RESUMO
The modifier of mdg4, mod(mdg4), locus in Drosophila melanogaster represents a new type of complex gene in which functional diversity is resolved by mRNA trans-splicing. A protein family of >30 transcriptional regulators, which are supposed to be involved in higher-order chromatin structure, is encoded by both DNA strands of this locus. Mutations in mod(mdg4) have been identified independently in a number of genetic screens involving position-effect variegation, modulation of chromatin insulators, apoptosis, pathfinding of nerve cells, and chromosome pairing, indicating pleiotropic effects. The unusual gene structure and mRNA trans-splicing are evolutionary conserved in the distantly related species Drosophila virilis. Chimeric mod(mdg4) transcripts encoded from nonhomologous chromosomes containing the splice donor from D. virilis and the acceptor from D. melanogaster are produced in transgenic flies. We demonstrate that a significant amount of protein can be produced from these chimeric mRNAs. The evolutionary and functional conservation of mod(mdg4) and mRNA trans-splicing in both Drosophila species is furthermore demonstrated by the ability of D. virilis mod(mdg4) transgenes to rescue recessive lethality of mod(mdg4) mutant alleles in D. melanogaster.
Assuntos
Sequência Conservada , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila/genética , Proteínas Recombinantes/genética , Trans-Splicing , Fatores de Transcrição/genética , Alelos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Cromatina , Evolução Molecular , Genes de Insetos , Genes Letais , Genes Recessivos , Genoma , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , TransgenesRESUMO
The modifier of mdg4 (mod[mdg4]) locus of Drosophila melanogaster (Dme) encodes chromatin proteins which are involved in position effect variegation, establishment of chromatin boundaries, nerve pathfinding, meiotic chromosome pairing and apoptosis. It was recently shown that mRNA trans-splicing is involved in the generation of at least 26 different mod(mdg4) transcripts. Here, we show that a similar complex mod(mdg4) locus exists in Drosophila pseudoobscura (Dps), Drosophila virilis (Dvi), Anopheles gambiae (Aga) and Bombyx mori (Bmo). As in D. melanogaster, most isoforms of these species contain a strongly conserved BTB/POZ domain (hereafter referred to as BTB domain) within the common N-terminal part and a Cys(2)His(2) motif containing FLYWCH domain within the isoform-specific C-terminal parts. By sequence comparison, we identified six novel isoforms in D. melanogaster and show that altogether 31 isoforms are perfectly conserved by sequence and position in the mod(mdg4) locus of the Drosophila species analyzed. We found significant differences in evolutionary speed of synonymous/nonsynonymous divergence between the various isoform specific exons. These results were extended by tree reconstruction analysis based on the evolved FLYWCH domains of predicted Mod(mdg4) proteins in Drosophila and Anopheles. Comparative analysis of mod(mdg4) gene structure in species of dipterans implicates that several internal inversions occurred making the mRNA trans-splicing mechanism indispensable for mod(mdg4) expression. Finally, we propose a model for the evolution of trans-splicing implementing effective regulation of many alternative gene products in a composite gene structure.
Assuntos
Processamento Alternativo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Evolução Molecular , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Anopheles/genética , Sítios de Ligação/genética , Bombyx/genética , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/genética , Dípteros/genética , Drosophila/genética , Lepidópteros/genética , Modelos Genéticos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
In Drosophila males, homologous chromosomes segregate by an unusual process involving physical connections not dependent on recombination. We have identified two meiotic proteins specifically required for this process. Stromalin in Meiosis (SNM) is a divergent member of the SCC3/SA/STAG family of cohesin proteins, and Modifier of Mdg4 in Meiosis (MNM) is one of many BTB-domain proteins expressed from the mod(mdg4) locus. SNM and MNM colocalize along with a repetitive rDNA sequence known to function as an X-Y pairing site to nucleolar foci during meiotic prophase and to a compact structure associated with the X-Y bivalent during prometaphase I and metaphase I. Additionally, MNM localizes to autosomal foci throughout meiosis I. These proteins are mutually dependent for their colocalization, and at least MNM requires the function of teflon, another meiotic gene. SNM and MNM do not colocalize with SMC1, suggesting that the homolog conjunction mechanism is independent of cohesin.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Segregação de Cromossomos/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Meiose/fisiologia , Fatores de Transcrição/genética , Animais , Proteínas de Ciclo Celular/genética , Divisão do Núcleo Celular/genética , Proteínas Cromossômicas não Histona/genética , Aberrações Cromossômicas , Pareamento Cromossômico/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Cromossomos/metabolismo , DNA Ribossômico/genética , Dípteros/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Éxons/genética , Feminino , Proteínas Fúngicas/genética , Masculino , Meiose/genética , Dados de Sequência Molecular , Mutação/genética , Não Disjunção Genética , Proteínas Nucleares/genética , Fenótipo , Filogenia , Isoformas de Proteínas/genética , Cromossomos Sexuais/genética , Fatores de Transcrição/fisiologia , CoesinasRESUMO
SU(VAR)3-9 like histone methyltransferases control heterochromatic domains in eukaryotes. In Arabidopsis, 10 SUVH genes encode SU(VAR)3-9 homologues where SUVH1, SUVH2 and SUVH4 (KRYPTONITE) represent distinct subgroups of SUVH genes. Loss of SUVH1 and SUVH4 causes weak reduction of heterochromatic histone H3K9 dimethylation, whereas in SUVH2 null plants mono- and dimethyl H3K9, mono- and dimethyl H3K27, and monomethyl H4K20, the histone methylation marks of Arabidopsis heterochromatin are significantly reduced. Like animal SU(VAR)3-9 proteins SUVH2 displays strong dosage-dependent effects. Loss of function suppresses, whereas overexpression enhances, gene silencing, causes ectopic heterochromatization and significant growth defects. Furthermore, modification of transgene silencing by SUVH2 is partially transmitted to the offspring plants. This epigenetic stability correlates with heritable changes in DNA methylation. Mutational dissection of SUVH2 indicates an implication of its N-terminus and YDG domain in directing DNA methylation to target sequences, a prerequisite for consecutive histone methylation. Gene silencing by SUVH2 depends on MET1 and DDM1, but not CMT3. In Arabidopsis, SUVH2 with its histone H3K9 and H4K20 methylation activity has a central role in heterochromatic gene silencing.
