RESUMO
Su(Hw) belongs to the class of proteins that organize chromosome architecture, determine promoter activity, and participate in formation of the boundaries/insulators between the regulatory domains. This protein contains a cluster of 12 zinc fingers of the C2H2 type, some of which are responsible for binding to the consensus site. The Su(Hw) protein forms complex with the Mod(mdg4)-67.2 and the CP190 proteins, where the last one binds to all known Drosophila insulators. To further study functioning of the Su(Hw)-dependent complexes, we used the previously described su(Hw)E8 mutation with inactive seventh zinc finger, which produces mutant protein that cannot bind to the consensus site. The present work shows that the Su(Hw)E8 protein continues to directly interact with the CP190 and Mod(mdg4)-67.2 proteins. Through interaction with Mod(mdg4)-67.2, the Su(Hw)E8 protein can be recruited into the Su(Hw)-dependent complexes formed on chromatin and enhance their insulator activity. Our results demonstrate that the Su(Hw) dependent complexes without bound DNA can be recruited to the Su(Hw) binding sites through the specific protein-protein interactions that are stabilized by Mod(mdg4)-67.2.
Assuntos
Cromatina , Proteínas de Drosophila , Drosophila melanogaster , Proteínas Repressoras , Fatores de Transcrição , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/química , Animais , Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Ligação Proteica , Proteínas Nucleares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dedos de Zinco , Proteínas Associadas aos MicrotúbulosRESUMO
Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems. Drosophila became the first model organism in which architectural proteins that determine the activity of insulators were described. In Drosophila, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and attP-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in cis and trans only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer-promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the D. melanogaster model.
Assuntos
Proteínas de Drosophila , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Cromatina/metabolismo , Cromatina/genética , Elementos Isolantes/genética , Sítios de Ligação , Ligação Proteica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Edição de Genes/métodos , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proteínas Associadas aos MicrotúbulosRESUMO
The Drosophila melanogaster dADD1 and dXNP proteins are orthologues of the ADD and SNF2 domains of the vertebrate ATRX (Alpha-Thalassemia with mental Retardation X-related) protein. ATRX plays a role in general molecular processes, such as regulating chromatin status and gene expression, while dADD1 and dXNP have similar functions in the Drosophila genome. Both ATRX and dADD1/dXNP interact with various protein partners and participate in various regulatory complexes. Disruption of ATRX expression in humans leads to the development of α-thalassemia and cancer, especially glioma. However, the mechanisms that allow ATRX to regulate various cellular processes are poorly understood. Studying the functioning of dADD1/dXNP in the Drosophila model may contribute to understanding the mechanisms underlying the multifunctional action of ATRX and its connection with various cellular processes. This review provides a brief overview of the currently available information in mammals and Drosophila regarding the roles of ATRX, dXNP, and dADD1. It discusses possible mechanisms of action of complexes involving these proteins.
Assuntos
Proteínas de Drosophila , Animais , Humanos , Talassemia alfa/genética , Cromatina/genética , Cromatina/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Drosophila/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mamíferos/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismoRESUMO
Drosophila CP190 and CP60 are transcription factors that are associated with centrosomes during mitosis. CP190 is an essential transcription factor and preferentially binds to housekeeping gene promoters and insulators through interactions with architectural proteins, including Su(Hw) and dCTCF. CP60 belongs to a family of transcription factors that contain the N-terminal MADF domain and the C-terminal BESS domain, which is characterized by the ability to homodimerize. In this study, we show that the conserved CP60 region adjacent to MADF is responsible for interacting with CP190. In contrast to the well-characterized MADF-BESS transcriptional activator Adf-1, CP60 is recruited to most chromatin sites through its interaction with CP190, and the MADF domain is likely involved in protein-protein interactions but not in DNA binding. The deletion of the Map60 gene showed that CP60 is not an essential protein, despite the strong and ubiquitous expression of CP60 at all stages of Drosophila development. Although CP60 is a stable component of the Su(Hw) insulator complex, the inactivation of CP60 does not affect the enhancer-blocking activity of the Su(Hw)-dependent gypsy insulator. Overall, our results indicate that CP60 has an important but redundant function in transcriptional regulation as a partner of the CP190 protein.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
