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A role for monoubiquitinated FANCD2 at telomeres in ALT cells.

Fan Q, Zhang F, Barrett B, Ren K, Andreassen PR - Nucleic Acids Res. (2009)

Bottom Line: In contrast, FANCD2 does not colocalize with telomeres or PML bodies in cells which express telomerase.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).

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.

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.

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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.
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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.

Mentions: ALT cells maintain telomeres through homologous recombination (15,16). Homologous recombination at ALT telomeres is shown schematically in Figure 8A, and a more detailed consideration of the molecular steps that may be involved can be found elsewhere (42,43). Because depletion of FANCA or FANCD2 results in the loss of detectable telomeres in ALT cells (Figure 7), we sought to determine whether FANCA and FANCD2 are required for telomeric recombination in ALT cells. Sister chromatid exchange at telomeres (T-SCE) can be measured by CO-FISH (34). According to the protocol that we utilized (shown schematically in Figure 8B), newly synthesized strands were degraded and only the pre-existing strands were detected by hybridization with a strand-specific telomere probe [Cy3-(CCCTAA)3]. In the absence of a T-SCE event, only a single signal at each end of the chromosome was observed. In contrast, a T-SCE event resulted in a signal that was split between the chromatids on a particular end of the chromosome (Figure 8B). Less frequently, both ends of a chromosome underwent T-SCE and each arm displayed a signal.Figure 8.


A role for monoubiquitinated FANCD2 at telomeres in ALT cells.

Fan Q, Zhang F, Barrett B, Ren K, Andreassen PR - Nucleic Acids Res. (2009)

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.
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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.
Mentions: ALT cells maintain telomeres through homologous recombination (15,16). Homologous recombination at ALT telomeres is shown schematically in Figure 8A, and a more detailed consideration of the molecular steps that may be involved can be found elsewhere (42,43). Because depletion of FANCA or FANCD2 results in the loss of detectable telomeres in ALT cells (Figure 7), we sought to determine whether FANCA and FANCD2 are required for telomeric recombination in ALT cells. Sister chromatid exchange at telomeres (T-SCE) can be measured by CO-FISH (34). According to the protocol that we utilized (shown schematically in Figure 8B), newly synthesized strands were degraded and only the pre-existing strands were detected by hybridization with a strand-specific telomere probe [Cy3-(CCCTAA)3]. In the absence of a T-SCE event, only a single signal at each end of the chromosome was observed. In contrast, a T-SCE event resulted in a signal that was split between the chromatids on a particular end of the chromosome (Figure 8B). Less frequently, both ends of a chromosome underwent T-SCE and each arm displayed a signal.Figure 8.

Bottom Line: In contrast, FANCD2 does not colocalize with telomeres or PML bodies in cells which express telomerase.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).

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.

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.

Show MeSH
Related in: MedlinePlus