A role for monoubiquitinated FANCD2 at telomeres in ALT cells - PubMed (original) (raw)

A role for monoubiquitinated FANCD2 at telomeres in ALT cells

Qiang Fan et al. Nucleic Acids Res. 2009 Apr.

Abstract

Both Fanconi anemia (FA) and telomere dysfunction are associated with chromosome instability and an increased risk of cancer. Because of these similarities, we have investigated whether there is a relationship between the FA protein, FANCD2 and telomeres. We find that FANCD2 nuclear foci colocalize with telomeres and PML bodies in immortalized telomerase-negative cells. These cells maintain telomeres by alternative lengthening of telomeres (ALT). In contrast, FANCD2 does not colocalize with telomeres or PML bodies in cells which express telomerase. Using a siRNA approach we find that FANCA and FANCL, which are components of the FA nuclear core complex, regulate FANCD2 monoubiquitination and the telomeric localization of FANCD2 in ALT cells. Transient depletion of FANCD2, or FANCA, results in a dramatic loss of detectable telomeres in ALT cells but not in telomerase-expressing cells. Furthermore, telomere loss following depletion of these proteins in ALT cells is associated with decreased homologous recombination between telomeres (T-SCE). Thus, the FA pathway has a novel function in ALT telomere maintenance related to DNA repair. ALT telomere maintenance is therefore one mechanism by which monoubiquitinated FANCD2 may promote genetic stability.

PubMed Disclaimer

Figures

Figure 1.

FANCD2 colocalizes with the telomere-binding protein TRF1 in ALT cells. (A) Images of FANCD2 foci (red) and TRF1 foci (green) in the telomerase-expressing line, HeLa, and in the ALT cell line, GM847. The position of the nucleus is shown by counterstaining with DAPI. (B) Quantification of the colocalization of FANCD2 foci with TRF1 foci. The percentage of three telomerase-expressing cell lines (HeLa, MCF7 and 293-EBNA) and three ALT cell lines (GM847, WI38/VA13 and U2OS) with five or more FANCD2 foci, or with two or more FANCD2 foci which colocalized with the telomeric protein TRF1, is shown. Two foci were used as the standard for colocalization, since telomeres can aggregate in ALT cells into a small number of bright telomeric foci (22,26,38). Each bar represents the average of three counts of at least 150 cells each ±SD. The levels of colocalization of FANCD2 with TRF1 in ALT cells were statistically different from those seen in telomerase-expressing cells (P < 0.01).

Figure 2.

FANCD2 colocalizes with TRF1 foci in ALT cells in a cell-cycle-specific manner. (A) Cell-cycle distributions of asynchronous U2OS ALT cells, or at various timepoints following double-thymidine synchronization, were determined by flow cytometric measurements of DNA content. (B and C) The percentage of cells with five or more FANCD2 foci (B), or with two or more FANCD2 foci colocalized with TRF1 foci (C), in asynchronous populations or at timepoints following release from double-thymidine synchronization. Each bar represents the average of three counts of 150 or more cells ±SD. (D) A representative field of GM847 ALT cells, treated with 1 µg/ml nocodazole for 5 h to arrest mitotic progression, contains an interphase cell and a cell with mitotic chromosomes detected by counterstaining with DAPI (blue). FANCD2 foci (red) were detected with E35 antibody.

Figure 3.

FANCD2 colocalizes with telomeric DNA repeats in ALT cells. (A) FANCD2 was detected with antibodies (green) and telomeric DNA was detected with a Cy3-PNA probe (red) in the ALT cell lines GM847 and U2OS. (B) TRF1 was detected with anti-TRF1 antibodies (green) and telomeric DNA (red) was detected with a Cy3-PNA probe in GM847 cells.

Figure 4.

FANCD2 colocalizes with PML in ALT bodies. (A) FANCD2 foci (red) and PML bodies (green) were detected in GM847 (ALT cell line) and in HeLa cells (telomerase-expressing cell line). (B) Quantification of the assembly of PML bodies and their colocalization with FANCD2 foci in various telomerase-expressing cell lines and ALT cell lines. The percentage of cells with five or more PML foci, or in which two or more FANCD2 foci colocalized with PML, is shown for each cell line. The levels of colocalization of FANCD2 foci with PML foci in ALT cells were statistically different from those observed in telomerase-expressing cells (P < 0.01). (C) The percentage of GM847 ALT cells with five or more FANCD2 foci, five or more PML foci, or with two or more colocalized FANCD2 and PML foci at 0 or 12 h following release from double-thymidine synchronization is shown. The levels of FANCD2 colocalization with PML foci at 0 and 12 h of release were statistically different (P < 0.01). Each bar represents the average of three counts of at least 150 cells each ±SD (B and C).

