Mammalian Ku86 mediates chromosomal fusions and apoptosis caused by critically short telomeres - PubMed (original) (raw)

Mammalian Ku86 mediates chromosomal fusions and apoptosis caused by critically short telomeres

Silvia Espejel et al. EMBO J. 2002.

Abstract

Here we analyze the functional interaction between Ku86 and telomerase at the mammalian telomere by studying mice deficient for both proteins. We show that absence of Ku86 prevents the end-to-end chromosomal fusions that result from critical telomere shortening in telomerase-deficient mice. In addition, Ku86 deficiency rescues the male early germ cell apoptosis triggered by short telomeres in these mice. Together, these findings define a role for Ku86 in mediating chromosomal instability and apoptosis triggered by short telomeres. In addition, we show here that Ku86 deficiency results in telomerase-dependent telomere elongation and in the fusion of random pairs of chromosomes in telomerase-proficient cells, suggesting a model in which Ku86 keeps normal-length telomeres less accessible to telomerase-mediated telomere lengthening and to DNA repair activities.

PubMed Disclaimer

Figures

None

Fig. 1. Generation of the different mutant mice. The genotypes printed in gray are those used for the analysis.

None

Fig. 2. (A) Telomere length distribution in primary MEFs. One TFU corresponds to 1 kb of TTAGGG repeats (Zijlmans et al., 1997). Arrows indicate telomeres with <1 kb of TTAGGG repeats. Average telomere length and the standard error, as well as the total number of telomeres analyzed, are indicated. Note that standard error and not standard deviation is shown. The distribution of the telomere length frequencies for each MEF is an indication of the standard deviation. Littermate MEFs are shown. (B) Quantification using Q-FISH of the percentage of telomeres with undetectable TTAGGG repeats out of the total number of telomeres shown in (A). Littermate MEFs are indicated. (C) Telomeric fluorescence (in arbitrary units, a.u.) using flow cytometry combined with telomeric Q-FISH (Flow-FISH). Littermate mice are indicated. (D) TRF analysis of primary BM cells or liver nuclei, as indicated, from different genotypes. Notice the upwards shift of TRF fragments in Terc+/+/Ku86–/– compared with the wild type. Littermate mice are indicated. (E) G-strand overhangs using liver nuclei visualized in the native gel after hybridization with a (CCCTAA)4 probe. Notice that upon treatment with mung bean nuclease (mbn), the G-strand-specific signal decreases. As control, the same gel was denatured and reprobed with the (CCCTAA)4 probe to visualize telomeres. Table I shows quantification of the G-strand overhang signals corrected by the denaturing gel hybridization signal for the 0 units of mbn lanes. Littermate mice are indicated.

None

Fig. 2. (A) Telomere length distribution in primary MEFs. One TFU corresponds to 1 kb of TTAGGG repeats (Zijlmans et al., 1997). Arrows indicate telomeres with <1 kb of TTAGGG repeats. Average telomere length and the standard error, as well as the total number of telomeres analyzed, are indicated. Note that standard error and not standard deviation is shown. The distribution of the telomere length frequencies for each MEF is an indication of the standard deviation. Littermate MEFs are shown. (B) Quantification using Q-FISH of the percentage of telomeres with undetectable TTAGGG repeats out of the total number of telomeres shown in (A). Littermate MEFs are indicated. (C) Telomeric fluorescence (in arbitrary units, a.u.) using flow cytometry combined with telomeric Q-FISH (Flow-FISH). Littermate mice are indicated. (D) TRF analysis of primary BM cells or liver nuclei, as indicated, from different genotypes. Notice the upwards shift of TRF fragments in Terc+/+/Ku86–/– compared with the wild type. Littermate mice are indicated. (E) G-strand overhangs using liver nuclei visualized in the native gel after hybridization with a (CCCTAA)4 probe. Notice that upon treatment with mung bean nuclease (mbn), the G-strand-specific signal decreases. As control, the same gel was denatured and reprobed with the (CCCTAA)4 probe to visualize telomeres. Table I shows quantification of the G-strand overhang signals corrected by the denaturing gel hybridization signal for the 0 units of mbn lanes. Littermate mice are indicated.

