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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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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).
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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).

Mentions: The FA protein FANCD2 forms nuclear foci during S phase in the absence of exogenous DNA damage (9). Whether S phase FANCD2 foci associate with specific substructures within the nucleus is unknown. Since another DNA damage response protein, NBS1, colocalizes with telomeres in non-ALT cells (36), we tested whether spontaneous FANCD2 foci colocalize with telomeres in cells which maintain telomere length by either of two different mechanisms: telomerase expression or ALT. In initial experiments, we assayed colocalization of FANCD2 foci with the telomere-binding protein TRF1 (37) (Figure 1). FANCD2 did not colocalize with TRF1 in the telomerase-expressing cell line HeLa (Figure 1A) or in proliferating primary human fibroblasts (GM00038) (data not shown). We did find, however, that FANCD2 colocalized with TRF1 in the ALT cell line, GM847 (Figure 1A). Telomeres aggregate (cluster) in ALT cells (22,26,38). As a result, ALT cells frequently have a smaller number of TRF1 foci, which are larger than those detected in telomerase-expressing cells. We found that FANCD2 also colocalized with the telomeric protein TRF2 in 35.9 ± 2.1% of GM847 ALT cells, but not in HeLa cells (1.3 ± 0.6%).Figure 1.


A role for monoubiquitinated FANCD2 at telomeres in ALT cells.

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

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).
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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).
Mentions: The FA protein FANCD2 forms nuclear foci during S phase in the absence of exogenous DNA damage (9). Whether S phase FANCD2 foci associate with specific substructures within the nucleus is unknown. Since another DNA damage response protein, NBS1, colocalizes with telomeres in non-ALT cells (36), we tested whether spontaneous FANCD2 foci colocalize with telomeres in cells which maintain telomere length by either of two different mechanisms: telomerase expression or ALT. In initial experiments, we assayed colocalization of FANCD2 foci with the telomere-binding protein TRF1 (37) (Figure 1). FANCD2 did not colocalize with TRF1 in the telomerase-expressing cell line HeLa (Figure 1A) or in proliferating primary human fibroblasts (GM00038) (data not shown). We did find, however, that FANCD2 colocalized with TRF1 in the ALT cell line, GM847 (Figure 1A). Telomeres aggregate (cluster) in ALT cells (22,26,38). As a result, ALT cells frequently have a smaller number of TRF1 foci, which are larger than those detected in telomerase-expressing cells. We found that FANCD2 also colocalized with the telomeric protein TRF2 in 35.9 ± 2.1% of GM847 ALT cells, but not in HeLa cells (1.3 ± 0.6%).Figure 1.

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