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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

Peo directly interacts with HOAP and is also likely to bind Moi and Ver.(A) Schematic of HOAP truncations used in GST pulldown experiments; R indicates the repeated segments found in the C-proximal half of the protein. (B) Bacterially purified GST-HOAP segments spanning the N proximal half of the protein precipitate bacterially expressed His-Peo, which is not pulled down by GST alone or the C-proximal half of HOAP. His-Peo was detected using our anti-Peo antibody; the two Peo bands are likely to correspond to different translation products generated in E. coli starting from the two closely spaced ATG codons present on peo cDNA (see Fig 2) (C) GST-HOAP, GST-Moi and GST-Ver precipitate Peo-FLAG from HeLa cell extracts. Peo-FLAG was detected with anti-FLAG antibody.
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pgen.1005260.g005: Peo directly interacts with HOAP and is also likely to bind Moi and Ver.(A) Schematic of HOAP truncations used in GST pulldown experiments; R indicates the repeated segments found in the C-proximal half of the protein. (B) Bacterially purified GST-HOAP segments spanning the N proximal half of the protein precipitate bacterially expressed His-Peo, which is not pulled down by GST alone or the C-proximal half of HOAP. His-Peo was detected using our anti-Peo antibody; the two Peo bands are likely to correspond to different translation products generated in E. coli starting from the two closely spaced ATG codons present on peo cDNA (see Fig 2) (C) GST-HOAP, GST-Moi and GST-Ver precipitate Peo-FLAG from HeLa cell extracts. Peo-FLAG was detected with anti-FLAG antibody.

Mentions: We next asked whether the Peo protein physically interacts with the terminin components. A preliminary experiment using the yeast two-hybrid assay suggested that Peo directly interacts with HOAP but not with HP1a (S1 Fig). To confirm this result we performed a GST pulldown assay using bacterially expressed 6His-Peo and GST-tagged HOAP polypeptides of different length. As shown in Fig 5A and 5B, the intact HOAP protein and the HOAP fragments containing the N-terminal region of the protein (aa 1–145) precipitated Peo, while a larger HOAP fragments including the 3 repeated segments of the protein (aa 109–343) failed to bind Peo. We then investigated whether Peo interacts with Moi and Ver. We performed GST pulldown experiments with extracts from human 293T cells expressing Peo-FLAG and either GST alone, GST-Moi, GST-Ver or GST-HOAP. We chose to express Drosophila tagged proteins in human cells because a heterogeneous cellular environment is likely to reduce the probability of indirect interactions among fly proteins. As shown in Fig 5C, Peo-FLAG is precipitated by GST-Moi, GST-Ver and GST HOAP but not GST alone. Collectively, these results provide strong evidence that Peo directly binds HOAP; in addition, they suggest that Peo directly interacts with Moi and Ver.


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)

Peo directly interacts with HOAP and is also likely to bind Moi and Ver.(A) Schematic of HOAP truncations used in GST pulldown experiments; R indicates the repeated segments found in the C-proximal half of the protein. (B) Bacterially purified GST-HOAP segments spanning the N proximal half of the protein precipitate bacterially expressed His-Peo, which is not pulled down by GST alone or the C-proximal half of HOAP. His-Peo was detected using our anti-Peo antibody; the two Peo bands are likely to correspond to different translation products generated in E. coli starting from the two closely spaced ATG codons present on peo cDNA (see Fig 2) (C) GST-HOAP, GST-Moi and GST-Ver precipitate Peo-FLAG from HeLa cell extracts. Peo-FLAG was detected with anti-FLAG antibody.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005260.g005: Peo directly interacts with HOAP and is also likely to bind Moi and Ver.(A) Schematic of HOAP truncations used in GST pulldown experiments; R indicates the repeated segments found in the C-proximal half of the protein. (B) Bacterially purified GST-HOAP segments spanning the N proximal half of the protein precipitate bacterially expressed His-Peo, which is not pulled down by GST alone or the C-proximal half of HOAP. His-Peo was detected using our anti-Peo antibody; the two Peo bands are likely to correspond to different translation products generated in E. coli starting from the two closely spaced ATG codons present on peo cDNA (see Fig 2) (C) GST-HOAP, GST-Moi and GST-Ver precipitate Peo-FLAG from HeLa cell extracts. Peo-FLAG was detected with anti-FLAG antibody.
Mentions: We next asked whether the Peo protein physically interacts with the terminin components. A preliminary experiment using the yeast two-hybrid assay suggested that Peo directly interacts with HOAP but not with HP1a (S1 Fig). To confirm this result we performed a GST pulldown assay using bacterially expressed 6His-Peo and GST-tagged HOAP polypeptides of different length. As shown in Fig 5A and 5B, the intact HOAP protein and the HOAP fragments containing the N-terminal region of the protein (aa 1–145) precipitated Peo, while a larger HOAP fragments including the 3 repeated segments of the protein (aa 109–343) failed to bind Peo. We then investigated whether Peo interacts with Moi and Ver. We performed GST pulldown experiments with extracts from human 293T cells expressing Peo-FLAG and either GST alone, GST-Moi, GST-Ver or GST-HOAP. We chose to express Drosophila tagged proteins in human cells because a heterogeneous cellular environment is likely to reduce the probability of indirect interactions among fly proteins. As shown in Fig 5C, Peo-FLAG is precipitated by GST-Moi, GST-Ver and GST HOAP but not GST alone. Collectively, these results provide strong evidence that Peo directly binds HOAP; in addition, they suggest that Peo directly interacts with Moi and Ver.

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