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1.
PLoS One ; 9(5): e96802, 2014.
Article in English | MEDLINE | ID: mdl-24820312

ABSTRACT

Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.


Subject(s)
Argonaute Proteins/metabolism , DNA Transposable Elements/genetics , Drosophila Proteins/metabolism , Fertility/physiology , Oogenesis/physiology , Solanum melongena/metabolism , Animals , Argonaute Proteins/genetics , Drosophila , Drosophila Proteins/genetics , Female , Fertility/genetics , Gene Silencing , Oogenesis/genetics , Solanum melongena/genetics
2.
PLoS One ; 7(9): e44253, 2012.
Article in English | MEDLINE | ID: mdl-22962605

ABSTRACT

Transposable elements (TEs) are a major source of genetic variability in genomes, creating genetic novelty and driving genome evolution. Analysis of sequenced genomes has revealed considerable diversity in TE families, copy number, and localization between different, closely related species. For instance, although the twin species Drosophila melanogaster and D. simulans share the same TE families, they display different amounts of TEs. Furthermore, previous analyses of wild type derived strains of D. simulans have revealed high polymorphism regarding TE copy number within this species. Several factors may influence the diversity and abundance of TEs in a genome, including molecular mechanisms such as epigenetic factors, which could be a source of variation in TE success. In this paper, we present the first analysis of the epigenetic status of four TE families (roo, tirant, 412 and F) in seven wild type strains of D. melanogaster and D. simulans. Our data shows intra- and inter-specific variations in the histone marks that adorn TE copies. Our results demonstrate that the chromatin state of common TEs varies among TE families, between closely related species and also between wild type strains.


Subject(s)
DNA Transposable Elements , Drosophila/genetics , Histones/genetics , Protein Processing, Post-Translational , Animals , Biological Evolution , Chromatin/metabolism , DNA Copy Number Variations , DNA Methylation , Drosophila/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Epigenesis, Genetic , Gene Expression , Genetic Variation , Genome, Insect , Genotype , Histones/metabolism , Species Specificity
3.
Genetics ; 185(4): 1183-92, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20457875

ABSTRACT

The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. Here we address the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. We show that the lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, we show that in the absence of JIL-1 kinase activity, Su(var)3-7 gets redistributed and upregulated on the chromosome arms. Reducing the dose of the Su(var)3-7 gene dramatically decreases this redistribution; however, the spreading of H3K9me2 to the chromosome arms was unaffected, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality. These observations suggest a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity.


Subject(s)
Chromosomal Position Effects , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Epistasis, Genetic , Protein Serine-Threonine Kinases/genetics , Animals , Animals, Genetically Modified , Chromosomes, Insect/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Histones/metabolism , Immunohistochemistry , Lysine/metabolism , Male , Methylation , Methyltransferases/genetics , Microscopy, Fluorescence , Mutation , Protein Serine-Threonine Kinases/metabolism
4.
PLoS Genet ; 4(5): e1000066, 2008 May 02.
Article in English | MEDLINE | ID: mdl-18451980

ABSTRACT

In Drosophila, dosage compensation augments X chromosome-linked transcription in males relative to females. This process is achieved by the Dosage Compensation Complex (DCC), which associates specifically with the male X chromosome. We previously found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. In this study, we examine the impact of change in levels of SU(VAR)3-7 on dosage compensation. We first demonstrate that the DCC makes the X chromosome a preferential target for heterochromatic markers. In addition, reduced or increased amounts of SU(VAR)3-7 result in redistribution of the DCC proteins MSL1 and MSL2, and of Histone 4 acetylation of lysine 16, indicating that a wild-type dose of SU(VAR)3-7 is required for X-restricted DCC targeting. SU(VAR)3-7 is also involved in the dosage compensated expression of the X-linked white gene. Finally, we show that absence of maternally provided SU(VAR)3-7 renders dosage compensation toxic in males, and that global amounts of heterochromatin affect viability of ectopic MSL2-expressing females. Taken together, these results bring to light a link between heterochromatin and dosage compensation.


Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dosage Compensation, Genetic , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Heterochromatin/genetics , Heterochromatin/metabolism , ATP-Binding Cassette Transporters/genetics , Animals , Animals, Genetically Modified , Crosses, Genetic , Eye Proteins/genetics , Female , Gene Expression , Genes, Insect , Male , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , X Chromosome/genetics , X Chromosome/metabolism
5.
Genetics ; 177(3): 1955-7, 2007 Nov.
Article in English | MEDLINE | ID: mdl-18039887

ABSTRACT

Mammalian G9a is a euchromatic histone H3 lysine 9 (H3K9) methyltransferase essential for development. Here, we characterize the Drosophila homolog of G9a, dG9a. We generated a dG9a deletion allele by homologous recombination. Analysis of this allele revealed that, in contrast to recent findings, dG9a is not required for fly viability.


Subject(s)
Drosophila/enzymology , Drosophila/genetics , Genes, Insect , Histone-Lysine N-Methyltransferase/genetics , Animals , Animals, Genetically Modified , Drosophila/growth & development , Gene Deletion , Histone Methyltransferases , Larva/enzymology , Larva/growth & development , Mutation , Phenotype , Protein Methyltransferases , Recombination, Genetic
6.
Chromosoma ; 115(2): 139-50, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16463146

ABSTRACT

The Drosophila protein SU(VAR)3-7 is essential for fly viability, chromosome structure, and heterochromatin formation. We report that searches in silico and in vitro for homologues of SU(VAR)3-7 were successful within, but not outside, the Drosophila genus. Protein sequence homology between the distant sibling species Drosophila melanogaster and Drosophila virilis is low, except for the general organization of the protein and three conserved motives: seven widely spaced zinc fingers in the N-terminal half and the BESS and BoxA motives in the C-terminal half of the protein. We have undertaken a fine functional dissection of SU(VAR)3-7 in vivo using transgenes encoding truncations of the protein. BESS mediates interaction of SU(VAR)3-7 with itself, and BoxA is required for specific heterochromatin association. Both are necessary for the silencing properties of SU(VAR)3-7. The seven zinc fingers, widely spaced over the N-terminal half of SU(VAR)3-7, are required for binding to polytene chromosomes. One finger is necessary and sufficient to determine the appropriate chromatin association of the C-terminal half of the protein. Conferring a function to each of the conserved motives allows us to better understand the mode of action of SU(VAR)3-7 in triggering heterochromatin formation and subsequent genomic silencing.


Subject(s)
DNA-Binding Proteins/physiology , Drosophila Proteins/physiology , Gene Silencing , Heterochromatin/physiology , Amino Acid Sequence , Animals , Conserved Sequence , DNA-Binding Proteins/chemistry , Drosophila , Drosophila Proteins/chemistry , Drosophila melanogaster , Evolution, Molecular , Heterochromatin/chemistry , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Zinc Fingers/physiology
7.
J Cell Sci ; 118(Pt 21): 5047-57, 2005 Nov 01.
Article in English | MEDLINE | ID: mdl-16234327

ABSTRACT

Loss of Su(var)3-7 or HP1 suppresses the genomic silencing of position-effect variegation, whereas over-expression enhances it. In addition, loss of Su(var)3-7 results in preferential male lethality. In polytene chromosomes deprived of Su(var)3-7, we observe a specific bloating of the male X chromosome, leading to shortening of the chromosome and to blurring of its banding pattern. In addition, the chromocenter, where heterochromatin from all polytene chromosomes fuses, appears decondensed. The same chromosomal phenotypes are observed as a result of loss of HP1. Mutations of Su(var)3-7 or of Su(var)2-5, the gene encoding HP1, also cause developmental defects, including a spectacular increase in size of the prothoracic gland and its polytene chromosomes. Thus, although structurally very different, the two proteins cooperate closely in chromosome organization and development. Finally, bloating of the male X chromosome in the Su(var)3-7 mutant depends on the presence of a functional dosage compensation complex on this chromosome. This observation reveals a new and intriguing genetic interaction between epigenetic silencing and compensation of dose.


