Monoubiquitinated FANCD2 Is Both Necessary and Sufficient for Mitomycin C Resistance in the Absence of a Functional Fanconi Anemia Core Complex.

Monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, is necessary for resistance to MMC/DEB-induced chromosomal breakage, which is the diagnostic feature of Fanconi anemia. The upstream Fanconi proteins FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, and FANCL are necessary for FANCD2 monoubiquitination and Fanconi pathway function. We posed the question whether FANCD2 monoubiquitination is sufficient to restore the Fanconi pathway function in the absence of the upstream FA proteins. We constructed a chimeric monoubiquitinated FANCD2 protein by fusing the FANCD2 cDNA sequence contiguously (in-frame) with a ubiquitin coding sequence. This FANCD2-ubiquition fusion protein (D2-ubi) was stably expressed in FANCD2−/−, FANCA−/−, FANCC−/−, and FANCG−/− human patient-derived cell lines. D2-ubi expression corrected the MMC hypersensitivity of these FA cell lines in the absence of a functional upstream FA core complex. By western blot analysis, the D2-ubi protein migrated at a slower rate than the endogenous monoubiquitinated FANCD2 protein, consistent with its higher molecular mass. An even slower migrating D2-ubi form (D2-ubi-L) was detectable in cells harboring an intact Fanconi core protein complex (i.e., FA-D2 cells) but was absent in cells lacking functional Fanconi proteins FANCA, FANCC, or FANCG. These results indicate the D2-ubi can be further monoubiquitinated on lysine 561, but only in the presence of an intact FA complex. Furthermore, D2-ubi-L was upregulated in response to DNA damage in transfected FA-D2 and wild type cells. Like endogenous FANCD2, D2-ubi formed nuclear foci in the presence of the upstream Fanconi core complex. In the absence of the FA core complex, D2-ubi was localized diffusely throughout the nucleus, but unlike endogenous FANCD2, D2-ubi was retained within the nucleus following detergent permeabilization of FA-A, FA-C, and FA-G cells. These results further support the epistatic relationship of the FA genes in a linear pathway. In summary, these data indicate that a constitutively monoubiquitinated FANCD2 polypeptide is able to bypass defects in the upstream FA core complex to restore Fanconi pathway function. These studies identify FANCD2 ubiquitination as a potential therapeutic target for patients lacking the upstream FA core complex.