CP190 is a co-factor in many Drosophila architectural proteins, being involved in the formation of active promoters and insulators. CP190 contains the N-terminal BTB/POZ (Broad-Complex, Tramtrack and Bric a brac/POxvirus and Zinc finger) domain and adjacent conserved regions involved in protein interactions. Here, we examined the functional roles of these domains of CP190 in vivo. The best-characterized architectural proteins with insulator functions, Pita, Su(Hw), and dCTCF, interacted predominantly with the BTB domain of CP190. Due to the difficulty of mutating the BTB domain, we obtained a transgenic line expressing a chimeric CP190 with the BTB domain of the human protein Kaiso. Another group of architectural proteins, M1BP, Opbp, and ZIPIC, interacted with one or both of the highly conserved regions in the N-terminal part of CP190. Transgenic lines of D. melanogaster expressing CP190 mutants with a deletion of each of these domains were obtained. The results showed that these mutant proteins only partially compensated for the functions of CP190, weakly binding to selective chromatin sites. Further analysis confirmed the essential role of these domains in recruitment to regulatory regions associated with architectural proteins. We also found that the N-terminal of CP190 was sufficient for recruiting Z4 and Chromator proteins and successfully achieving chromatin opening. Taken together, our results and the results of previous studies showed that the N-terminal region of CP190 is a platform for simultaneous interaction with various DNA-binding architectural proteins and transcription complexes.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Humanos , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Nucleares/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Cromatina/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited cooperatively to chromatin through interactions with the DNA-binding architectural protein Su(Hw). While Mod(mdg4)-67.2 interacts only with Su(Hw), CP190 interacts with many other architectural proteins. In spite of the fact that CP190 is critical for the activity of Su(Hw) insulators, interaction between these proteins has not been studied yet. Therefore, we have performed a detailed analysis of domains involved in the interaction between the Su(Hw) and CP190. The results show that the BTB domain of CP190 interacts with two adjacent regions at the N-terminus of Su(Hw). Deletion of either region in Su(Hw) only weakly affected recruiting of CP190 to the Su(Hw) sites in the presence of Mod(mdg4)-67.2. Deletion of both regions in Su(Hw) prevents its interaction with CP190. Using mutations in vivo, we found that interactions with Su(Hw) and Mod(mdg4)-67.2 are essential for recruiting of CP190 to the Su(Hw) genomic sites.
Assuntos
Domínio BTB-POZ , Proteínas de Drosophila/metabolismo , Elementos Isolantes , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Animais , Domínio BTB-POZ/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Genéticos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fenótipo , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genéticaRESUMO
Recent data suggest that insulators organize chromatin architecture in the nucleus. The best characterized Drosophila insulator, found in the gypsy retrotransposon, contains 12 binding sites for the Su(Hw) protein. Enhancer blocking, along with Su(Hw), requires BTB/POZ domain proteins, Mod(mdg4)-67.2 and CP190. Inactivation of Mod(mdg4)-67.2 leads to a direct repression of the yellow gene promoter by the gypsy insulator. Here, we have shown that such repression is regulated by the level of the EAST protein, which is an essential component of the interchromatin compartment. Deletion of the EAST C-terminal domain suppresses Su(Hw)-mediated repression. Partial inactivation of EAST by mutations in the east gene suppresses the enhancer-blocking activity of the gypsy insulator. The binding of insulator proteins to chromatin is highly sensitive to the level of EAST expression. These results suggest that EAST, one of the main components of the interchromatin compartment, can regulate the activity of chromatin insulators.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos Isolantes , Fosfoproteínas/metabolismo , Proteínas Repressoras/genética , Alelos , Animais , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Masculino , Mutação , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/metabolismoRESUMO