Assuntos
Arabidopsis/fisiologia , Inativação Gênica/fisiologia , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Agrobacterium tumefaciens , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Western Blotting , Cruzamentos Genéticos , DNA (Citosina-5-)-Metiltransferases/metabolismo , Análise Mutacional de DNA , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/fisiologia , Vetores Genéticos , Genótipo , Histona-Lisina N-Metiltransferase/genética , Imuno-Histoquímica , Luciferases , Metilação , Microscopia Eletrônica , Microscopia de Fluorescência , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Transfecção , Transgenes/genéticaRESUMO
The modifier of mdg4 (mod(mdg4)) gene of Drosophila melanogaster has been identified in many different genetic assays. It has been independently identified through mutations isolated for their effects on position effect variegation (PEV), the properties of insulator sequences, correct pathfinding of growing nerve cells, meiotic pairing of chromosomes, or apoptosis. Molecular analysis of the mod(mdg4) locus revealed that it encodes a family of at least 26 protein isoforms. Inspired by the fact that some mod(mdg4) transcripts are encoded by both antiparallel DNA strands, it was shown that mRNA trans splicing is the mechanism used by this locus to produce mature transcripts. All Mod(mdg4) protein isoforms share a common N-terminal region of 402 amino acids, which includes the conserved BTB/POZ domain. However, the isoforms differ in their C-terminal ends. Most of the C-termini contain a conserved Cys2His2 protein motif, which we have named the FLYWCH motif. Genetic and immunological data indicate that mod(mdg4) encodes a family of related chromatin proteins. Recent results indicate a functional correlation between the large number of different isoforms and the pleiotropic mutant phenotypes of most mod(mdg4) mutations. We discuss the putative function of Mod(mdg4) proteins as chromatin modulators involved in higher order chromatin domains. We also provide evidence for the evolutionary conservation of several of the isoforms and the unusual structure of the locus.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Expressão Gênica/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos , Animais , Cromatina , Evolução Molecular , Elementos Isolantes , Mutação , Trans-SplicingRESUMO
In position-effect variegation (PEV) genes become silenced by heterochromatisation. Genetic dissection of this process has been performed by means of dominant suppressor [Su(var)] and enhancer [E(var)] mutations. Selective genetic screens allowed mass isolation of more than 380 PEV modifier mutations identifying about 150 genes. Genetic fine structure studies revealed unique dosage dependent effects. Most of the haplo-dependent Su(var) and E(var) genes do not display triplo-dependent effects. Several Su(var) loci with triplo-dependent opposite enhancer effects have been identified and shown to encode heterochromatin-associated proteins. From these the evolutionary conserved histone H3 lysine 9 methyltransferase SU(VAR)3-9 plays a central role in heterochromatic gene silencing. Molecular function of most PEV modifier genes is still unknown also including genes identified with mutations displaying lethal interaction to heterochromatin. Their analysis should contribute to further understanding of processes connected with regulation of higher order chromatin structure and epigenetic programming.
Assuntos
Drosophila/genética , Inativação Gênica , Heterocromatina/genética , Animais , Cromatina , Metiltransferases , Mutação , FenótipoRESUMO
Su(var)3-9 is a dominant modifier of heterochromatin-induced gene silencing. Like its mammalian and Schizosaccharomyces pombe homologues, Su(var) 3-9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9). In Su(var)3-9 null mutants, H3-K9 methylation at chromocentre heterochromatin is strongly reduced, indicating that SU(VAR)3-9 is the major heterochromatin-specific HMTase in Drosophila. SU (VAR)3-9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, SU(VAR)3-7. Notably, SU(VAR)3-9-HP1 interaction is interdependent and governs distinct localization patterns of both proteins. In Su(var)3-9 null mutants, concentration of HP1 at the chromocentre is nearly lost without affecting HP1 accumulation at the fourth chromosome. By contrast, in HP1 null mutants SU(VAR)3-9 is no longer restricted at heterochromatin but broadly dispersed across the chromosomes. Despite this interdependence, Su(var)3-9 dominates the PEV modifier effects of HP1 and Su(var)3-7 and is also epistatic to the Y chromosome effect on PEV. Finally, the human SUV39H1 gene is able to partially rescue Su(var)3-9 silencing defects. Together, these data indicate a central role for the SU(VAR)3-9 HMTase in heterochromatin-induced gene silencing in Drosophila.