Abstract
AbstractFanconi anemia (FA) is an autosomal recessive disease marked by bone marrow failure, birth defects, and cancer. The FA proteins FANCA, FANCC, FANCE, FANCF, FANCG, and FANCL participate in a core complex. We previously have shown that several members of this complex bind to chromatin until mitosis and that this binding increases after DNA damage. The purpose of the present study was to determine the dynamics of complex movement between cytoplasm and nuclear compartments. Fluorescent-tagged versions of FANCA, FANCC, and FANCG colocalize in cytoplasm and nucleus, chiefly in chromatin. At the G1-S border, the FA core complex exists as foci on chromatin, progressively diffusing and migrating to the nuclear periphery and becoming completely excluded from condensed chromosomes by mitosis. Chromatin fiber analysis shows FA proteins diffusely staining along chromatin fibers during G1-S and S phase. Treatment with the DNA cross-linker mitomycin C results in a diffusion of foci and increased binding of complex proteins to chromatin, as well as diffuse and increased complex binding to chromatin fibers. These data are consistent with the idea that the FA proteins function at the level of chromatin during S phase to regulate and maintain genomic stability.
Highlights
One group has shown that BRCA2/FANCD1 binds to FANCG.[37]
In order to determine if the fluorescent versions of the FANCA, FANCC, and FANCG were functional, we transfected the fluorescent and nonfluorescent tagged constructs into Fanconi anemia (FA)-A (HSC72), FA-C (HSC536N), and FA-G (EUFA143) mutant cells, respectively
We confirm and extend these findings by showing that this is an S-phase–specific process by (1) demonstrating that the FA core complex forms chromatin foci at the G1-S border and at the beginning of S phase, (2) showing that these foci diffuse in response to DNA damage and during S phase, and (3) showing that the proteins exit the nucleus by the onset of mitosis as a multiprotein complex
Summary
Treatment with the DNA cross-linker mitomycin C results in a diffusion of foci and increased binding of complex proteins to chromatin, as well as diffuse and increased complex binding to chromatin fibers. These data are consistent with the idea that the FA proteins function at the level of chromatin during S phase to regulate and maintain genomic stability. Our work has established that FA proteins bind to chromatin.[39] Immunoblotting experiments revealed that the increased FA proteins were bound to chromatin after DNA damage and that the complex underwent egress from the nucleus at mitosis. Recent work has implicated FANCD2 in chromatin, reinforcing the importance of the FA core complex, since it is required for FANCD2 monoubiquitination.[40,41]
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