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The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres.

Cenci G, Ciapponi L, Marzullo M, Raffa GD, Morciano P, Raimondo D, Burla R, Saggio I, Gatti M - PLoS Genet. (2015)

Bottom Line: The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment.However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells.We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Biotecnologie, Sapienza-Università di Roma, Roma, Italy; Istituto Pasteur Fondazione Cenci Bolognetti, Sapienza-Università di Roma, Roma, Italy.

ABSTRACT
Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.

No MeSH data available.


Related in: MedlinePlus

Aphidicolin (APH) does not induce TFs in Drosophila brain cells.(A) Experimental scheme used. Larval brains were dissected in saline (Dissect), incubated for 1.5 hours in saline plus APH, briefly rinsed in saline (Wash), and then transferred to 3 ml of APH-free Schneider’s medium supplemented with 10% FBS; Colch, addition of colchicine; Fix, fixation. Wild type and peoh/peoh control brains were not treated with APH but processed like the APH-treated brains; they were fixed after 2 hours incubation in the medium following colchicine addition. (B) Results of the experiments outlined in A.
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pgen.1005260.g011: Aphidicolin (APH) does not induce TFs in Drosophila brain cells.(A) Experimental scheme used. Larval brains were dissected in saline (Dissect), incubated for 1.5 hours in saline plus APH, briefly rinsed in saline (Wash), and then transferred to 3 ml of APH-free Schneider’s medium supplemented with 10% FBS; Colch, addition of colchicine; Fix, fixation. Wild type and peoh/peoh control brains were not treated with APH but processed like the APH-treated brains; they were fixed after 2 hours incubation in the medium following colchicine addition. (B) Results of the experiments outlined in A.

Mentions: Collectively our results suggest three possibilities: (i) that any impairment of Drosophila telomere replication results in telomere fusion, (ii) that Peo is required for a specific step of telomere replication and that an incorrect execution of this steps results in fusigenic lesions, or (iii) that Peo plays a dual function being independently required for telomere replication and telomere capping. To discriminate between these possibilities we treated wild type and peoh/peoh mutant brains with the DNA polymerase inhibitor aphidicolin (APH), which is known to impair human telomere replication [45–47]. As shown in Fig 11, control and mutant brains were treated in three different ways. Dissected brains were incubated for 1.5 hours in 110 mM APH in NaCl 0.7%. They were then washed, transferred into 3 ml of APH-free saline and fixed 1, 2 or 3 hours after the end of the APH treatment; in all cases, 1 hour before fixation, we added colchicine to the saline to collect metaphases. None of the APH treatments caused TFs in wild type brains or increased the TF frequency in peoh/peoh mutants. We note that the APH treatment was highly effective as it caused a strong reduction in the frequency of mitoses in brains fixed 1 h after the end of the APH treatment (Fig 11B).


The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres.

Cenci G, Ciapponi L, Marzullo M, Raffa GD, Morciano P, Raimondo D, Burla R, Saggio I, Gatti M - PLoS Genet. (2015)

Aphidicolin (APH) does not induce TFs in Drosophila brain cells.(A) Experimental scheme used. Larval brains were dissected in saline (Dissect), incubated for 1.5 hours in saline plus APH, briefly rinsed in saline (Wash), and then transferred to 3 ml of APH-free Schneider’s medium supplemented with 10% FBS; Colch, addition of colchicine; Fix, fixation. Wild type and peoh/peoh control brains were not treated with APH but processed like the APH-treated brains; they were fixed after 2 hours incubation in the medium following colchicine addition. (B) Results of the experiments outlined in A.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481407&req=5

pgen.1005260.g011: Aphidicolin (APH) does not induce TFs in Drosophila brain cells.(A) Experimental scheme used. Larval brains were dissected in saline (Dissect), incubated for 1.5 hours in saline plus APH, briefly rinsed in saline (Wash), and then transferred to 3 ml of APH-free Schneider’s medium supplemented with 10% FBS; Colch, addition of colchicine; Fix, fixation. Wild type and peoh/peoh control brains were not treated with APH but processed like the APH-treated brains; they were fixed after 2 hours incubation in the medium following colchicine addition. (B) Results of the experiments outlined in A.
Mentions: Collectively our results suggest three possibilities: (i) that any impairment of Drosophila telomere replication results in telomere fusion, (ii) that Peo is required for a specific step of telomere replication and that an incorrect execution of this steps results in fusigenic lesions, or (iii) that Peo plays a dual function being independently required for telomere replication and telomere capping. To discriminate between these possibilities we treated wild type and peoh/peoh mutant brains with the DNA polymerase inhibitor aphidicolin (APH), which is known to impair human telomere replication [45–47]. As shown in Fig 11, control and mutant brains were treated in three different ways. Dissected brains were incubated for 1.5 hours in 110 mM APH in NaCl 0.7%. They were then washed, transferred into 3 ml of APH-free saline and fixed 1, 2 or 3 hours after the end of the APH treatment; in all cases, 1 hour before fixation, we added colchicine to the saline to collect metaphases. None of the APH treatments caused TFs in wild type brains or increased the TF frequency in peoh/peoh mutants. We note that the APH treatment was highly effective as it caused a strong reduction in the frequency of mitoses in brains fixed 1 h after the end of the APH treatment (Fig 11B).

Bottom Line: The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment.However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells.We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Biotecnologie, Sapienza-Università di Roma, Roma, Italy; Istituto Pasteur Fondazione Cenci Bolognetti, Sapienza-Università di Roma, Roma, Italy.

ABSTRACT
Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.

No MeSH data available.


Related in: MedlinePlus