RESUMEN
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.
Asunto(s)
Proteínas Argonautas/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/metabolismo , Fertilidad/fisiología , Oogénesis/fisiología , Solanum melongena/metabolismo , Animales , Proteínas Argonautas/genética , Drosophila , Proteínas de Drosophila/genética , Femenino , Fertilidad/genética , Silenciador del Gen , Oogénesis/genética , Solanum melongena/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Compensación de Dosificación (Genética) , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Animales Modificados Genéticamente , Cruzamientos Genéticos , Proteínas del Ojo/genética , Femenino , Expresión Génica , Genes de Insecto , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cromosoma X/genética , Cromosoma X/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Silenciador del Gen , Heterocromatina/fisiología , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Proteínas de Unión al ADN/química , Drosophila , Proteínas de Drosophila/química , Drosophila melanogaster , Evolución Molecular , Heterocromatina/química , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Dedos de Zinc/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Compensación de Dosificación (Genética)/genética , Proteínas de Drosophila/genética , Silenciador del Gen , Mutagénesis Sitio-Dirigida , Cromosoma X/genética , Animales , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Sobrepuestos , Heterocromatina/metabolismo , Interfase/genética , Masculino , Mitosis/genética , Fenotipo , Unión Proteica/genética , Recombinación Genética , Supresión GenéticaRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Drosophila/biosíntesis , Heterocromatina/química , Proteínas Represoras/biosíntesis , Animales , Cromatina/metabolismo , Bandeo Cromosómico , Cromosomas/ultraestructura , Drosophila , Femenino , Heterocromatina/ultraestructura , Histonas/metabolismo , Homocigoto , Masculino , Metilación , Oxazinas/metabolismo , Cromosoma X/ultraestructuraRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Animales , Cromosomas , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Eucromatina/metabolismo , Femenino , Silenciador del Gen , Heterocromatina/metabolismo , Masculino , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Recombinación Genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Codón de Terminación/genética , Cruzamientos Genéticos , Femenino , Heterocigoto , Masculino , Mutagénesis , Oligodesoxirribonucleótidos/química , Dedos de ZincRESUMEN
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.