The looping model of enhancer-promoter interactions predicts that these specific long-range interactions are supported by a certain class of proteins. In particular, the Drosophila transcription factor Zeste was hypothesized to facilitate long-distance associations between enhancers and promoters. We have re-examined the role of Zeste in supporting long-range interactions between an enhancer and a promoter using the white gene as a model system. The results show that Zeste binds to the upstream white promoter region and the enhancer that is responsible for white activation in the eyes. We have confirmed the previous finding that Zeste is not required for the activity of the eye enhancer and the promoter when they are located in close proximity to each other. However, inactivation of Zeste markedly affects the enhancer-promoter communication in transgenes when the eye enhancer and the white promoter are separated by a 3-kb spacer or the yellow gene. Zeste is also required for insulator bypass by the eye enhancer. Taken together, these results show that Zeste can support specific long-range interactions between enhancers and promoters.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Regiões Promotoras Genéticas/fisiologia , Animais , Drosophila melanogaster , Elementos Isolantes , Transgenes/fisiologiaRESUMO
In the present study, we set out to investigate cytogenetic changes in the progeny of two normal human fibroblast cell strains after exposure to sparsely or densely ionizing irradiation (X-rays or 9.8 MeV u(-1) carbon ions). The cells were regularly subcultured up to senescence. The transition to senescence was determined by measurement of population doubling numbers and senescence associated (SA) beta-galactosidase activity. Chromosomal changes (structural aberrations, tetraploidy) were investigated by solid staining. In temporal proximity to senescence, we observed for all populations of the two fibroblasts cell strains an increase in the fraction of cells with structural and numerical aberrations. The observed changes in the yield of structural chromosomal aberrations were similar for the progeny of controls and irradiated cells, except that a previous irradiation with a high, fractionated X-ray dose resulted in a stronger increase. Noteworthy, delayed tetraploidy in the descendants of irradiated cells exceeded the level in control cells. In addition, tetraploidy and the time of onset of senescence were significantly correlated for all populations, regardless of a preceding radiation exposure. However, the time of the onset of senescence depends on previous exposure to radiation. We conclude that the occurrence of tetraploidy is associated with senescence independently of exposure to radiation.
Assuntos
Senescência Celular/efeitos da radiação , Aberrações Cromossômicas , Carbono , Proliferação de Células , Células Cultivadas , Fibroblastos/efeitos da radiação , Prepúcio do Pênis/citologia , Instabilidade Genômica , Íons Pesados , Humanos , Masculino , Poliploidia , beta-Galactosidase/análiseRESUMO
Much of the research on insulators in Drosophila has been done with transgenic constructs using the white gene (mini-white) as reporter. Hereby we report that the sequence between the white and CG32795 genes in Drosophila melanogaster contains an insulator of a novel kind. Its functional core is within a 368 bp segment almost contiguous to the white 3'UTR, hence we name it as Wari (white-abutting resident insulator). Though Wari contains no binding sites for known insulator proteins and does not require Su(Hw) or Mod(mdg4) for its activity, it can equally well interact with another copy of Wari and with unrelated Su(Hw)-dependent insulators, gypsy or 1A2. In its natural downstream position, Wari reinforces enhancer blocking by any of the three insulators placed between the enhancer and the promoter; again, Wari-Wari, Wari-gypsy or 1A2-Wari pairing results in mutual neutralization (insulator bypass) when they precede the promoter. The distressing issue is that this element hides in all mini-white constructs employed worldwide to study various insulators and other regulatory elements as well as long-range genomic interactions, and its versatile effects could have seriously influenced the results and conclusions of many works.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Genes de Insetos , Genes Reporter , Elementos Isolantes , Animais , Mapeamento Cromossômico , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras GenéticasRESUMO
The Su(Hw) insulator found in the gypsy retrotransposon is the most potent enhancer blocker in Drosophila melanogaster. However, two such insulators in tandem do not prevent enhancer-promoter communication, apparently because of their pairing interaction that results in mutual neutralization. Furthering our studies of the role of insulators in the control of gene expression, here we present a functional analysis of a large set of transgenic constructs with various arrangements of regulatory elements, including two or three insulators. We demonstrate that their interplay can have quite different outcomes depending on the order of and distance between elements. Thus, insulators can interact with each other over considerable distances, across interposed enhancers or promoters and coding sequences, whereby enhancer blocking may be attenuated, cancelled, or restored. Some inferences concerning the possible modes of insulator action are made from collating the new data and the relevant literature, with tentative schemes illustrating the regulatory situations in particular model constructs.