Subject(s)
Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Dosage Compensation, Genetic/genetics , Drosophila Proteins/genetics , Gene Silencing , Mutagenesis, Site-Directed , X Chromosome/genetics , Animals , Chromosomal Proteins, Non-Histone/deficiency , Chromosomal Proteins, Non-Histone/physiology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Drosophila Proteins/deficiency , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , Female , Gene Expression Regulation, Developmental , Genes, Overlapping , Heterochromatin/metabolism , Interphase/genetics , Male , Mitosis/genetics , Phenotype , Protein Binding/genetics , Recombination, Genetic , Suppression, Genetic
8.
J Cell Sci ; 117(Pt 25): 6239-47, 2004 Dec 01.
Article in English | MEDLINE | ID: mdl-15564384

ABSTRACT

The Su(var)3-7 protein is essential for fly viability, and several lines of evidence support its key importance in heterochromatin formation: it binds to pericentric heterochromatin, it potently suppresses variegation and it interacts with HP1. However, the mode of action of Su(var)3-7 is poorly understood. Here we investigate in vivo the consequences of increased Su(var)3-7 expression on fly viability and chromatin structure. A large excess of Su(var)3-7 induces lethality, whereas lower doses permit survival and cause spectacular changes in the morphology of polytene chromosomes in males, and to a lesser extent in females. The male X is always the most affected chromosome: it becomes highly condensed and shortened, and its characteristic banding pattern is modified. In addition, Su(var)3-7 was found over the complete length of all chromosomes. This event coincides with the appearance of heterochromatin markers such as histone H3K9 dimethylation and HP1 at many sites on autosomes and, more strikingly, on the male X chromosome. These two features are strictly dependent on the histone-methyltransferase Su(var)3-9, whereas the generalised localisation of Su(var)3-7 is not. These data provide evidence for a dose-dependent regulatory role of Su(var)3-7 in chromosome morphology and heterochromatin formation. Moreover they show that Su(var)3-7 expression is sufficient to induce Su(var)3-9-dependent ectopic heterochromatinisation and suggest a functional link between Su(var)3-7 and the histone-methyltransferase Su(var)3-9.


Subject(s)
DNA-Binding Proteins/biosynthesis , Drosophila Proteins/biosynthesis , Heterochromatin/chemistry , Repressor Proteins/biosynthesis , Animals , Chromatin/metabolism , Chromosome Banding , Chromosomes/ultrastructure , Drosophila , Female , Heterochromatin/ultrastructure , Histones/metabolism , Homozygote , Male , Methylation , Oxazines/metabolism , X Chromosome/ultrastructure
9.
Development ; 129(17): 3975-82, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12163401

ABSTRACT

An increase in the dose of the heterochromatin-associated Su(var)3-7 protein of Drosophila augments the genomic silencing of position-effect variegation. We have expressed a number of fragments of the protein in flies to assign functions to the different domains. Specific binding to pericentric heterochromatin depends on the C-terminal half of the protein. The N terminus, containing six of the seven widely spaced zinc fingers, is required for binding to bands on euchromatic arms, with no preference for pericentric heterochromatin. In contrast to the enhancing properties of the full-length protein, the N terminus half has no effect on heterochromatin-dependent position-effect variegation. In contrast, the C terminus moiety suppresses variegation. This dominant negative effect on variegation could result from association of the fragment with the wild type endogenous protein. Indeed, we have found and mapped a domain of self-association in this C-terminal half. Furthermore, a small fragment of the C-terminal region actually depletes pericentric heterochromatin from endogenous Su(var)3-7 and has a very strong suppressor effect. This depletion is not followed by a depletion of HP1, a companion of Su(var)3-7. This indicates that Su(var)3-7 does not recruit HP1 to heterochromatin. We propose in conclusion that the association of Su(var)3-7 to heterochromatin depends on protein-protein interaction mediated by the C-terminal half of the sequence, while the silencing function requires also the N-terminal half containing the zinc fingers.