None

Fig. 2. (A) Telomere length distribution in primary MEFs. One TFU corresponds to 1 kb of TTAGGG repeats (Zijlmans et al., 1997). Arrows indicate telomeres with <1 kb of TTAGGG repeats. Average telomere length and the standard error, as well as the total number of telomeres analyzed, are indicated. Note that standard error and not standard deviation is shown. The distribution of the telomere length frequencies for each MEF is an indication of the standard deviation. Littermate MEFs are shown. (B) Quantification using Q-FISH of the percentage of telomeres with undetectable TTAGGG repeats out of the total number of telomeres shown in (A). Littermate MEFs are indicated. (C) Telomeric fluorescence (in arbitrary units, a.u.) using flow cytometry combined with telomeric Q-FISH (Flow-FISH). Littermate mice are indicated. (D) TRF analysis of primary BM cells or liver nuclei, as indicated, from different genotypes. Notice the upwards shift of TRF fragments in Terc+/+/Ku86–/– compared with the wild type. Littermate mice are indicated. (E) G-strand overhangs using liver nuclei visualized in the native gel after hybridization with a (CCCTAA)4 probe. Notice that upon treatment with mung bean nuclease (mbn), the G-strand-specific signal decreases. As control, the same gel was denatured and reprobed with the (CCCTAA)4 probe to visualize telomeres. Table I shows quantification of the G-strand overhang signals corrected by the denaturing gel hybridization signal for the 0 units of mbn lanes. Littermate mice are indicated.

None

Fig. 3. (A) Frequencies of RT-like fusions per metaphase in primary MEFs from different genotypes. White bars, fusions showing TTAGGG signals at the fusion point and involving non-homologous pairs of chromosomes (NH). Black bars, fusions with undetectable TTAGGG signals at the fusion point and involving homologous pairs of chromosomes (H); these fusions are largely rescued in the absence of Ku86 in late-generation G3 and G4 telomerase-deficient mice. Littermate MEFs are indicated. (B) Top: complete SKY of a metaphase from a G4 Terc–/–/Ku86–/– primary MEF. The corresponding spectral image, the DAPI banding and the classified spectrum are shown for illustrative purposes. Arrowheads point to two RT-like fusions with involvement of non-homologous chromosomes and to one acentric fragment. Bottom: detail from a G4 Terc–/–/Ku86–/– metaphase showing chromosome breaks and gaps from a G4 Terc–/–/Ku86–/– primary MEF. (C) Representative examples of chromosomal aberrations detected by combined Q-FISH and SKY analyses on primary MEFs from the indicated genotypes. Notice that the fusions associated with Ku86 deficiency always show TTAGGG signals at the fusion point and involve random pairs of chromosomes, whereas a fraction of the fusions associated with critical telomere shortening are characterized by lacking TTAGGG signals at the fusion point and involving homologous pairs of chromosomes.

None

Fig. 3. (A) Frequencies of RT-like fusions per metaphase in primary MEFs from different genotypes. White bars, fusions showing TTAGGG signals at the fusion point and involving non-homologous pairs of chromosomes (NH). Black bars, fusions with undetectable TTAGGG signals at the fusion point and involving homologous pairs of chromosomes (H); these fusions are largely rescued in the absence of Ku86 in late-generation G3 and G4 telomerase-deficient mice. Littermate MEFs are indicated. (B) Top: complete SKY of a metaphase from a G4 Terc–/–/Ku86–/– primary MEF. The corresponding spectral image, the DAPI banding and the classified spectrum are shown for illustrative purposes. Arrowheads point to two RT-like fusions with involvement of non-homologous chromosomes and to one acentric fragment. Bottom: detail from a G4 Terc–/–/Ku86–/– metaphase showing chromosome breaks and gaps from a G4 Terc–/–/Ku86–/– primary MEF. (C) Representative examples of chromosomal aberrations detected by combined Q-FISH and SKY analyses on primary MEFs from the indicated genotypes. Notice that the fusions associated with Ku86 deficiency always show TTAGGG signals at the fusion point and involve random pairs of chromosomes, whereas a fraction of the fusions associated with critical telomere shortening are characterized by lacking TTAGGG signals at the fusion point and involving homologous pairs of chromosomes.

