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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

Involvement of individual telomeres in fusion events in different TF mutants.The vertical axes of the graphs show the frequencies (%) with which individual telomeres are involved in TFs. The red columns indicate the expected frequencies assuming a random involvement of telomeres in fusion events. The columns in the graph correspond to numbered genotypes in the box, and the numbers in parentheses next to each genotype indicates the observed TF frequencies. At least 122 fused telomeres from at least 4 brains were scored for each genotype. The Su(var)205 mutant (# 4) is a Su(var)20504/Su(var)20505 heteroallelic combination. The observed number of "heterochromatic telomeres" involved in TFs in each peo mutant combination is significantly higher than would be expected by chance (p < 0.001 in Chi-square test). In contrast, in all other mutants, this number is significantly lower than the expected one (p < 0.001 in Chi-square test). All observed and expected values and the statistical analysis are presented in detail in S1 Table.
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pgen.1005260.g003: Involvement of individual telomeres in fusion events in different TF mutants.The vertical axes of the graphs show the frequencies (%) with which individual telomeres are involved in TFs. The red columns indicate the expected frequencies assuming a random involvement of telomeres in fusion events. The columns in the graph correspond to numbered genotypes in the box, and the numbers in parentheses next to each genotype indicates the observed TF frequencies. At least 122 fused telomeres from at least 4 brains were scored for each genotype. The Su(var)205 mutant (# 4) is a Su(var)20504/Su(var)20505 heteroallelic combination. The observed number of "heterochromatic telomeres" involved in TFs in each peo mutant combination is significantly higher than would be expected by chance (p < 0.001 in Chi-square test). In contrast, in all other mutants, this number is significantly lower than the expected one (p < 0.001 in Chi-square test). All observed and expected values and the statistical analysis are presented in detail in S1 Table.

Mentions: Although peo mutants show a high DTA/STA ratio as the other TF mutants, they exhibit a specific pattern of TFs. The analysis of the peoh mutant that shows ~1 TF/cell allowed a very precise definition of the telomeres involved in fusion events. In peoh homozygous brains of males and females, nearly all TFs involved the telomeres associated with the heterochomatic regions of the chromosomes, namely those of the entirely heterochromatic Y chromosome (YS and YL), the telomere of the right arm of the X chromosome (XR) and the fourth chromosome telomeres (Fig 1A). Of the two telomeres of the fourth chromosome only one is associated with constitutive heterochromatin (4L) but we were not able to distinguish between 4L and 4R, as the DAPI-stained fourth chromosomes appear as brightly fluorescent dots in which the chromosome arms are not discernible. High frequencies of TFs between heterochromatin-associated telomeres (henceforth abbreviated with Ha-telomeres) were also observed in peoh/Df and peoh/peo1 brains (Fig 3). We note that we classified as DTAs all TFs between Ha-telomeres, because the close apposition of the sister chromatids in heterochromatin does not allow a distinction between STAs and DTAs. Thus, the apparent lack of STAs observed in weak peo mutants (Fig 1B) might not reflect a real absence of this type of TFs.


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)

Involvement of individual telomeres in fusion events in different TF mutants.The vertical axes of the graphs show the frequencies (%) with which individual telomeres are involved in TFs. The red columns indicate the expected frequencies assuming a random involvement of telomeres in fusion events. The columns in the graph correspond to numbered genotypes in the box, and the numbers in parentheses next to each genotype indicates the observed TF frequencies. At least 122 fused telomeres from at least 4 brains were scored for each genotype. The Su(var)205 mutant (# 4) is a Su(var)20504/Su(var)20505 heteroallelic combination. The observed number of "heterochromatic telomeres" involved in TFs in each peo mutant combination is significantly higher than would be expected by chance (p < 0.001 in Chi-square test). In contrast, in all other mutants, this number is significantly lower than the expected one (p < 0.001 in Chi-square test). All observed and expected values and the statistical analysis are presented in detail in S1 Table.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005260.g003: Involvement of individual telomeres in fusion events in different TF mutants.The vertical axes of the graphs show the frequencies (%) with which individual telomeres are involved in TFs. The red columns indicate the expected frequencies assuming a random involvement of telomeres in fusion events. The columns in the graph correspond to numbered genotypes in the box, and the numbers in parentheses next to each genotype indicates the observed TF frequencies. At least 122 fused telomeres from at least 4 brains were scored for each genotype. The Su(var)205 mutant (# 4) is a Su(var)20504/Su(var)20505 heteroallelic combination. The observed number of "heterochromatic telomeres" involved in TFs in each peo mutant combination is significantly higher than would be expected by chance (p < 0.001 in Chi-square test). In contrast, in all other mutants, this number is significantly lower than the expected one (p < 0.001 in Chi-square test). All observed and expected values and the statistical analysis are presented in detail in S1 Table.
Mentions: Although peo mutants show a high DTA/STA ratio as the other TF mutants, they exhibit a specific pattern of TFs. The analysis of the peoh mutant that shows ~1 TF/cell allowed a very precise definition of the telomeres involved in fusion events. In peoh homozygous brains of males and females, nearly all TFs involved the telomeres associated with the heterochomatic regions of the chromosomes, namely those of the entirely heterochromatic Y chromosome (YS and YL), the telomere of the right arm of the X chromosome (XR) and the fourth chromosome telomeres (Fig 1A). Of the two telomeres of the fourth chromosome only one is associated with constitutive heterochromatin (4L) but we were not able to distinguish between 4L and 4R, as the DAPI-stained fourth chromosomes appear as brightly fluorescent dots in which the chromosome arms are not discernible. High frequencies of TFs between heterochromatin-associated telomeres (henceforth abbreviated with Ha-telomeres) were also observed in peoh/Df and peoh/peo1 brains (Fig 3). We note that we classified as DTAs all TFs between Ha-telomeres, because the close apposition of the sister chromatids in heterochromatin does not allow a distinction between STAs and DTAs. Thus, the apparent lack of STAs observed in weak peo mutants (Fig 1B) might not reflect a real absence of this type of TFs.

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