Coordinate nuclear targeting of the FANCD2 and FANCI proteins via a FANCD2 nuclear localization signal

Abstract

Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.

Figure 1. The amino terminal 58 amino acids of FANCD2 contain a highly conserved nuclear localization signal, which facilitates nuclear expression of GFP.

(A) A Clustal Omega multiple sequence alignment of the amino terminus of FANCD2 demonstrates strong evolutionary conservation of several blocks of basic amino acids of this region, indicated by blue bars. The red box indicates the highly conserved K4, R5, and R6 residues selected for mutagenesis. (B) The amino terminal 58 amino acids of FANCD2 are required to promote exclusive nuclear expression of GFP. HeLa cells were transiently transfected with wild type GFP (GFP-WT), amino acids 1-27 (D2-1-27-GFP), 24-55 (D2-24-55-GFP), or 1-58 (D2-1-58-GFP) of FANCD2 fused to GFP followed by analysis by inverted fluorescence microscopy.

Figure 2. The FANCD2 NLS is required for the nuclear localization of FANCD2.

(A) Schematic diagram of the FANCD2 constructs generated in this study. In addition to the NLS, FANCD2 harbors a CUE (for coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain [47], a PCNA-interaction motif or PIP-box [19], and a carboxy-terminus EDGE motif [10]. (B) FA-D2 cells stably expressing FANCD2-WT and FANCD2-∆N57 were incubated in the absence (NT) or presence of 40 nM MMC for 24 h. Cells were then incubated in the absence (-Pre-Perm) or presence (+Pre-Perm) of pre-permeablization buffer, fixed, stained with mouse monoclonal anti-V5 antibody (green) and counterstained with DAPI (blue). (C) FA-D2 cells stably expressing LacZ, FANCD2-WT, FANCD2-∆N57, FANCD2-∆N100, and FANCD2-3N were incubated in the absence (NT) or presence of MMC for 24 h, fixed, and stained with rabbit polyclonal anti-FANCD2 antibody, and counterstained with phalloidin and DAPI. At least 300 cells were scored for cytoplasmic (Cyto.), nuclear (Nucl.), and both cytoplasmic and nuclear (Both) localization of FANCD2.

Figure 3. The FANCD2 NLS is required for the nuclear localization of a subset of FANCI.

(A) FA-D2 patient cells or FA-D2 cells stably expressing FANCD2-WT were incubated in the absence (NT) or presence of MMC for 24 h, fixed, stained with rabbit polyclonal anti-FANCD2 or anti-FANCI antibody and counterstained with phalloidin and DAPI. AF-488, Alexa Fluor 488. (B) FA-D2 cells stably expressing LacZ, FANCD2-WT, FANCD2-∆N57, FANCD2-∆N100, and FANCD2-3N were incubated in the absence (NT) or presence of MMC for 24 h, fixed, and stained with rabbit polyclonal anti-FANCI antibody, and counterstained with phalloidin and DAPI. At least 300 cells were scored for cytoplasmic (Cyto.), nuclear (Nucl.), and both cytoplasmic and nuclear (Both) localization of FANCI. (C) COS-7 cells were transiently transfected with no DNA, FANCI-GFP, FANCI-GFP plus FANCD2-V5-WT, or FANCI-GFP plus FANCD2-V5-∆N57. Whole-cell lysates were immunoprecipitated with anti-V5 or anti-GFP antibodies and immune complexes immunoblotted with anti-GFP and anti-V5 antibodies.

Figure 4. The FANCD2 NLS is required for efficient FANCD2 and FANCI monoubiquitination and chromatin association.

(A) FA-D2 cells stably expressing LacZ, FANCD2-WT, FANCD2-K561R, FANCD2-∆N57, FANCD2-∆N100 and FANCD2-3N were incubated in the absence and presence of 250 nM MMC for 18 h, and whole-cell lysates were immunoblotted with antibodies to FANCD2, V5, FANCI and α-tubulin. The FANCD2 and FANCI L/S ratios are the ratios of monoubiquitinated to nonubiquitinated protein, and were calculated by measuring protein band intensities using ImageJ image processing and analysis software (http://rsb.info.nih.gov/ij/). (B and C) FA-D2 cells stably expressing FANCD2-WT, FANCD2-∆N57, FANCD2-∆N100 and FANCD2-3N were treated as above and cell pellets were fractionated into soluble (S) and chromatin-associated (C) fractions. Fractions were immunoblotted with antibodies against V5, FANCI, α-tubulin and H2A. W, unfractionated whole cell extract.

Figure 5. The FANCD2 NLS mutants fail to correct the ICL sensitivity of FA-D2 patient cells.

(A) FA-D2 cells stably expressing LacZ, FANCD2-WT, FANCD2-∆N57, FANCD2-∆N100, FANCD2-3N, or FANCD2-K561R were incubated in the absence or presence of 8 or 16 nM MMC for 24 h and the numbers of chromosome aberrations including gaps and breaks, dicentrics, and complex chromosome aberrations, including radial formations, were scored. Metaphase spreads were analyzed using a Zeiss AxioImager.A1 upright epifluorescent microscope with AxioVision LE 4.6 image acquisition software. At least 80 metaphases were scored per treatment. Error bars represent the standard error of the means. (B) FA-D2 cells stably expressing LacZ, FANCD2-WT and FANCD2-∆N57 were incubated in the absence (NT) or presence of 40 nM MMC for 18 h, and allowed to recover for 0, 4.5 or 7 h. Cells were then fixed, stained with rabbit polyclonal anti-DNA-PK_CS pS2056, and counterstained with DAPI. At least 300 cells were scored for nuclei with > 5 DNA-PK_CS foci. Error bars represent the standard error of the means from two independent experiments. ***, p < 0.001.

Figure 6. FANCD2-dependent and -independent mechanisms of FANCI nuclear localization.

We propose that a subset of FANCI (blue) associates with FANCD2 (red) in the cytoplasm, and that the ID2 heterodimer is transported to the nucleus via an importin α/β (brown)-mediated transport mechanism, using the amino terminal FANCD2 NLS (light green). Nuclear ID2 binds to DNA (orange) and is also phosphorylated by the ATM/ATR kinases (dark green). One or both of these events may trigger ID2 complex restructuring, facilitating FANCD2 and FANCI monoubiquitination by FANCL (black), UBE2T (yellow) and the FA core complex (not shown).