Figure 5.

FANCA and FANCL are required for the colocalization of FANCD2 foci with TRF1 foci in ALT cells. (A) U2OS (ALT) cells were transfected with siRNAs directed against either FANCA or FANCD2, or with a control siRNA directed against GFP. In an independent experiment, U2OS (ALT) cells were transfected with siRNAs directed against FANCL or GFP. Depletion of FANCA or FANCD2 (left panel), or FANCL (right panel) was assayed on immunoblots. Cells were left untreated or were exposed to HU for 24 h. The ratio of monoubiquitinated (-L) to non-ubiquitinated (-S) FANCD2 is indicated (L/S ratio). Due to the low levels of FANCD2 protein present following transfection with the siRNA directed against FANCD2 (siFANCD2), it was not possible to accurately measure the L/S ratio for these cells and this value was not determined (N.D.). Antibodies directed against FANCA also recognized a non-specific band (N.S.). Immunoblots for actin are shown as a loading control. (B) Quantification of the percentage of U2OS cells, transfected with siRNAs directed against GFP, FANCA or FANCL, which had five or more FANCD2 foci in untreated populations or following exposure to 0.5 µM MMC for 24 h (left). Quantification of the percentage of U2OS cells, transfected with siRNAs directed against GFP, FANCA, or FANCA, which had two or more FANCD2 foci colocalized with TRF1 foci is also shown (right panel in figure part). Cells were left untreated or were exposed to 0.5 µM MMC for 24 h. (C) Quantification of the percentage of U2OS cells, transfected with siRNAs directed against GFP, FANCA or FANCL, which had two or more FANCD2 foci colocalized with TRF1 foci, at 0 or 9 h following release from double-thymidine synchronization. Each bar represents the average of three counts of 150 or more cells ±SD, except that three counts from two experiments were included for cells transfected with siGFP. The behavior of FANCD2 foci was statistically different in cells transfected with siFANCA or siFANCL, as compared to controls transfected with siGFP (P < 0.01) (B and C).

Figure 6.

ATR is required for the colocalization of FANCD2 foci with TRF1 foci in ALT cells following induction of DNA damage. (A) U2OS ALT cells were transfected with a siRNA directed against ATR or with a control siRNA directed against GFP. Immunoblots for ATR and FANCD2 demonstrate ATR depletion and effects on FANCD2 monoubiquitination, respectively. Cells were either left untreated or were exposed to HU for 24 h. The ratio of monoubiquitinated (-L) to non-ubiquitinated (-S) FANCD2 is indicated (L/S ratio). An immunoblot for actin is shown as a loading control. (B) Quantification of the percentage of U2OS cells, transfected with siRNAs directed against either GFP or ATR, which had five or more FANCD2 foci following treatment with 0.5 mM MMC for 24 h. (C) Quantification of the percentage of U2OS cells, transfected with siRNAs directed against either GFP or ATR, which had two or more FANCD2 foci colocalized with telomeres detected with antibodies to TRF1 following treatment with 0.5 mM MMC for 24 h. Each bar represents the average of three counts of 150 or more cells ±SD, and differences in the behavior of FANCD2 foci were statistically different in cells transfected with siGFP and siATR (P < 0.01) (B and C). (D) ATR (red) and TRF1 (green) display a similar pattern of nuclear foci in untreated GM847 ALT cells. Colocalization is demonstrated by merged images (not shown). There is an apparent non-specific signal detected by anti-ATR antibodies outside of the nucleus.

Figure 7.