None

Fig. 3. (A) Frequencies of RT-like fusions per metaphase in primary MEFs from different genotypes. White bars, fusions showing TTAGGG signals at the fusion point and involving non-homologous pairs of chromosomes (NH). Black bars, fusions with undetectable TTAGGG signals at the fusion point and involving homologous pairs of chromosomes (H); these fusions are largely rescued in the absence of Ku86 in late-generation G3 and G4 telomerase-deficient mice. Littermate MEFs are indicated. (B) Top: complete SKY of a metaphase from a G4 Terc–/–/Ku86–/– primary MEF. The corresponding spectral image, the DAPI banding and the classified spectrum are shown for illustrative purposes. Arrowheads point to two RT-like fusions with involvement of non-homologous chromosomes and to one acentric fragment. Bottom: detail from a G4 Terc–/–/Ku86–/– metaphase showing chromosome breaks and gaps from a G4 Terc–/–/Ku86–/– primary MEF. (C) Representative examples of chromosomal aberrations detected by combined Q-FISH and SKY analyses on primary MEFs from the indicated genotypes. Notice that the fusions associated with Ku86 deficiency always show TTAGGG signals at the fusion point and involve random pairs of chromosomes, whereas a fraction of the fusions associated with critical telomere shortening are characterized by lacking TTAGGG signals at the fusion point and involving homologous pairs of chromosomes.

None

Fig. 4. (A) Quantification of TUNEL+ cells per 100 tubules of a total of 3–4 different mice of each of the indicated genotypes. (B) Apoptosis in DAPI-stained (blue) Ku86-deficient and Ku86-proficient Terc–/– testis cryosections with progressive telomere shortening. Fluorescein isothiocyanate- labeled TUNEL+ cells (in green) are detected at low frequency in testes from wild-type or G1 Terc–/– mice, irrespective of their Ku86 status. Increased TUNEL+ cells are seen in G3 Terc–/– testes with critically short telomeres. In contrast, the frequency of TUNEL+ cells in G3 Terc–/–/Ku86–/– testis is similar to that of wild-type testis. Magnification, ×20. Hematoxylin–eosin staining of G3 Terc–/– seminiferous tubules is also shown. Notice the paucity of germ cells and the presence of Sertoli-only tubules in a G3 Terc–/–/Ku86+/+ testis, while germ cell maturation is preserved in a G3 Terc–/–/Ku86–/– counterpart. Magnification, ×10.

None

Fig. 4. (A) Quantification of TUNEL+ cells per 100 tubules of a total of 3–4 different mice of each of the indicated genotypes. (B) Apoptosis in DAPI-stained (blue) Ku86-deficient and Ku86-proficient Terc–/– testis cryosections with progressive telomere shortening. Fluorescein isothiocyanate- labeled TUNEL+ cells (in green) are detected at low frequency in testes from wild-type or G1 Terc–/– mice, irrespective of their Ku86 status. Increased TUNEL+ cells are seen in G3 Terc–/– testes with critically short telomeres. In contrast, the frequency of TUNEL+ cells in G3 Terc–/–/Ku86–/– testis is similar to that of wild-type testis. Magnification, ×20. Hematoxylin–eosin staining of G3 Terc–/– seminiferous tubules is also shown. Notice the paucity of germ cells and the presence of Sertoli-only tubules in a G3 Terc–/–/Ku86+/+ testis, while germ cell maturation is preserved in a G3 Terc–/–/Ku86–/– counterpart. Magnification, ×10.

None

Fig. 5. (A) The histograms show the frequency distribution of the number of TRF1 spots per meiotic cell (n = 100 cells). (B) TRF1 distribution in pachytene meiocytes. The TRF1 signal (green) localizes at the end of paired meiotic chromosomes at the pachytene stage (red).

None

Fig. 5. (A) The histograms show the frequency distribution of the number of TRF1 spots per meiotic cell (n = 100 cells). (B) TRF1 distribution in pachytene meiocytes. The TRF1 signal (green) localizes at the end of paired meiotic chromosomes at the pachytene stage (red).

Similar articles

Cited by

References

    1. Bailey S.M., Meyne,J., Chen,D.J., Kurimasa,A., Li,G.C., Lehnert,B.E. and Goodwin,E.H. (1999) DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. Proc. Natl Acad. Sci. USA, 96, 14899–14904. - PMC - PubMed
    1. Bailey S.M., Conforth,M.N., Kurimasa,A., Chen,D.J. and Goodwin,E.H. (2001) Strand-specific postreplicative processing of mammalian telomeres. Science, 293, 2462–2465. - PubMed
    1. Baumann P. and Cech,T.R. (2001) Pot1, the putative telomere end-binding protein in fission yeast and humans. Science, 292, 1171–1175. - PubMed
    1. Bianchi A. and de Lange,T. (1999) Ku binds telomeric DNA in vitro. J. Biol. Chem., 274, 21223–21227. - PubMed
    1. Bilaud T., Brun,C., Ancelin,K., Koering,C.E., Laroche,T. and Gilson,E. (1997) Telomeric localization of TRF2, a novel human telobox protein. Nature Genet., 17, 236–239. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources