RESUMEN
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.
Asunto(s)
Cromatina , Proteínas de Drosophila , Drosophila melanogaster , Proteínas Represoras , Factores de Transcripción , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/química , Animales , Cromatina/metabolismo , Factores de Transcripción/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Unión Proteica , Proteínas Nucleares/metabolismo , Proteínas de Unión al ADN/metabolismo , Dedos de Zinc , Proteínas Asociadas a MicrotúbulosRESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Sistemas CRISPR-Cas , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Cromatina/metabolismo , Cromatina/genética , Elementos Aisladores/genética , Sitios de Unión , Unión Proteica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Edición Génica/métodos , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Proteínas Asociadas a MicrotúbulosRESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Humanos , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Nucleares/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Cromatina/metabolismo , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Animales , Humanos , Talasemia alfa/genética , Cromatina/genética , Cromatina/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Drosophila/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mamíferos/metabolismo , Proteína Nuclear Ligada al Cromosoma X/genética , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Alelos , Animales , Drosophila melanogaster/genética , Exones , Femenino , Regulación de la Expresión Génica , Masculino , Mutación , Ovario/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
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).
Asunto(s)
Proteínas de Drosophila/metabolismo , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Biología Computacional , Proteínas de Unión al ADN , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Elementos Aisladores , Unión Proteica , Proteínas Represoras/químicaRESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Animales , Sitios de Unión , ADN/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Nucleares/química , Unión ProteicaRESUMEN
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.
Asunto(s)
Dominio BTB-POZ , Proteínas de Drosophila/metabolismo , Elementos Aisladores , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Animales , Dominio BTB-POZ/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Modelos Genéticos , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fenotipo , Unión Proteica , Proteínas Represoras/química , Proteínas Represoras/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Elementos Aisladores , Animales , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Complejos Multiproteicos/metabolismo , Fenotipo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de ProteínasRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos Aisladores , Fosfoproteínas/metabolismo , Proteínas Represoras/genética , Alelos , Animales , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Masculino , Mutación , Fenotipo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas Represoras/metabolismoRESUMEN
Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.
Asunto(s)
Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Elementos Aisladores/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/fisiología , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Senescencia Celular/efectos de la radiación , Aberraciones Cromosómicas , Carbono , Proliferación Celular , Células Cultivadas , Fibroblastos/efectos de la radiación , Prepucio/citología , Inestabilidad Genómica , Iones Pesados , Humanos , Masculino , Poliploidía , beta-Galactosidasa/análisisRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Elementos de Facilitación Genéticos/fisiología , Regiones Promotoras Genéticas/fisiología , Animales , Drosophila melanogaster , Elementos Aisladores , Transgenes/fisiologíaRESUMEN
Chromatin insulators are thought to restrict the action of enhancers and silencers. The best-known insulators in Drosophila require proteins such as Suppressor of Hairy wing (Su(Hw)) and Modifier of mdg4 (Mod(mdg4)) to be functional. The insulator-related proteins apparently colocalize as nuclear speckles in immunostained cells. It has been asserted that these speckles are 'insulator bodies' of many Su(Hw)-insulator DNA sites held together by associated proteins, including Mod(mdg4). As we show here using flies, larvae and S2 cells, a mutant Mod(mdg4) protein devoid of the Q-rich domain supports the function of Su(Hw)-dependent insulators and efficiently binds to correct insulator sites on the chromosome, but does not form or enter the Su(Hw)-marked nuclear speckles; conversely, the latter accumulate another (C-truncated) Mod(mdg4) mutant that cannot interact with Su(Hw) or with the genuine insulators. Hence, it is not the functional genomic insulators but rather aggregated proteins that make the so-called 'insulator bodies'.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Elementos Aisladores , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Drosophila/citología , Drosophila/embriología , Drosophila/genética , Embrión no Mamífero , Inmunohistoquímica , Larva , Masculino , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Transgenes , Alas de Animales/embriologíaRESUMEN
In Drosophila melanogaster, broken chromosome ends behave as real telomeres and are believed to be covered with telomere-specific chromatin. It has been shown previously that the telomeric chromatin represses normal activity of enhancers that regulate yellow expression in wings and body cuticle. In this paper, we have found that a modified yellow promoter is fully active in the wing and body cuticle when it is located at the chromosome end, which is evidence that the telomeric chromatin does not repress transcription. Substitution of the yellow core promoter region, including TATA and Inr, with the promoter regions of the eve, hsp70 (TATA-containing), and white (TATA-less) promoters does not affect the ability of the promoter to be cis- or trans-activated by the yellow enhancers if the heterologous promoter is located at a distance of about 6 kb from the chromosome end. The best characterized Drosophila insulator found in the gypsy retrotransposon can specifically repress the yellow promoter at a distance when one component of the insulator complex, Mod(mdg4)-67.2 protein, is inactive. We have also found that, in the mod(mdg4) mutant background, the gypsy insulator can repress the heterologous promoters, indicating that the core promoter elements are not critical for specificity of repression. However, long-distance functional enhancer-promoter and gypsy-promoter interactions were suppressed when the distance between the yellow promoter and the end of the deficient chromosome was less than 6 kb. These results suggest that Drosophila telomeric chromatin does not generally repress transcription but is somehow involved in suppression of some long-distance interactions between regulatory elements.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Retroelementos , Factores de Transcripción/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Southern Blotting , Cromatina/genética , Cromosomas/genética , Cartilla de ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , Genes de Insecto , Genes Reporteros , Elementos Aisladores , Animales , Mapeo Cromosómico , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Regiones Promotoras GenéticasRESUMEN
There is ample evidence that the enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other chromosome where the enhancers are inactive or deleted, which is indicative of a high specificity of the enhancer-promoter interaction in yellow. In this paper, we have found that the yellow sequence from -100 to -69 is essential for stimulation of the heterologous eve (TATA-containing) and white (TATA-less) promoters by the yellow enhancers from a distance. However, the presence of this sequence is not required when the yellow enhancers are directly fused to the heterologous promoters or are activated by the yeast GAL4 activator. Unexpectedly, the same promoter proximal region defines previously described promoter-specific, long-distance repression of the yellow promoter by the gypsy insulator on the mod(mdg4) ( u1 ) background. These finding suggest that proteins bound to the -100 to -69 sequence are essential for communication between the yellow promoter and upstream regulatory elements.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Animales , Cruzamientos Genéticos , Drosophila melanogaster , Proteínas de Insectos/metabolismo , Modelos Genéticos , Modelos Moleculares , Mutación , Pigmentación , Proteínas Recombinantes/genética , Secuencias Reguladoras de Ácidos Nucleicos , Retroelementos , TransgenesRESUMEN
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.
Asunto(s)
Drosophila melanogaster/genética , Elementos de Facilitación Genéticos/fisiología , Elementos Aisladores/fisiología , Regiones Promotoras Genéticas/fisiología , Retroelementos/genética , Animales , Regulación de la Expresión GénicaRESUMEN
In most eukaryotes, telomeres are composed of simple repetitive sequences renewable by telomerase. By contrast, Drosophila telomeres comprise arrays of non-LTR retrotransposons HeT-A, TART, and TAHRE belonging to three different families. However, closer inspection reveals that the two quite different telomere systems share quite a few components and regulatory circuits. Here we present the current knowledge on Drosophila telomeres and discuss the possible mechanisms of telomere length control.