Depletion of FANCA or FANCD2 results in an increased frequency of chromosome ends in ALT cells which are undetectable by in situ hybridization with a probe for telomeric DNA. (A) Representative images for chromosomes assayed with a Cy3-labeled telomeric probe (PNA) (red) or stained with DAPI (blue) in U2OS (ALT) or HeLa (telomerase-expressing) cells transfected with siRNAs against GFP (control). The telomeric signal was more uniform in HeLa cells than in U2OS cells. Abnormally long telomeres and some telomere ends which were undetectable with Cy3-PNA probe are indicated in the images for U2OS cells by ⁁ and * symbols, respectively. (B and C) The percentage of signal-free chromosome ends, quantified from images obtained from metaphase spreads of HeLa or U2OS cells (B) or GM847 cells (C). U2OS and HeLa cells were transfected with siRNAs against GFP (control), FANCA or FANCD2. GM847 cells were transduced with shRNAs against FANCA or a scrambled control (shScr). Cells were examined at 4 days following transfection or transduction. In each case, over 4000 telomeres were examined. For each value, the average of four counts of five or more metaphases each is shown with the SD. Levels of signal-free telomeres in U2OS or GM847 ALT cells that contained si/shRNAs against FANCA or FANCD2 were statistically different (P < 0.01) from cells transfected/transduced with control si/shRNAs. (D) Histogram showing the distribution of telomere lengths in HeLa and U2OS cells, as determined by flow FISH using Cy3-OO-(CCCTAA)3. Results obtained in HeLa (black line) and U2OS (grey line) cells are overlayed for purposes of comparison. (E) Histograms of telomere lengths (relative fluorescence units) in U2OS ALT cells transfected with a control siRNA directed against GFP (left, dark blue line) or a siRNA directed against FANCA (center, light blue line). An overlay of these results is shown at right. Different settings for flow cytometry were utilized in figure parts D and E.

Figure 8.

FANCA and FANCD2 are involved in telomere sister chromatid exchange (T-SCE) in ALT cells but not in telomerase-expressing cells. (A) Schematic of homologous recombination at telomeres. In normal cells, the 3′ telomeric overhang loops back and invades duplex DNA in the same telomere to form a T-loop that prevents the telomere from being recognized as a DNA double-strand break. In ALT cells, a 3′ overhang of telomeres that is not protected in a T-loop can initiate homologous recombination by invading another telomere. Because telomeres are composed of 5′-TTAGGG-3′ repeats, strand invasion can occur anywhere within the other telomere. Replication can lengthen both strands of the invading telomere. Recombination can be completed by resolution of the Holliday junction. (B) Schematic of the CO-FISH protocol utilized. Newly synthesized strands were labeled with BrdU (dashed lines), and the strands were nicked, following treatment with Hoecsht33258 and UV radiation, and nicked strands digested with Exonuclease III. This left only the parental DNA strands (solid lines). Telomeres were detected with a strand-specific Cy3-labeled probe (PNA) by FISH (indicated by red spot). This diagram also shows a signal split between sister chromatids, indicating T-SCE. (C) Representative images of CO-FISH for U2OS cells transfected with siRNAs against GFP (control) or FANCD2. Telomeres remaining after digestion of newly replicated strands were detected with a Cy3-labeled telomeric probe (PNA) (red) and the entire chromosome was stained with DAPI (blue), as shown in merged images. Representative T-SCE events are indicated by yellow dots. More T-SCE events were observed in cells treated with siGFP than in those treated with siFANCD2. Some chromosome ends lacked detectable telomeres, and not all T-SCE events are visible in the merged images shown. (D and E) The number of T-SCE events per 100 chromosomes is shown for U2OS (ALT) or HeLa (telomerase-expressing) cells (D), or GM847 cells (E). (F and G) The number of T-SCE events per 100 chromosome ends detectable with the telomere probe is shown for U2OS or HeLa cells (F), or GM847 cells (G). U2OS and HeLa cells were transiently transfected with siRNAs that targeted GFP, FANCA or FANCD2, and GM847 cells were transduced with shRNAs directed against FANCA or a scrambled control (shScr) (D–G). Cells were analyzed 4 days after transfection or transduction. Values represent the average of four groups of nine metaphases each ±SD (D–G). A minimum of 2000 chromosomes were examined for each sample. Levels of T-SCE in U2OS or GM847 ALT cells depleted of FANCA or FANCD2 were statistically different (P < 0.01) from levels in cells transfected or transduced with control shRNAs.

References

1. Alter BP. Cancer in Fanconi anemia, 1927–2001. Cancer. 2003;97:425–440. - PubMed
1. Taniguchi T, D'Andrea AD. Molecular pathogenesis of Fanconi anemia: recent progress. Blood. 2006;107:4223–4233. - PubMed
1. Garcia-Higuera I, Taniguchi T, Ganesan S, Meyn MS, Timmers C, Hejna J, Grompe M, D'Andrea AD. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol. Cell. 2001;7:249–262. - PubMed
1. Thompson LH. Unraveling the Fanconi anemia-DNA repair connection. Nat. Genet. 2005;37:921–922. - PubMed
1. Grompe M, van de Vrugt H. The Fanconi family adds a fraternal twin. Dev. Cell. 2007;12:661–662. - PubMed

A role for monoubiquitinated FANCD2 at telomeres in ALT cells - PubMed (original) (raw)