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Fanconi anemia (FA) is an autosomal recessive disease characterized by a variety of congenital abnormalities. Cells from FA patients show chromosomal instability and are hypersensitive to DNA cross-linking agents, though the basic cellular defect in FA is not known. The FANCA gene encodes a protein with an Mr of 162 kDa and with unknown function. The cellular localization of the FANCA protein has been controversial, and has been shown in different reports to be exclusively cytoplasmic and predominantly nuclear. In the present study, we further confirm that FANCA localizes primarily to the nucleus. Fusions of FANCA with the green fluorescent protein (GFP) showed a strong nuclear signal and a weak cytoplasmic signal in several cell types. Confocal laser microscopy confirmed that FANCA is evenly distributed throughout the nucleus. We also examined regions in FANCA that participate in its nuclear import. FANCA contains two bipartite nuclear localization signal (NLS) motifs at the extreme N-terminus. Deletion of amino acids N-terminal to the NLS motifs had no effect on the nuclear localization of FANCA or on its ability to correct mitomycin C sensitivity in an FA-A cell line, while deletion of both motifs impeded but did not prevent nuclear import. Deletions of 75, 90 and 150 residues from the N-terminus yielded a mixture of cells with only a cytoplasmic signal, and with both a nuclear and cytoplasmic signal. Deletion of the N-terminal 250 amino acids was required to block nuclear localization completely. Fusion of GFP to the N-terminal 250 amino acids showed a localization pattern similar to FANCA. Mutant forms of FANCA with deletions of the C-terminal 70 or 260 residues localized to the cytoplasm, although the C-terminal 260 amino acids alone lacked NLS activity. The results show that nuclear localization of FANCA involves several functional regions.

Fanconi anemia (FA) is one of several genetic diseases with characteristic cellular hypersensitivity to DNA crosslinking agents which suggest that FA proteins may function as part of DNA repair processes. At the clinical level, FA is characterized by bone marrow failure that affects children at an early age. The clinical phenotype is heterogeneous and includes various congenital malformations as well as cancer predisposition. FA patients are distributed into eight complementation groups suggesting a complex molecular pathway. Three of the eight possible FA genes have been cloned, although their function(s) have not been identified. FA cells are highly sensitive to DNA crosslinking agents (mitomycin C (MMC) and diepoxybutane), with some variability between cell lines. Sensitivity to monofunctional alkylating agents has been reported in some cases, although these studies were performed with genetically unclassified FA cells. To further analyse and characterize the newly identified FA complementation groups, we tested their sensitivity to UV radiation, monofunctional and bifunctional alkylating agents and to the X-ray mimetic drug bleomycin. We found that FA complementation groups D to H show increased sensitivity to the X-ray mimetic drug bleomycin. Furthermore, the single known FA-H cell line shows increased sensitivity to ethylethane sulfonate (EMS), methylmethane sulfonate (MMS) in addition to the characteristic sensitivity to crosslinking agents, suggesting a broader spectrum of drug sensitivities in FA cells.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies1. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents1. Eight complementation groups (A-H) have been distinguished2, each group possibly representing a distinct FA gene3. The genes mutated in patients of complementation groups A (FANCA; Refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control9,10. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

In previous studies we have described a 5.0 kb Hin dIII fragment downstream of muscle exon 1 that exhibits properties consistent with a muscle-specific transcriptional enhancer. The goal of this study has been to identify the sequence elements responsible for muscle-specific enhancer activity. Functional studies indicated that this enhancer is active in pre- and post-differentiated H9C2(2-1) myoblasts but functions poorly in L6 and C2C12 myotubes. The core enhancer region was delimited to a 195 bp Spe I- Acc I fragment and sequence analysis identified three MEF-1/E box and two MEF-2/AT-rich motifs as potential muscle-specific regulatory domains. EMSA competition and DNase footprinting indicated that sequences within a 30 bp region containing single adjoining MEF-1/E box and MEF-2/AT-rich motifs are target binding sites for trans -acting factors expressed in H9C2(2-1) myotubes but not in L6 or C2C12 myotubes. Site-specific mutations within these motifs resulted in a significant reduction in enhancer activity in H9C2(2-1) myotubes. These results suggest that the mechanisms governing DMD gene expression in muscle are similar to those identified in other muscle-specific genes. However, the myogenic profile of enhancer activity and trans -acting factor binding suggests a more specialized role for this enhancer that is consistent with its potential involvement in dystrophin gene regulation in cardiac muscle.

