RESUMEN
Compensatory mutations offer clues to deciphering the role of a particular protein in cellular processes. Here we investigate an unknown compensatory mutation, present in the BEAFNP6377 fly line, that provides sufficient rescue of the defective ovary phenotype caused by null BEAF alleles to allow maintenance of fly stocks lacking the chromatin domain insulator proteins Boundary Element-Associated Factors BEAF-32A and BEAF-32B. We call this mutation Tofu. We employ both classical genetics and genomic sequencing to attempt to identify the mutation. We find evidence that points to a mutation in a predicted Polycomb response element upstream of the ribbon gene, which may lead to aberrant rib expression.
RESUMEN
The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) as candidate redundant factors.
Asunto(s)
Proteínas de Drosophila , Elementos Aisladores , Animales , Ensamble y Desensamble de Cromatina , Nucleosomas/genética , Nucleosomas/metabolismo , Nucleasa Microcócica/genética , Nucleasa Microcócica/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Drosophila/genética , Drosophila/metabolismo , Cromatina/genética , Cromatina/metabolismoRESUMEN
Chromatin domain insulators are thought to help partition the genome into genetic units called topologically associating domains (TADs). In Drosophila, TADs are often separated by inter-TAD regions containing active housekeeping genes and associated insulator binding proteins. This raises the question of whether insulator binding proteins are involved primarily in chromosomal TAD architecture or gene activation, or if these two activities are linked. The Boundary Element-Associated Factor of 32 kDa (BEAF-32, or BEAF for short) is usually found in inter-TADs. BEAF was discovered based on binding to the scs' insulator, and is important for the insulator activity of scs' and other BEAF binding sites. There are divergent promoters in scs' with a BEAF binding site by each. Here, we dissect the scs' insulator to identify DNA sequences important for insulator and promoter activity, focusing on the half of scs' with a high affinity BEAF binding site. We find that the BEAF binding site is important for both insulator and promoter activity, as is another sequence we refer to as LS4. Aside from that, different sequences play roles in insulator and promoter activity. So while there is overlap and BEAF is important for both, insulator and promoter activity can be separated.
Asunto(s)
Cromatina/genética , Cromosomas/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , Elementos Aisladores , Regiones Promotoras Genéticas , Animales , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas del Ojo/metabolismo , FemeninoRESUMEN
BEAF (Boundary Element-Associated Factor) was originally identified as a Drosophila melanogaster chromatin domain insulator-binding protein, suggesting a role in gene regulation through chromatin organization and dynamics. Genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, suggesting a role in promoter function. This would be a nontraditional role for an insulator-binding protein. To gain insight into molecular mechanisms of BEAF function, we identified interacting proteins using yeast two-hybrid assays. Here, we focus on the transcription factor Serendipity δ (Sry-δ). Interactions were confirmed in pull-down experiments using bacterially expressed proteins, by bimolecular fluorescence complementation, and in a genetic assay in transgenic flies. Sry-δ interacted with promoter-proximal BEAF both when bound to DNA adjacent to BEAF or > 2-kb upstream to activate a reporter gene in transient transfection experiments. The interaction between BEAF and Sry-δ was detected using both a minimal developmental promoter (y) and a housekeeping promoter (RpS12), while BEAF alone strongly activated the housekeeping promoter. These two functions for BEAF implicate it in playing a direct role in gene regulation at hundreds of BEAF-associated promoters.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/metabolismo , Genes Esenciales , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas del Ojo/genética , Unión Proteica , Proteínas Ribosómicas/genética , Factores de Transcripción/genéticaRESUMEN
An engineered phiC31 "Disintegrase" able to make an attP site in Drosophila out of an attR-attL pair is described. This was used to generate attP sites at genomic locations where a mini-white (mini-w) transgene was subject to chromosomal position effects (CPE). The first step was random genomic integration of a P-element-based transposon with an insulated mini-w transgene. We then removed the upstream insulator using FLP recombinase to detect CPE. Next mini-w and the downstream insulator were "dis-integrated" leaving behind an attP site. The location is marked by a yellow+ transgene that is flanked by loxP sites, so it can also be removed. Using this system, we generated 10 new attP landing platforms. Three of these showing strong activating CPE were selected for further analysis. We show that the attP sites are functional by integrating in plasmids with attB sites. The CPE is recapitulated and can be blocked by insulators. We show that a dimerized 215 bp fragment of the 500 bp BEAF-dependent scs' insulator containing a high affinity BEAF binding site blocks the CPE, while a monomer of the sequence is less effective. This indicates that two BEAF binding sites make a stronger insulator than a single site. This system could be useful for generating attP sites at prescreened sites for other purposes, such as studying CPE in embryos or other tissues or for use with "trapped" enhancers of interest.