Assuntos
Drosophila melanogaster/genética , Elementos Facilitadores Genéticos/fisiologia , Elementos Isolantes/fisiologia , Regiões Promotoras Genéticas/fisiologia , Retroelementos/genética , Animais , Regulação da Expressão GênicaRESUMO
Suppressor of Hairy-wing [Su(Hw)] is one of the best characterized architectural proteins in Drosophila and recruits the CP190 and Mod(mdg4)-67.2 proteins to chromatin, where they form a well-known insulator complex. Recently, HP1 and insulator partner protein 1 (HIPP1), a homolog of the human co-repressor Chromodomain Y-Like (CDYL), was identified as a new partner for Su(Hw). Here, we performed a detailed analysis of the domains involved in the HIPP1 interactions with Su(Hw)-dependent complexes. HIPP1 was found to directly interact with the Su(Hw) C-terminal region (aa 720-892) and with CP190, but not with Mod(mdg4)-67.2. We have generated Hipp1 null mutants (HippΔ1) and found that the loss of Hipp1 does not affect the enhancer-blocking or repression activities of the Su(Hw)-dependent complex. However, the simultaneous inactivation of both HIPP1 and Mod(mdg4)-67.2 proteins resulted in reduced CP190 binding with Su(Hw) sites and significantly altered gypsy insulator activity. Taken together, these results suggested that the HIPP1 protein stabilized the interaction between CP190 and the Su(Hw)-dependent complex.
Assuntos
Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Alelos , Animais , Drosophila melanogaster/genética , Éxons , Feminino , Regulação da Expressão Gênica , Masculino , Mutação , Ovário/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Suppressor of Hairy-wing [Su(Hw)] is a DNA-binding architectural protein that participates in the organization of insulators and repression of promoters in Drosophila. This protein contains acidic regions at both ends and a central cluster of 12 zinc finger domains, some of which are involved in the specific recognition of the binding site. One of the well-described in vivo function of Su(Hw) is the repression of transcription of neuronal genes in oocytes. Here, we have found that the same Su(Hw) C-terminal region (aa 720-892) is required for insulation as well as for promoter repression. The best characterized partners of Su(Hw), CP190 and Mod(mdg4)-67.2, are not involved in the repression of neuronal genes. Taken together, these results suggest that an unknown protein or protein complex binds to the C-terminal region of Su(Hw) and is responsible for the direct repression activity of Su(Hw).
Assuntos
Proteínas de Drosophila/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Biologia Computacional , Proteínas de Ligação a DNA , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Elementos Isolantes , Ligação Proteica , Proteínas Repressoras/químicaRESUMO
Su(Hw) belongs to the class of proteins that organize chromosome architecture and boundaries/insulators between regulatory domains. This protein contains a cluster of 12 zinc finger domains most of which are responsible for binding to three different modules in the consensus site. Su(Hw) forms a complex with CP190 and Mod(mdg4)-67.2 proteins that binds to well-known Drosophila insulators. To understand how Su(Hw) performs its activities and binds to specific sites in chromatin, we have examined the previously described su(Hw)f mutation that disrupts the 10th zinc finger (ZF10) responsible for Su(Hw) binding to the upstream module. The results have shown that Su(Hw)f loses the ability to interact with CP190 in the absence of DNA. In contrast, complete deletion of ZF10 does not prevent the interaction between Su(Hw)Δ10 and CP190. Having studied insulator complex formation in different mutant backgrounds, we conclude that both association with CP190 and Mod(mdg4)-67.2 partners and proper organization of DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators.
Assuntos
Cromatina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Animais , Sítios de Ligação , DNA/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Nucleares/química , Ligação ProteicaRESUMO
Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Their transposition to broken chromosome ends has been implicated in chromosome healing and telomere elongation. We have developed a genetic system which enables the determination of the frequency of telomere elongation events and their mechanism. The frequency differs among lines with different genotypes, suggesting that several genes are in control. Here we show that the Su(var)2-5 gene encoding heterochromatin protein 1 (HP1) is involved in regulation of telomere length. Different Su(var)2-5 mutations in the heterozygous state increase the frequency of HeT-A and TART attachment to the broken chromosome end by more than a hundred times. The attachment occurs through either HeT-A/TART transposition or recombination with other telomeres. Terminal DNA elongation by gene conversion is greatly enhanced by Su(var)2-5 mutations only if the template for DNA synthesis is on the same chromosome but not on the homologous chromosome. The Drosophila lines bearing the Su(var)2-5 mutations maintain extremely long telomeres consisting of HeT-A and TART for many generations. Thus, HP1 plays an important role in the control of telomere elongation in D. melanogaster.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Produtos do Gene gag , Telômero/metabolismo , Animais , Sequência de Bases , Proteínas Cromossômicas não Histona/genética , Aberrações Cromossômicas , Cruzamentos Genéticos , DNA/genética , DNA/metabolismo , Proteínas de Drosophila/genética , Feminino , Conversão Gênica , Duplicação Gênica , Regulação da Expressão Gênica , Genes de Insetos/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Mutação , Mapeamento Físico do Cromossomo , Telômero/genética , Moldes GenéticosRESUMO
The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, we have performed a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190. We found that the N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, we have shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Elementos Isolantes , Animais , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Complexos Multiproteicos/metabolismo , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de ProteínasRESUMO
Chromosome ends in Drosophila melanogaster can be elongated either by terminal attachment of the telomere-specific retrotransposons HeT-A and TART or by terminal gene conversion. Here we show that a decrease in Ku70 or Ku80 gene dosage causes a sharp increase in the frequency of HeT-A and TART attachments to a broken chromosome end and in terminal DNA elongation by gene conversion. Loss of Ku80 has more pronounced effects than loss of Ku70. However, lower Ku70 concentration reduces the stability of terminally deficient chromosomes. Our results suggest a role of the end-binding Ku complex in the accessibility and length regulation of Drosophila telomeres.
