Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2665210&req=5

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.

Mentions: The assembly of FANCD2 foci in response to DNA damage or replication stress is regulated by the ATR checkpoint kinase in telomerase-expressing cells (10). Depletion of ATR in U2OS ALT cells using a siRNA we have described previously (10), inhibited FANCD2 monoubiquitination in HU-treated cells (Figure 6A). But depletion of ATR did not inhibit FANCD2 monoubiquitination in untreated cells. Quantification shows that depletion of ATR resulted in a decrease in the percentage of cells treated with MMC that had FANCD2 foci (Figure 6B) and in the percentage of cells in which FANCD2 foci colocalized with TRF1 foci (Figure 6C). In cells treated with MMC, 45.5 ± 1.4 and 6.3 ± 1.7% of U2OS cells transfected with siGFP and siATR, respectively, displayed colocalization of FANCD2 and TRF1 foci. But depletion of ATR did not alter the number of TRF1 foci (data not shown). Thus, the DNA damage-induced assembly of FANCD2 foci at telomeres in ALT cells requires ATR.Figure 6.


A role for monoubiquitinated FANCD2 at telomeres in ALT cells.

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

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665210&req=5

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.
Mentions: The assembly of FANCD2 foci in response to DNA damage or replication stress is regulated by the ATR checkpoint kinase in telomerase-expressing cells (10). Depletion of ATR in U2OS ALT cells using a siRNA we have described previously (10), inhibited FANCD2 monoubiquitination in HU-treated cells (Figure 6A). But depletion of ATR did not inhibit FANCD2 monoubiquitination in untreated cells. Quantification shows that depletion of ATR resulted in a decrease in the percentage of cells treated with MMC that had FANCD2 foci (Figure 6B) and in the percentage of cells in which FANCD2 foci colocalized with TRF1 foci (Figure 6C). In cells treated with MMC, 45.5 ± 1.4 and 6.3 ± 1.7% of U2OS cells transfected with siGFP and siATR, respectively, displayed colocalization of FANCD2 and TRF1 foci. But depletion of ATR did not alter the number of TRF1 foci (data not shown). Thus, the DNA damage-induced assembly of FANCD2 foci at telomeres in ALT cells requires ATR.Figure 6.

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