FANCC suppresses short telomere-initiated telomere sister chromatid exchange

Abstract

Telomere shortening has been linked to rare human disorders that present with bone marrow failure including Fanconi anemia (FA). FANCC is one of the most commonly mutated FA genes in FA patients and the FANCC subtype tends to have a relatively early onset of bone marrow failure and hematologic malignancies. Here, we studied the role of Fancc in telomere length regulation in mice. Deletion of Fancc (Fancc(-/-)) did not affect telomerase activity, telomere length or telomeric end-capping in a mouse strain possessing intrinsically long telomeres. However, ablation of Fancc did exacerbate telomere attrition when murine bone marrow cells experienced high cell turnover after serial transplantation. When Fancc(-/-) mice were crossed into a telomerase reverse transcriptase heterozygous or null background (Tert(+/-) or Tert(-/-)) with short telomeres, Fancc deficiency led to an increase in the incidence of telomere sister chromatid exchange. In contrast, these phenotypes were not observed in Tert mutant mice with long telomeres. Our data indicate that Fancc deficiency accelerates telomere shortening during high turnover of hematopoietic cells and promotes telomere recombination initiated by short telomeres.

Figure 1.

Telomere lengths are comparable between wild-type and Fancc^−/− bone marrow cells derived from C57BL/6 mice. Q-FISH analysis of bone marrow cells derived from wild-type and Fancc^−/− mice (n = 6). (A) Representative metaphase spreads of wild-type (Bi) and Fancc^−/− (Bii) bone marrow cells showing DAPI staining (blue) and telomere fluorescence signals (red). There was no significant difference in the mean telomere signal intensities and distribution of telomere signal intensities between two genotypes in overlapping histogram (Biii) and box-plot (C).

Figure 2.

Average telomere lengths in wild-type and Fancc^−/− hematopoietic cells derived from C57BL/6 mice. Flow-FISH analysis of average telomere signal intensity of bone marrow cells, splenocytes and thymocytes derived from wild-type and Fancc^−/− mice. Error bars represent the standard error from different mice of each genotype (n = 5).

Figure 3.

Telomerase activity and Tert and Terc RNA levels in wild-type and Fancc^−/− mouse bone marrow cells derived from C57BL/6 mice. qT-PCR analysis indicates a comparable telomerase activity (A), and Tert and Terc RNA levels (B) in wild-type and Fancc^−/− mouse bone marrow cells (n = 6). Error bars represent the standard error obtained from different mice of each genotype. P-values are not significant between wild-type and Fancc^−/− mouse bone marrow cells.

Figure 4.

Accelerated telomere attrition is observed in Fancc^−/− bone marrow cells after two serial bone marrow transplantations. Q-FISH analysis of wild-type and Fancc^−/− bone marrow cells after two serial bone marrow transplantations (n = 4). (A) Representative metaphase spreads of wild-type and Fancc^−/− bone marrow cells showing DAPI staining (upper panel, blue) and telomere fluorescence signals (upper panel, red; lower panel, black). There was a decrease in telomere signal intensities in Fancc^−/− cells (Bii) in comparison to wild-type cells (Bi), shown here as shift of the dynamic range in overlapping histogram (Biii) and box-plot (C).

Figure 5.

Telomerase activity in wild-type and Fancc^−/− bone marrow cells after two serial bone marrow transplantations. qT-PCR analysis indicates a comparable telomerase activity in serially transplanted wild-type and Fancc^−/− mouse bone marrow cells (n = 4). Error bars represent the standard error from different mice of each genotype.

Figure 6.

Critically short telomeres are detectable in G2 Tert^−/− Fancc^+/+ and Tert^−/− Fancc^−/− mutant mice. Q-FISH analysis of bone marrow cells derived from G2 Tert^−/− Fancc^+/+ and Tert^−/− Fancc^−/− mice (n = 6). Representative metaphase spreads of G2 Tert^−/− Fancc^+/+ and Tert^−/− Fancc^−/− mouse bone marrow cells showing DAPI staining (upper panel, blue) and telomere fluorescence signals (upper panel, red; lower panel, black). SFEs are detectable in G2 Tert^−/− Fancc^+/+ and Tert^−/− Fancc^−/− mice (see arrows).

Figure 7.

Fancc deletion increases the frequencies of T-SCE in late generation Tert mutant mice. CO-FISH analysis of mouse bone marrow cells with indicated genotypes. (A) A schematic representation of CO-FISH procedure. In brief, newly synthesized strands are removed, leaving parental strands to be detected by fluorescent-labeled telomeric C-rich or G-rich probes. A chromosome with more than two telomere signals is considered to be positive for T-SCE. (B) Representative metaphase spreads of G2 Tert^−/− Fancc^+/+ and Tert^−/− Fancc^−/− mouse bone marrow cells showing DAPI staining (blue), leading strand telomere fluorescence signals (red) and lagging strand telomere fluorescence signals (green). Arrows indicate T-SCEs. (C) The frequencies of T-SCEs in HG1, HG5 and G2 Fancc^+/+ and Fancc^−/− mice. Error bars represent standard errors from different mice of each genotype (n = 3).

Figure 8.

The frequencies of genome SCEs are comparable between HG5 Tert^+/− Fancc^+/+ and Tert^+/− Fancc^−/− mouse bone marrow cells. (A) Representative metaphase spreads of bone marrow cells with indicated genotypes, showing genome SCE events. (B) (i) The incidences of genome SCEs are comparable between Tert^+/− Fancc^+/+ and Tert^+/− Fancc^−/− bone marrow cells (n = 4). Arrows indicate genome SCE events. (ii) The frequencies are derived from the number of SCE events divided by total number of chromosomes (%).