells accounts for the molecular pathogenesis of the bone-marrow failure characteristic of this disease. In fact there is evidence that at least some of these proteins are multifunctional 2 and participate in canonical signaling pathways in hematopoietic cells. [2][3][4][5][6][7][8] Fanconi anemia, complementation group C (FANCC)-deficient cells, for example, are hypersensitive to the apoptotic effects of tumor necrosis factor-␣ (TNF-␣). [4][5][6][7][8][9] In addition, FA cells overproduce TNF-␣ for reasons that have not yet been fully explained. [10][11][12] Most importantly, there is clear evidence that overproduction of and hypersensitivity to TNF-␣ in hematopoietic cells of Fancc Ϫ/Ϫ mice results in bone marrow hypoplasia 13,14 and that long-term ex vivo exposure of murine Fancc Ϫ/Ϫ hematopoietic cells to both growth factors and TNF-␣ results in the evolution of cytogenetically marked preleukemic clones. 9 Therefore, the hematopoietic phenotype of FA may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. We designed gene expression microarray experiments by using marrow cells from both patients with FA and normal volunteers in part to seek potential clues to the mechanisms by which FA cells overproduce TNF-␣.Recognizing that transcriptomal analysis would not reveal aspects of the FA phenotype that were controlled translationally or posttranslationally, we also conducted a proteomics analysis. We based our experimental design on an accepted function of the FA "nuclear core complex," that is, its capacity to facilitate monoubiquitinylation of both Fanconi anemia, complementation group I and Fanconi anemia, complementation group D2 (FANCD2). 15,16 Although it is clear that monoubiquitinylation, at least of FANCD2, is required for the avoidance of genotoxicity, 17 it seemed to us unlikely that 8 individual FA genes encoding the "core complex proteins" should have evolved to control the monoubiquitinylation of merely 1 or 2 nuclear proteins. Therefore, reasoning that ubiquitinylation of a variety of other proteins might also be influenced by the core FA proteins, we designed a proteomics survey of ubiquitinylated proteins in FA-C cells and isogenic controls. We reasoned that this approach might lead to the identification of other proteins underubiquitinylated in mutant cells. As reported herein, the gene expression microarray analysis revealed a significant overrepresentation of overexpressed ubiquitin pathway genes in the mutant cells. We therefore took into account the alternative possibility that some ubiquitinylated proteins might be found uniquely in the mutant cells.Indeed, one such protein, Toll-like receptor 8 (TLR8), did appear in the ubiquitin-positive fractions only in FANCC-mutant cells. Given that TLR8 activ...

Fanconi anemia (FA) is an autosomal recessive chromosome instability syndrome characterized by progressive bone marrow (BM) failure, skeletal defects, and increased susceptibility to malignancy. FA cells are hypersensitive to DNA cross-linking agents, oxygen and have cell cycle abnormalities. To develop an animal model of the disease we generated mice homozygous for a targeted deletion of exon 9 of the murine FA complementation group C gene (fac). Mutant mice had normal neonatal viability and gross morphology, but their cells had the expected chromosome breakage and DNA cross-linker sensitivity. Surprisingly, male and female mutant mice had reduced numbers of germ cells and females had markedly impaired fertility. No anemia was detectable in the peripheral blood during the first year of life, but the colony forming capacity of marrow progenitor cells was abnormal in vitro in mutant mice. Progenitor cells from fac knock-out mice were hypersensitive to interferon gamma. This previously unrecognized phenotype may form the basis for BM failure in human FA.

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Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus (FAC −/−) are hypersensitive to the mitotic inhibitory effects of interferon (IFN-γ). We tested the hypothesis that HPC from the bone marrow of Fanconi group C children are similarly hypersensitive and that the fas pathway is involved in affecting programmed cell death in response to low doses of IFN-γ. In normal human and murine HPC, IFN-γ primed the fas pathway and induced both fas and interferon response factor-1 (IRF-1) gene expression. These IFN-γ-induced apoptotic responses in HPC from the marrow of a child with FA of the C group (FA-C) and in FAC −/− mice occurred at significantly lower IFN doses (by an order of magnitude) than did the apoptotic responses of normal HPC. Treatment of FA-C CD34+ cells with low doses of recombinant IFN-γ, inhibited growth of colony forming unit granulocyte-macrophage and burst-forming unit erythroid, while treatment with blocking antibodies to fas augmented clonal growth and abrogated the clonal inhibitory effect of IFN-γ. Transfer of the normal FAC gene into FA-C B-cell lines prevented mitomycin C–induced apoptosis, but did not suppress fas expression or inhibit the primed fas pathway. However, the kinetics of Stat1-phosphate decay in IFN-γ–treated cells was prolonged in mutant cells and was normalized by transduction of the normal FAC gene. Therefore, the normal FAC protein serves, in part, to modulate IFN-γ signals. HPC bearing inactivating mutations of FAC fail to normally modulate IFN-γ signals and, as a result, undergo apoptosis executed through the fas pathway.