Asunto(s)
Sitios de Ligazón Microbiológica , Efectos de la Posición Cromosómica , Drosophila/genética , Ingeniería Genética/métodos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Animales Modificados Genéticamente , Bacteriófagos , Sitios de Unión , Ojo Compuesto de los Artrópodos/metabolismo , Elementos Transponibles de ADN , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Enzimas , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Femenino , GenomaRESUMEN
Chromatin organization is crucial for nuclear functions such as gene regulation, DNA replication and DNA repair. Insulator binding proteins, such as the Drosophila Boundary Element-Associated Factor (BEAF), are involved in chromatin organization. To further understand the role of BEAF, we detected cis- and trans-interaction partners of four BEAF binding regions (viewpoints) using 4C (circular chromosome conformation capture) and analyzed their association with different genomic features. Previous genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, so our viewpoints were selected to reflect this. Our 4C data show the interaction partners of our viewpoints are highly variable and generally enriched for active chromatin marks. The most consistent association was with housekeeping genes, a feature in common with our viewpoints. Fluorescence in situ hybridization indicated that the long-distance interactions occur even in the absence of BEAF. These data are most consistent with a model in which BEAF is redundant with other factors found at active promoters. Our results point to principles of long-distance interactions made by active chromatin, supporting a previously proposed model in which condensed chromatin is sticky and associates into topologically associating domains (TADs) separated by active chromatin. We propose that the highly variable long-distance interactions we detect are driven by redundant factors that open chromatin to promote transcription, combined with active chromatin filling spaces between TADs while packing of TADs relative to each other varies from cell to cell.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Elementos Aisladores/genética , Activación Transcripcional/fisiología , Animales , Sitios de Unión , Cromatina/genética , Cromatina/fisiología , Ensamble y Desensamble de Cromatina/genética , Mapeo Cromosómico , Cromosomas/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas del Ojo/fisiología , Regulación de la Expresión Génica/genética , Genoma de los Insectos , Hibridación Fluorescente in Situ , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN/métodos , Sitio de Iniciación de la TranscripciónRESUMEN
Data implicate the Drosophila 32 kDa Boundary Element-Associated Factors BEAF-32A and BEAF-32B in both chromatin domain insulator element function and promoter function. They might also function as an epigenetic memory by remaining bound to mitotic chromosomes. Both proteins are made from the same gene. They differ in their N-terminal 80 amino acids, which contain single DNA-binding BED fingers. The remaining 200 amino acids are identical in the two proteins. The structure and function of the middle region of 120 amino acids is unknown, while the C-terminal region of 80 amino acids has a putative leucine zipper and a BESS domain and mediates BEAF-BEAF interactions. Here we report a further characterization of BEAF. We show that the BESS domain alone is sufficient to mediate BEAF-BEAF interactions, although the presence of the putative leucine zipper on at least one protein strengthens the interactions. BEAF-32B is sufficient to rescue a null BEAF mutation in flies. Using mutant BEAF-32B rescue transgenes, we show that the middle region and the BESS domain are essential. In contrast, the last 40 amino acids of the middle region, which is poorly conserved among Drosophila species, is dispensable. Deleting the putative leucine zipper results in a hypomorphic mutant BEAF-32B protein. Finally, we document the dynamics of BEAF-32A-EGFP and BEAF-32B-mRFP during mitosis in embryos. A subpopulation of both proteins appears to remain on mitotic chromosomes and also on the mitotic spindle, while much of the fluorescence is dispersed during mitosis. Differences in the dynamics of the two proteins are observed in syncytial embryos, and both proteins show differences between syncytial and later embryos. This characterization of BEAF lays a foundation for future studies into molecular mechanisms of BEAF function.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas del Ojo/genética , Femenino , Técnicas de Inactivación de Genes , Genes de Insecto , Elementos Aisladores , Masculino , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
