UBE2T, the Fanconi anemia core complex, and FANCD2 are recruited independently to chromatin: a basis for the regulation of FANCD2 monoubiquitination

Abstract

The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.

FIG. 1.

Functional separation of the FA core complex assembly and its E3 ubiquitin ligase activity. (A) Schematic representation of the genomic locus of FANCL with the exon configuration in correlation with the domain structure and the gene disruption construct with exons 2 to 6 removed. S, StuI. Southern blot analysis was performed on StuI/KpnI-digested genomic DNA from heterozygous and homozygous genotypes. WT, wild-type locus; Δ, gene knockout locus. The position of the probe used is indicated by the bar. (B) Sensitivity curves of the indicated cell lines. Cells were treated with various concentrations of cisplatin for 72 h and analyzed for survival in an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. Standard errors of the means are given from three independent experiments. (C) Immunoblot analysis of FANCD2 in subfractionated ΔFANCL cells either untreated (−) or treated with 150 ng/ml MMC (+). D2, FANCD2; D2-Ub, monoubiquitinated FANCD2; NEX, high-salt nuclear extract; CHEX, solubilized chromatin extract. (D) Nuclear extracts of the indicated cell lines (WT/C-TAP, ΔFANCL/C-TAP, and ΔFANCL/C-TAP + L357) were separated by size exclusion chromatography, and fractions were analyzed for FANCC-TAP and TAP-FANCL357 by immunoblot analysis. (E) Size exclusion chromatography of nuclear extracts of the indicated cell lines and immunoblot analyses for TAP-FANCC. (F) Subfractionation of either MMC-treated (+) or untreated (-) cells into high-salt NEX and CHEX and immunoblot analysis for FANCD2 and TAP-FANCL. D2, FANCD2; D2-Ub, monoubiquitinated FANCD2. Histone H3 was used as a CHEX loading control. (G) Coimmunoprecipitation of wild-type and mutant TAP-FANCL and immunoblot analysis for FANCG. Nontagged wild-type cells were used as a negative immunoprecipitation control. FANCG antibody cross-reacts with the protein A domain of TAP-tag. WB, Western blot; S/N, supernatant of nonprecipitated proteins; P, fraction of precipitated proteins; Ig-H, immunoglobulin heavy chain.

FIG. 2.

Generation of a cell line expressing a multitagged FA core complex. (A) Schematic of the genomic locus of FANCB with the configuration of the last four exons. In situ tagging of the 3′ with 3× HA tag cDNA. wt, wild type; B, BamHI. Homologous targeting was detected by Southern blot analysis of BamHI-digested genomic DNA. The position of the probe used is indicated by the bar. (B) Sensitivity curves of the indicated cell lines. Cells were analyzed after 72 h of exposure to various concentrations of cisplatin. Standard errors of the means are given from three independent experiments. (C) MMC-induced FANCD2 monoubiquitination of the multitagged FA core complex strains (C/B/F) (clone 1 and clone 15). D2, FANCD2; D2-Ub, monoubiquitinated FANCD2. (D) Nuclear extracts of the multitagged FA core complex cell lines were separated by size exclusion chromatography, and the elution profiles for tagged complex proteins were analyzed by immunoblot detection. Peak fractions were subjected to TAP immunoprecipitation. Portions (10%) of the input material (I), nonprecipitated fraction (SN), and precipitated fraction were analyzed by immunoblotting for FANCM, FANCB-HA, FANCC-TAP, FANCG, and FANCF-FLAG.

FIG. 3.

The FA core complex is constitutively assembled throughout the cell cycle. (A) Cells expressing the multitagged FA core complex were arrested at mitosis by nocodazole treatment and released into fresh medium. Synchronization was verified by FACS analysis. (B) At the indicated time points, whole-cell lysates of synchronized cell populations were subjected to TAP immunoprecipitation (IP), and the presence of FA core complex components was analyzed by immunoblotting. ΔFANCL cell lysate was used as a negative IP control. Equal input was verified by immunoblotting for tubulin and FANCC-TAP. (C) FANCD2 immunoblot of total cell lysates from the indicated time points after nocodazole release. (D) Nocodazole-arrested cells were released into fresh medium supplemented with hydroxyurea. High-salt nuclear extracts (NEX) and soluble chromatin extracts (CHEX) of cells in G₁ phase (2 h after release) and S phase (7 h after release) were used to perform TAP immunoprecipitations. (E) FANCD2 monoubiquitination was monitored in NEX and CHEX from the indicated cell cycle phases. (F) Coprecipitated proteins were identified by immunoblot analyses. Equal input was verified by immunoblotting for topoisomerase 2 (NEX) and histone H3 (CHEX). AS, asynchronous cells; G₁, G₁ phase synchronized; S, S phase synchronized; ΔL, ΔFANCL cell extracts used as a negative IP control.

FIG. 4.

The overall size of the FA core complex is not markedly affected during the cell cycle or after DNA damage. (A) Size exclusion chromatography of high-salt nuclear extracts (NEX) and soluble chromatin extracts (CHEX) of G₁- and S-phase-synchronized cells expressing the multitagged FA core complex (Fig. 3) and immunoblot detection for FANCC-TAP. (B) Size exclusion chromatography of NEX and CHEX, either MMC treated (+MMC) or untreated (-MMC), and immunoblot detection for FANCC-TAP.

FIG. 5.

UBE2T is constitutively localized to chromatin. (A) Schematic presentation of the genomic locus of UBE2T with the exon configuration in correlation with the domain structure and the gene disruption construct, with exons 4 to 6 removed. B, BamHI. Southern blot analysis of BamHI-digested genomic DNA from heterozygous (+/−) and homozygous (−/−) genotypes. wt, wild type locus; Δ, gene knockout locus. The position of the probe used is indicated by the bar. (B) Sensitivity curves of two independent ΔUBE2T cell line clones (cl6 and cl9). Cells were treated with increasing concentrations of cisplatin for 72 h and analyzed for survival. Standard errors of the means are given for three independent experiments. (C) Nuclear extracts of wild-type (WT) and ΔUBE2T cells were separated by size exclusion chromatography, and fractions were immunoblotted for FANCG. (D) Cellular subfractionation of wild-type and ΔUBE2T cells either untreated (−) or MMC treated (+). NEX, nuclear extract; CHEX, chromatin extract. Immunoblot detection of FANCG, histone H3 (H3), and FANCD2 was performed. (E) UBE2T distribution into nuclear extracts (N) and chromatin extracts (C) in asynchronous cells (Asy), mitosis-arrested cells (M), and synchronized cells from the G₁-to-S-phase transition. Results show immunoblot detection of UBE2T and FANCD2. D2, FANCD2; D2-Ub, monoubiquitinated FANCD2. (F) Cellular subfractionation of the multitagged FA core complex cell line either untreated (−) or treated with MMC (+). Immunoblot detection of FANCC-TAP, FANCG, FANCF-FLAG, UBE2T, and histone H3 (H3) is shown. (G) Model for the S phase and DNA damage-induced activation of the FA core complex (see Discussion for details).