The 14 kb muscle isoform of the Duchenne muscular dystrophy (DMD) gene is expressed primarily in skeletal and cardiac muscle. Transcription of the muscle isoform is induced as myoblasts differentiate into multinucleated myotubes and transcript levels are increased a further 10-fold in mature skeletal muscle. In previous studies we have demonstrated that the core muscle promoter of the human DMD gene contains sequences that regulate the induction of DMD gene expression with myoblast differentiation. However, direct injection studies have indicated that the activity of the core muscle promoter in mature skeletal muscle is 30-fold lower than in immature myotubes. This discrepancy between endogenous transcript levels and core promoter activity suggested that additional transcriptional elements are involved in the regulation of DMD gene expression in muscle. In this report we present evidence for the existence of a muscle-specific enhancer within intron 1 of the human DMD gene. Functional analysis of HindIII fragments from within a 36 kb region surrounding muscle exon 1 of the human DMD gene resulted in the identification of a 5.0 kb fragment within muscle intron 1 that consistently provided high levels of reporter gene expression in both immature and mature skeletal muscle. Sequences within this 5 kb fragment were shown to be functionally independent of position and orientation and to be inactive in fibroblasts, properties that are consistent with the definition of a muscle-specific enhancer. Although this enhancer provided a 30-fold increase in transcription from a SV40 viral promoter in mature skeletal muscle, only a 3-fold increase was observed from the DMD core muscle promoter. Intron 1 enhancer activity alone is therefore insufficient to account for the discrepancy between endogenous transcript levels and core muscle promoter activity in immature and mature skeletal muscle and points to the existence of additional enhancer elements in other regions of the DMD gene. This report provides the first evidence for the involvement of a transcriptional enhancer in DMD gene regulation in muscle and impacts on our understanding of the functional consequences of mutations at the 5'-end of gene. In this regard, deletions in this region in X-linked dilated cardiomyopathy patients provides indirect evidence for a role for this enhancer in regulating DMD gene expression in cardiac muscle.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by a diversity of clinical symptoms including skeletal abnormalities, progressive bone marrow failure and a marked predisposition to cancer1–4. FA cells exhibit chromosomal instability and hyper-responsiveness to the clastogenic5 and cytotoxic6 effects of bifunctional alkylating (cross-linking) agents, such as diepoxybutane (DEB) and mitomycin C (MMC). Five complementation groups (A–E) have been distinguished on the basis of somatic cell hybridization experiments7–9, with group FA-A accounting for over 65% of the cases analysed10,11. A cDNA for the group C gene (FAC) was reported12 and localized to chromosome 9q22.3 (ref. 8). Genetic map positions were recently reported for two more FA genes, FAA (16q24.3)13 and FAD (3p22–26)14. Here we report the isolation of a cDNA representing the FAA gene, following an expression cloning method similar to the one used to clone the FAC gene12. The 5.5-kb cDNA has an open reading frame of 4,368 nucleotides. In contrast to the 63-kD cytosolic protein encoded by the FAC gene, the predicted FAA protein (Mr 162,752) contains two overlapping bipartite nuclear localization signals and a partial leucine zipper consensus, which are suggestive of a nuclear localization.

Fanconi anaemia (FA) is a chromosomal instability syndrome with autosomal recessive inheritance. We have identified the gene mutated in Fanconi anaemia group F patients by complementation cloning. FANCF has no introns and encodes a polypeptide with homology to the prokaryotic RNA binding protein ROM.