It was reported that a chromosome with the BEAF(NP6377) (NP6377) allele leads to a loss of cell polarity and neoplastic growth in Drosophila melanogaster when homozygous (Gurudatta et al., 2012). We had previously generated the BEAF(AB-KO) (AB-KO) allele by homologous recombination and did not note these phenotypes (Roy et al., 2007). Both alleles are null mutations. It was unclear why two null alleles of the same gene would give different phenotypes. To resolve this, we performed genetic tests to explore the possibility that the chromosome with the NP6377 allele contained other, second site mutations that might account for the different phenotypes. We found that the chromosome with NP6377 has at least two additional mutations. At least one of these, possibly in combination with the NP6377 allele, is presumably responsible for the reported effects on gene expression, cell polarity and neoplastic growth.
Asunto(s)
Polaridad Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Proteínas del Ojo/genética , Regulación de la Expresión Génica , Genes de Insecto/genética , Elementos Aisladores/genética , Neoplasias/patología , Alelos , Animales , Cromosomas de Insectos/genética , Drosophila melanogaster/genética , Homocigoto , Meiosis/genética , Mutación/genética , Recombinación Genética/genéticaRESUMEN
The Drosophila Suppressor of Hairy-wing [Su(Hw)] protein is a globally expressed, multi-zinc finger (ZnF) DNA-binding protein. Su(Hw) forms a classic insulator when bound to the gypsy retrotransposon and is essential for female germline development. These functions are genetically separable, as exemplified by Su(Hw)(f) that carries a defective ZnF10, causing a loss of insulator but not germline function. Here, we completed the first genome-wide analysis of Su(Hw)-binding sites (SBSs) in the ovary, showing that tissue-specific binding is not responsible for the restricted developmental requirements for Su(Hw). Mapping of ovary Su(Hw)(f) SBSs revealed that female fertility requires binding to only one third of the wild-type sites. We demonstrate that Su(Hw)(f) retention correlates with binding site affinity and partnership with Modifier of (mdg4) 67.2 protein. Finally, we identify clusters of co-regulated ovary genes flanked by Su(Hw)(f) bound sites and show that loss of Su(Hw) has limited effects on transcription of these genes. These data imply that the fertility function of Su(Hw) may not depend upon the demarcation of transcriptional domains. Our studies establish a framework for understanding the germline Su(Hw) function and provide insights into how chromatin occupancy is achieved by multi-ZnF proteins, the most common transcription factor class in metazoans.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/genética , Proteínas Represoras/metabolismo , Animales , Sitios de Unión , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Femenino , Regulación de la Expresión Génica , Genoma de los Insectos , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Oogénesis/genética , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Proteínas Represoras/genética , Dedos de ZincRESUMEN
In a screen based on a rough eye phenotype caused by a dominant negative form of the BEAF-32A and BEAF-32B insulator proteins, we previously identified 17 proteins that genetically interact with BEAF. Eleven of these are developmental transcription factors, seven of which are encoded by the Antennapedia complex (ANT-C). While investigating potential reasons for the genetic interactions, we obtained evidence that BEAF plays a role in the regulation of genes in the ANT-C. BEAF does not localize near the transcription start sites of any genes in the ANT-C, indicating that BEAF does not locally affect regulation of these genes. Although BEAF affects chromatin structure or dynamics, we also found no evidence for a general change in binding to polytene chromosomes in the absence of BEAF. However, because we were unable to detect proteins encoded by ANT-C genes in salivary glands, the DREF and MLE proteins were used as proxies to examine binding. This does not rule out limited effects at particular binding sites or the possibility that BEAF might directly interact with certain transcription factors to affect their binding. In contrast, the embryonic expression levels and patterns of four examined ANT-C genes were altered (bcd, Dfd, ftz, pb). A control gene, Dref, was not affected. A full understanding of the regulation of ANT-C genes during development will have to take the role of BEAF into account.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas del Ojo/metabolismo , Animales , Animales Modificados Genéticamente , Proteína con Homeodominio Antennapedia/metabolismo , Cromosomas de Insectos , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/ultraestructura , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Microscopía Electrónica de RastreoRESUMEN
Gene replacement by homologous recombination is a powerful technique for generating mutations in Drosophila. while using this technique for the BEAF gene, we encountered non-targeted lethal mutations on the target chromosome that complicated the analysis of the BEAF mutations until they were discovered and removed by meiotic recombination. Subsequent experiments indicated that the gene-targeting method leads to a modest but significant three-fold increase in the rate of production of non-targeted lethal mutations. It is important to be aware of this phenomenon when using this method.