Subject(s)
DNA-Binding Proteins/physiology , Drosophila Proteins/physiology , Drosophila melanogaster/physiology , Animals , Chromosomes , Cloning, Molecular , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Euchromatin/metabolism , Female , Gene Silencing , Heterochromatin/metabolism , Male , Protein Binding , Protein Structure, Tertiary
10.
Genetics ; 161(3): 1125-36, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12136016

ABSTRACT

The Su(var)3-7 gene, a haplo-suppressor and triplo-enhancer of position-effect variegation (PEV), encodes a zinc finger heterochromatin-associated protein. To understand the role of this protein in heterochromatin and genomic silencing, mutations were generated by homologous recombination. The donor fragment contained a yellow(+) gene and 7.6 kb of the Su(var)3-7 gene inserted between two FRTs. The Su(var)3-7 sequence contained three stop codons flanking an I-SceI cut site located in the 5' half of the gene. Using two different screening approaches, we obtained an allelic series composed of three mutant alleles. The three mutations are dominant suppressors of PEV. One behaves as a null mutation and results in a maternal-effect recessive lethal phenotype that can be rescued by a zygotic paternal wild-type gene. A P transposon zygotically expressing a Su(var)3-7 full-length cDNA also rescues the mutant phenotype. One hypomorphic allele is viable and the pleiotropic phenotype showed by adult flies indicates that rapidly and late dividing cells seem the most affected by reduced amounts of Su(var)3-7 protein. All three mutants were characterized at the molecular level. Each expresses a portion of the Su(var)3-7 protein that is unable to enter the nucleus and bind chromatin.


Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Recombination, Genetic , Animals , Animals, Genetically Modified , Base Sequence , Codon, Terminator/genetics , Crosses, Genetic , Female , Heterozygote , Male , Mutagenesis , Oligodeoxyribonucleotides/chemistry , Zinc Fingers
11.
Int J Dev Biol ; 46(1): 167-71, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11902679

ABSTRACT

In a search for homologues of the dominant modifier of position-effect variegation Su(var)3-7, we have identified one ORF in Drosophila melanogaster. The 359 amino acid deduced protein is much shorter than the 1169 amino acid protein Su(var)3-7. Surprisingly, the two genes are very close to each other at 87E on the polytene chromosome map, and are transcribed divergently. The triplet coding for the N-terminus amino acid of the new gene lies only 368 base pairs from the start of transcription of Su(var)3-7. This opposite orientation of the homologue has led us to name it Ravus. The N-terminus of the Ravus protein contains only one of the seven unusual zinc fingers of Su(var)3-7. A second region of similarity encodes an acidic domain. Finally, there is a block of high similarity near the C-terminus of the two proteins. It corresponds to a new conserved protein domain, BESS, found also in the BEAF and Stonewall Drosophila proteins. We have constructed a tagged Ravus protein, and have expressed it as a heat-shock inducible transgene. Ravus associates in vivo with polytene chromosomes but, in contrast to the heterochromatin-associated protein Su(var)3-7, does not show specificity for the chromocenter. Ravus does not seem either to modify the genomic silencing of position-effect variegation, as over-expression of the transgene does not affect the variegated phenotype of a number of rearrangements tested.


Subject(s)
Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Amino Acid Sequence , Animals , Chromosomal Proteins, Non-Histone/biosynthesis , Drosophila Proteins/biosynthesis , Expressed Sequence Tags , Heterochromatin/chemistry , Models, Genetic , Molecular Sequence Data , Mutation , Open Reading Frames , Phenotype , Sequence Homology, Amino Acid , Transgenes
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