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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

Mutations in peo affect DNA replication in brain cell nuclei.(A) Analysis of EdU-treated brain cell nuclei reveal 3 gross incorporation patterns: S1 nuclei incorporate EdU in most of the nucleus but not in the DAPI stained chromocenter and are presumably in early S; S2 nuclei incorporate EdU in both the chromocenter and the rest of the nucleus and are likely to be in mid-S; S3 nuclei incorporate EdU only in the chromocenter an are thus in late S. (B) peo mutant brains exhibit a significant reduction in the frequency of EdU positive nuclei compared to wild type (wt), ver or woc mutant brains (** significant in Student's t test with p <0.01). The frequencies of EdU positive nuclei in ver and woc brains are not significantly different from the wild type frequency (C) Mutations in peo do not alter the relative frequencies of S1, S2 and S3, nuclei suggesting that Peo is not required for a specific step of the S phase. The frequencies and types of EdU labeled nuclei in each sample (wild type, peoh/peoh and peo1/peo1) were obtained by examining at least 5,000 nuclei from at least 6 brains.
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pgen.1005260.g009: Mutations in peo affect DNA replication in brain cell nuclei.(A) Analysis of EdU-treated brain cell nuclei reveal 3 gross incorporation patterns: S1 nuclei incorporate EdU in most of the nucleus but not in the DAPI stained chromocenter and are presumably in early S; S2 nuclei incorporate EdU in both the chromocenter and the rest of the nucleus and are likely to be in mid-S; S3 nuclei incorporate EdU only in the chromocenter an are thus in late S. (B) peo mutant brains exhibit a significant reduction in the frequency of EdU positive nuclei compared to wild type (wt), ver or woc mutant brains (** significant in Student's t test with p <0.01). The frequencies of EdU positive nuclei in ver and woc brains are not significantly different from the wild type frequency (C) Mutations in peo do not alter the relative frequencies of S1, S2 and S3, nuclei suggesting that Peo is not required for a specific step of the S phase. The frequencies and types of EdU labeled nuclei in each sample (wild type, peoh/peoh and peo1/peo1) were obtained by examining at least 5,000 nuclei from at least 6 brains.

Mentions: We have recently found that AKTIP/Ft1 is required for proper telomeric DNA replication [43]. This finding suggested that peo mutations could also impair DNA replication and specifically affect heterochromatic telomeres, which are likely to replicate at the end of the S phase together with the bulk of heterochromatin (reviewed in [38]). To test this possibility we examined DNA replication in brain cells by analyzing the incorporation of the EdU (5-ethynyl-2’-deoxyuridine) analog of thymidine. Brains were incubated in saline containing 10 mM EdU for 1 h, immediately fixed and then stained with the Click-It Alexa Fluor method to detect EdU (see Methods). In wild type brains, 10% of the nuclei were actively replicating their DNA and incorporated EdU, whereas in peoh/peoh and peo1/peo1 brains the frequency of EdU-labeled nuclei dropped to 7% and 5.5%, respectively (Fig 9A and 9B). In contrast, in woc and ver mutants, the frequency of EdU-positive nuclei was not significantly different from wild type controls, suggesting that the DNA replication defect observed in peo mutants is a specific outcome of the reduced peo activity and not a general consequence of impaired telomere protection.


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)

Mutations in peo affect DNA replication in brain cell nuclei.(A) Analysis of EdU-treated brain cell nuclei reveal 3 gross incorporation patterns: S1 nuclei incorporate EdU in most of the nucleus but not in the DAPI stained chromocenter and are presumably in early S; S2 nuclei incorporate EdU in both the chromocenter and the rest of the nucleus and are likely to be in mid-S; S3 nuclei incorporate EdU only in the chromocenter an are thus in late S. (B) peo mutant brains exhibit a significant reduction in the frequency of EdU positive nuclei compared to wild type (wt), ver or woc mutant brains (** significant in Student's t test with p <0.01). The frequencies of EdU positive nuclei in ver and woc brains are not significantly different from the wild type frequency (C) Mutations in peo do not alter the relative frequencies of S1, S2 and S3, nuclei suggesting that Peo is not required for a specific step of the S phase. The frequencies and types of EdU labeled nuclei in each sample (wild type, peoh/peoh and peo1/peo1) were obtained by examining at least 5,000 nuclei from at least 6 brains.
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pgen.1005260.g009: Mutations in peo affect DNA replication in brain cell nuclei.(A) Analysis of EdU-treated brain cell nuclei reveal 3 gross incorporation patterns: S1 nuclei incorporate EdU in most of the nucleus but not in the DAPI stained chromocenter and are presumably in early S; S2 nuclei incorporate EdU in both the chromocenter and the rest of the nucleus and are likely to be in mid-S; S3 nuclei incorporate EdU only in the chromocenter an are thus in late S. (B) peo mutant brains exhibit a significant reduction in the frequency of EdU positive nuclei compared to wild type (wt), ver or woc mutant brains (** significant in Student's t test with p <0.01). The frequencies of EdU positive nuclei in ver and woc brains are not significantly different from the wild type frequency (C) Mutations in peo do not alter the relative frequencies of S1, S2 and S3, nuclei suggesting that Peo is not required for a specific step of the S phase. The frequencies and types of EdU labeled nuclei in each sample (wild type, peoh/peoh and peo1/peo1) were obtained by examining at least 5,000 nuclei from at least 6 brains.
Mentions: We have recently found that AKTIP/Ft1 is required for proper telomeric DNA replication [43]. This finding suggested that peo mutations could also impair DNA replication and specifically affect heterochromatic telomeres, which are likely to replicate at the end of the S phase together with the bulk of heterochromatin (reviewed in [38]). To test this possibility we examined DNA replication in brain cells by analyzing the incorporation of the EdU (5-ethynyl-2’-deoxyuridine) analog of thymidine. Brains were incubated in saline containing 10 mM EdU for 1 h, immediately fixed and then stained with the Click-It Alexa Fluor method to detect EdU (see Methods). In wild type brains, 10% of the nuclei were actively replicating their DNA and incorporated EdU, whereas in peoh/peoh and peo1/peo1 brains the frequency of EdU-labeled nuclei dropped to 7% and 5.5%, respectively (Fig 9A and 9B). In contrast, in woc and ver mutants, the frequency of EdU-positive nuclei was not significantly different from wild type controls, suggesting that the DNA replication defect observed in peo mutants is a specific outcome of the reduced peo activity and not a general consequence of impaired telomere protection.

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