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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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Related in: MedlinePlus

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.
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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.

Mentions: As an alternative measure of the colocalization of FANCD2 foci with telomeres in ALT cells, we labeled cells with FANCD2 antibodies and detected telomeric repeat DNA by simultaneous FISH (37,39). We found that FANCD2 foci colocalized with telomeric DNA in two different ALT cell lines, GM847 and U2OS (Figure 3A). In contrast, FANCD2 foci did not colocalize with telomeric DNA in telomerase-expressing HeLa cells (data not shown). Furthermore, TRF1 uniformly colocalized with telomeric DNA in interphase GM847 cells (Figure 3B), and in HeLa cells (data not shown), validating the use of TRF1 antibody in subsequent figures for the identification of telomeres by immunofluorescence microscopy. Consistent with a recent report (40), we also found that TRF1 strongly colocalized with telomeric DNA following the induction of DNA damage (data not shown).Figure 3.


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

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

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.
Mentions: As an alternative measure of the colocalization of FANCD2 foci with telomeres in ALT cells, we labeled cells with FANCD2 antibodies and detected telomeric repeat DNA by simultaneous FISH (37,39). We found that FANCD2 foci colocalized with telomeric DNA in two different ALT cell lines, GM847 and U2OS (Figure 3A). In contrast, FANCD2 foci did not colocalize with telomeric DNA in telomerase-expressing HeLa cells (data not shown). Furthermore, TRF1 uniformly colocalized with telomeric DNA in interphase GM847 cells (Figure 3B), and in HeLa cells (data not shown), validating the use of TRF1 antibody in subsequent figures for the identification of telomeres by immunofluorescence microscopy. Consistent with a recent report (40), we also found that TRF1 strongly colocalized with telomeric DNA following the induction of DNA damage (data not shown).Figure 3.

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