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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 expression and localization in polytene chromosomes.(A) Western blotting showing the levels Peo expression in larval brains. The affinity purified anti-Peo antibody reacts with 3 bands of the apparent molecular weights of 32, 28 and 25 kDa, which are likely to correspond to the 3 Peo isoforms (PA, PB and PC; see Fig 2 and FlyBase). (B) Quantification of the intensities of the 3 bands (see Materials and Methods) after normalization to the tubulin band (Tub) used as loading control; bars show the mean values of 3 experiments ± SEM. In peo1/Df and peo1527/Df brains the 3 bands are reduced by 60–70% compared to +/Df brains used as control; ** significantly different from +/Df with p < 0.01 in the Student's t test. In peoh/Df brains the band intensity reduction is modest, ranging from 10 to 20%. (C) Immunostaining of wild type (WT), +/Df, peoh/Df and peo1/Df polytene chromosomes showing that Peo accumulates in the nucleolus and decorates many chromosome bands. (D) Fluorescence quantification (± SEM) of chromosome arms (but not of the nucleolus; see Materials and Methods) showed that the fluorescence intensities of peoh/Df and peo1/Df chromosomes are both significantly lower than those of either +/Df or wild type chromosomes. * and *** significant with p < 0.05 and p < 0.001 in the Student's t test, respectively.
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pgen.1005260.g008: Peo expression and localization in polytene chromosomes.(A) Western blotting showing the levels Peo expression in larval brains. The affinity purified anti-Peo antibody reacts with 3 bands of the apparent molecular weights of 32, 28 and 25 kDa, which are likely to correspond to the 3 Peo isoforms (PA, PB and PC; see Fig 2 and FlyBase). (B) Quantification of the intensities of the 3 bands (see Materials and Methods) after normalization to the tubulin band (Tub) used as loading control; bars show the mean values of 3 experiments ± SEM. In peo1/Df and peo1527/Df brains the 3 bands are reduced by 60–70% compared to +/Df brains used as control; ** significantly different from +/Df with p < 0.01 in the Student's t test. In peoh/Df brains the band intensity reduction is modest, ranging from 10 to 20%. (C) Immunostaining of wild type (WT), +/Df, peoh/Df and peo1/Df polytene chromosomes showing that Peo accumulates in the nucleolus and decorates many chromosome bands. (D) Fluorescence quantification (± SEM) of chromosome arms (but not of the nucleolus; see Materials and Methods) showed that the fluorescence intensities of peoh/Df and peo1/Df chromosomes are both significantly lower than those of either +/Df or wild type chromosomes. * and *** significant with p < 0.05 and p < 0.001 in the Student's t test, respectively.

Mentions: To determine the subcellular localization of Peo we raised a rabbit polyclonal antibody against the entirety of Peo and affinity purified it against a GST-Peo fusion protein (see Material and Methods). Western blotting analysis showed that this antibody recognizes 3 bands of ~ 32, ~28 and ~ 25 kDa. In extracts from peo1/Df, and peo1527/Df larval brains, these 3 bands were reduced by approximately 70% compared to +/Df controls (Fig 8A and 8B). Thus, the band with the highest molecular weight is likely to correspond to the longest Peo isoform, while the other two bands might correspond to the shorter isoforms (see Fig 2). In peoh/Df mutant brains, the Peo bands were reduced by approximately 20% with respect to +/Df brains, consistent with the relatively low TF frequency observed in peoh mutants (Fig 1). These results indicate that peo1 and peo1527 are strong hypomorphs compared to the weaker peoh allele.


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 expression and localization in polytene chromosomes.(A) Western blotting showing the levels Peo expression in larval brains. The affinity purified anti-Peo antibody reacts with 3 bands of the apparent molecular weights of 32, 28 and 25 kDa, which are likely to correspond to the 3 Peo isoforms (PA, PB and PC; see Fig 2 and FlyBase). (B) Quantification of the intensities of the 3 bands (see Materials and Methods) after normalization to the tubulin band (Tub) used as loading control; bars show the mean values of 3 experiments ± SEM. In peo1/Df and peo1527/Df brains the 3 bands are reduced by 60–70% compared to +/Df brains used as control; ** significantly different from +/Df with p < 0.01 in the Student's t test. In peoh/Df brains the band intensity reduction is modest, ranging from 10 to 20%. (C) Immunostaining of wild type (WT), +/Df, peoh/Df and peo1/Df polytene chromosomes showing that Peo accumulates in the nucleolus and decorates many chromosome bands. (D) Fluorescence quantification (± SEM) of chromosome arms (but not of the nucleolus; see Materials and Methods) showed that the fluorescence intensities of peoh/Df and peo1/Df chromosomes are both significantly lower than those of either +/Df or wild type chromosomes. * and *** significant with p < 0.05 and p < 0.001 in the Student's t test, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005260.g008: Peo expression and localization in polytene chromosomes.(A) Western blotting showing the levels Peo expression in larval brains. The affinity purified anti-Peo antibody reacts with 3 bands of the apparent molecular weights of 32, 28 and 25 kDa, which are likely to correspond to the 3 Peo isoforms (PA, PB and PC; see Fig 2 and FlyBase). (B) Quantification of the intensities of the 3 bands (see Materials and Methods) after normalization to the tubulin band (Tub) used as loading control; bars show the mean values of 3 experiments ± SEM. In peo1/Df and peo1527/Df brains the 3 bands are reduced by 60–70% compared to +/Df brains used as control; ** significantly different from +/Df with p < 0.01 in the Student's t test. In peoh/Df brains the band intensity reduction is modest, ranging from 10 to 20%. (C) Immunostaining of wild type (WT), +/Df, peoh/Df and peo1/Df polytene chromosomes showing that Peo accumulates in the nucleolus and decorates many chromosome bands. (D) Fluorescence quantification (± SEM) of chromosome arms (but not of the nucleolus; see Materials and Methods) showed that the fluorescence intensities of peoh/Df and peo1/Df chromosomes are both significantly lower than those of either +/Df or wild type chromosomes. * and *** significant with p < 0.05 and p < 0.001 in the Student's t test, respectively.
Mentions: To determine the subcellular localization of Peo we raised a rabbit polyclonal antibody against the entirety of Peo and affinity purified it against a GST-Peo fusion protein (see Material and Methods). Western blotting analysis showed that this antibody recognizes 3 bands of ~ 32, ~28 and ~ 25 kDa. In extracts from peo1/Df, and peo1527/Df larval brains, these 3 bands were reduced by approximately 70% compared to +/Df controls (Fig 8A and 8B). Thus, the band with the highest molecular weight is likely to correspond to the longest Peo isoform, while the other two bands might correspond to the shorter isoforms (see Fig 2). In peoh/Df mutant brains, the Peo bands were reduced by approximately 20% with respect to +/Df brains, consistent with the relatively low TF frequency observed in peoh mutants (Fig 1). These results indicate that peo1 and peo1527 are strong hypomorphs compared to the weaker peoh allele.

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