Asunto(s)
Drosophila/genética , Marcación de Gen , Mutagénesis , Recombinación Genética , Animales , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas del Ojo/genéticaRESUMEN
Insulator elements play a role in gene regulation that is potentially linked to nuclear organization. Boundary element-associated factors (BEAFs) 32A and 32B associate with hundreds of sites on Drosophila polytene chromosomes. We hybridized DNA isolated by chromatin immunoprecipitation to genome tiling microarrays to construct a genome-wide map of BEAF binding locations. A distinct difference in the association of 32A and 32B with chromatin was noted. We identified 1,820 BEAF peaks and found that more than 85% were less than 300 bp from transcription start sites. Half are between head-to-head gene pairs. BEAF-associated genes are transcriptionally active as judged by the presence of RNA polymerase II, dimethylated histone H3 K4, and the alternative histone H3.3. Forty percent of these genes are also associated with the polymerase negative elongation factor NELF. Like NELF-associated genes, most BEAF-associated genes are highly expressed. Using quantitative reverse transcription-PCR, we found that the expression levels of most BEAF-associated genes decrease in embryos and cultured cells lacking BEAF. These results provide an unexpected link between BEAF and transcription, suggesting that BEAF plays a role in maintaining most associated promoter regions in an environment that facilitates high transcription levels.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas del Ojo , Transcripción Genética , Animales , Sitios de Unión , Factor de Unión a CCCTC , Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica , Genoma de los Insectos , Histonas/metabolismo , Elementos Aisladores , Análisis por Micromatrices , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer-promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest. Here, we combined chromatin immunoprecipitations and computational approaches to break down the binding signature of the Drosophila boundary element-associated factor (BEAF) subfamily. We identify a dual-core BEAF binding signature at 1,720 sites genome-wide, defined by five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. Dual-cores are tightly linked to hundreds of genes highly enriched in cell-cycle and chromosome organization/segregation annotations. siRNA depletion of BEAF from cells leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses in BEAF-depleted cells show that BEAF controls the expression of dual core-associated genes, including key cell-cycle and chromosome segregation regulators. beaf mutants that impair its insulating function by preventing proper interactions of BEAF complexes with the dual-cores produce similar effects in embryos. Chromatin immunoprecipitations show that BEAF regulates transcriptional activity by restricting the deposition of methylated histone H3K9 marks in dual-cores. Our results reveal a novel role for BEAF chromatin dual-cores in regulating a distinct set of genes involved in chromosome organization/segregation and the cell cycle.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Animales , Sitios de Unión , Proteínas de Ciclo Celular/genética , Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Metilación de ADN , Drosophila/genética , Drosophila/metabolismo , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The Drosophila BEAF-32A and BEAF-32B proteins bind to the scs' insulator and to hundreds of other sites on Drosophila chromosomes. These two proteins are encoded by the same gene. We used ends-in homologous recombination to generate the null BEAF(AB-KO) allele and also isolated the BEAF(A-KO) allele that eliminates production of only the BEAF-32A protein. We find that the BEAF proteins together are essential, but BEAF-32B alone is sufficient to obtain viable flies. Our results show that BEAF is important for both oogenesis and development. Maternal or zygotic BEAF is sufficient to obtain adults, although having only maternal BEAF impairs female fertility. In the absence of all BEAF, a few fertile but sickly males are obtained. Using both a chromosomal position-effect assay and an enhancer-blocking assay, we find that BEAF is necessary for scs' insulator function. Lack of BEAF causes a disruption of male X polytene chromosome morphology. However, we did not find evidence that dosage compensation was affected. Position-effect variegation of the w(m4h) allele and different variegating y transgenes was enhanced by the knockout mutation. Combined with the effects on male X polytene chromosomes, we conclude that BEAF function affects chromatin structure or dynamics.