Assuntos
Antígenos Nucleares/genética , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Telômero/genética , Animais , Antígenos Nucleares/fisiologia , Cruzamentos Genéticos , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Conversão Gênica , Dosagem de Genes , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Heterozigoto , Autoantígeno Ku , RNA Mensageiro , Telômero/metabolismo , Transcrição GênicaRESUMO
Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Terminally deleted chromosomes can be maintained for many generations. Thus, broken chromosome ends behave as real telomeres. It was previously shown that gene conversion may extend the broken ends. Here we found that the frequency of terminal DNA elongation by gene conversion strongly depends on the genotype. A dominant E(tc) (Enhancer of terminal gene conversion) mutation markedly increases the frequency of this event but does not significantly influence the frequency of HeT-A and TART attachment to the broken chromosome end and recombination between directly repeated sequences at the end of the truncated chromosome. The E(tc) mutation was mapped to the 91-93 region on chromosome 3. Drosophila lines that bear the E(tc) mutation for many generations have telomeres, consisting of HeT-A and TART elements, that are longer than those found in wild-type lines. Thus, the E(tc) mutation plays a significant role in the control of telomere elongation in D. melanogaster.
Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Conversão Gênica , Produtos do Gene gag , Telômero/genética , Animais , Mapeamento Cromossômico , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Proteínas de Insetos/metabolismo , Modelos Genéticos , Recombinação GenéticaRESUMO
The best-characterized insulator in Drosophila melanogaster is the Su(Hw)-binding region contained within the gypsy retrotransposon. In the y(2) mutant, Su(Hw) protein partially inhibits yellow transcription by blocking the function of transcriptional enhancers located distally from the yellow promoter with respect to gypsy. Previously we have shown that yellow enhancers can overcome inhibition by a downstream insulator in the y(rh1) allele, when a second gypsy element is located upstream of the enhancers. To understand how two insulators neutralize each other, we isolated various deletions that terminate in the regulatory region of the y(rh1) allele. To generate these alleles we used DNA elongation by gene conversion of the truncated chromosomes at the end of the yellow regulatory region. We found that gypsy insulator can function at the end of the truncated chromosome. Addition of the gypsy insulator upstream of the yellow enhancers overcomes the enhancer-blocking activity of the gypsy insulator inserted between the yellow enhancers and promoter. These results suggest that the gypsy insulators do not form separate transcriptional domains that delimit the interactions between enhancers and promoters.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Proteínas de Insetos/genética , Regiões Promotoras Genéticas , Retroelementos , Animais , Sequência de Bases , Cruzamentos Genéticos , Feminino , Masculino , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Transposable element P of Drosophila melanogaster is one of the best-characterized eukaryotic transposons. Successful transposition requires the interaction between transposase complexes at both termini of the P element. Here we found that insertion of one or two copies of the Su(Hw) insulator in the P transposon reduces the frequency of its transposition. Inactivation of a Mod(mdg4) component of the Su(Hw) insulator suppresses the insulator effect. Thus, the Su(Hw) insulator can modulate interactions between transposase complexes bound to the ends of the P transposon in germ cells.