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas del Ojo/genética , Mutación de Línea Germinal , Recombinación Genética , Animales , Cromatina/genética , Cromatina/ultraestructura , Femenino , Fertilidad/genética , Genoma , Masculino , Mutagénesis Sitio-Dirigida , Ovario/citología , Ovario/fisiología , Reacción en Cadena de la PolimerasaRESUMEN
Chromatin domain insulators are thought to insulate adjacent genes, including their regulatory elements, from each other by organizing chromatin into functionally independent domains. Thus insulators should play a global role in gene regulation by keeping regulatory domains separated. However, this has never been demonstrated. We previously designed and characterized a transgene that is under GAL4 UAS control and encodes a dominant-negative form of the Boundary Element-Associated Factors BEAF-32A and BEAF-32B. The BID transgene encodes the BEAF self-interaction domain but lacks a DNA binding domain. Expression of BID in eye imaginal discs leads to a rough eye phenotype. Here we screen for dominant mutations that modify this eye phenotype. This assay provides evidence for cross-talk between different classes of insulators, and for a broad role of the BEAF proteins in maintaining patterns of gene expression during eye development. Most identified genes encode other insulator binding proteins, transcription factors involved in head development, or general transcription factors. Because it is unlikely that insulator function is limited to eye development, the present results support the hypothesis that insulators play a widespread role in maintaining global transcription programs.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas del Ojo/genética , Genes de Insecto , Animales , Animales Modificados Genéticamente , Mapeo Cromosómico , Drosophila/crecimiento & desarrollo , Proteínas del Huevo/genética , Ojo/crecimiento & desarrollo , Ojo/ultraestructura , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Femenino , Regulación del Desarrollo de la Expresión Génica , Pruebas Genéticas , Masculino , Microscopía Electrónica de Rastreo , Mutación , Fenotipo , Factores de Transcripción/genéticaRESUMEN
Binding sites for the Drosophila boundary element-associated factors BEAF-32A and -32B are required for the insulator activity of the scs' insulator. BEAF binds to hundreds of sites on polytene chromosomes, indicating that BEAF-utilizing insulators are an important class in Drosophila. To gain insight into the role of BEAF in flies, we designed a transgene encoding a dominant-negative form of BEAF under GAL4 UAS control. This BID protein encompasses the BEAF self-interaction domain. Evidence is provided that BID interacts with BEAF and interferes with scs' insulator activity and that BEAF is the major target of BID in vivo. BID expression during embryogenesis is lethal, implying that BEAF is required during early development. Expression of BID in eye imaginal discs leads to a rough-eye phenotype, and this phenotype is rescued by a third copy of the BEAF gene. Expression of BID in salivary glands leads to a global disruption of polytene chromatin structure, and this disruption is largely rescued by an extra copy of BEAF. BID expression also enhances position-effect variegation (PEV) of the w(m4h) allele and a yellow transgene inserted into the pericentric heterochromatin of chromosome 2R, while a third copy of the BEAF gene suppresses PEV of both genes. These results support the hypothesis that BEAF-dependent insulators function by affecting chromatin structure or dynamics.
Asunto(s)
Cromatina/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Proteínas del Ojo/metabolismo , Elementos Aisladores , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Cromatina/metabolismo , Cromosomas/metabolismo , Proteínas de Unión al ADN/genética , Dimerización , Drosophila/metabolismo , Drosophila/ultraestructura , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Inmunoprecipitación , Elementos Aisladores/genética , Elementos Aisladores/fisiología , Mitosis , Modelos Genéticos , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , TransgenesRESUMEN
Insulators are DNA elements that establish independent transcriptional domains within eukaryotic genomes. The Drosophila scs and scs' insulators localize near the borders of a structural domain in the polytene chromosomes, known as a puff, produced by transcription of the 87A heat shock protein (hsp) genes. It has been suggested that scs and scs' are boundary elements that delimit this decondensed chromatin domain, reflecting the mechanism by which these sequences act to constrain regulatory interactions. This model was tested using transposons that carried a yellow gene to assess enhancer blocking and an hsp70-lacZ gene to examine the structure of a heat shock puff in the presence and absence of insulators. We found that although scs and scs' blocked enhancer function, these sequences did not prevent the spread of decondensation resulting from hsp70-lacZ transcription. Further analysis of the endogenous 87A locus demonstrated that scs and scs' reside within, not at, the borders of the puff. Taken together, our studies suggest that scs and scs' are not boundary elements that block the propagation of an altered chromatin state associated with puff formation. We propose that these insulators may have a direct role in limiting regulatory interactions in the gene-dense 87A region.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromosomas/genética , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Elementos Silenciadores Transcripcionales/genética , Transcripción Genética/genética , Animales , Cromatina/química , Cromatina/metabolismo , Cromosomas/química , Cromosomas/metabolismo , Proteínas de Drosophila/genética , Genes de Insecto/genética , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Conformación de Ácido NucleicoRESUMEN
Polycomb group (PcG) proteins maintain transcriptional repression during development, likely by creating repressive chromatin states. The Extra Sex Combs (ESC) and Enhancer of Zeste [E(Z)] proteins are partners in an essential PcG complex, but its full composition and biochemical activities are not known. A SET domain in E(Z) suggests this complex might methylate histones. We purified an ESC-E(Z) complex from Drosophila embryos and found four major subunits: ESC, E(Z), NURF-55, and the PcG repressor, SU(Z)12. A recombinant complex reconstituted from these four subunits methylates lysine-27 of histone H3. Mutations in the E(Z) SET domain disrupt methyltransferase activity in vitro and HOX gene repression in vivo. These results identify E(Z) as a PcG protein with enzymatic activity and implicate histone methylation in PcG-mediated silencing.
Asunto(s)
Proteínas Cromosómicas no Histona , Proteínas de Drosophila/metabolismo , N-Metiltransferasa de Histona-Lisina , Proteínas de Homeodominio , Metiltransferasas/metabolismo , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción , Animales , Proteínas de Unión al ADN/genética , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Silenciador del Gen , Histona Metiltransferasas , Proteínas de Insectos/aislamiento & purificación , Lisina/metabolismo , Proteínas Nucleares/aislamiento & purificación , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteína Metiltransferasas , Recombinación Genética , Proteínas Represoras/aislamiento & purificación , Proteína 4 de Unión a RetinoblastomaRESUMEN
The insulating properties required to delimit higher-order chromosomal domains have been shown to be shared by a variety of chromatin boundary elements (BEs). Boundary elements have been described in several species, from yeast to human, and we have previously reported the existence of a class of chromatin BEs in Drosophila melanogaster whose insulating activity requires the DNA-binding protein BEAF (boundary element-associated factor). Here we focus on the characterization of a moderately repeated 1.2 kb DNA sequence that encompasses boundary element 28 (BE28). We show that it directionally blocks enhancer/promoter communication in transgenic flies. This sequence contains a BEAF-binding sequence juxtaposed to an AT-rich sequence that harbors a strong nuclease-hypersensitive site. Using a combination of DNA-protein and protein blotting techniques, we found that this region is recognized by the A+T-binding D1 non-histone chromosomal protein of D. melanogaster, and we provide evidence that D1 and BEAF physically interact. In addition, the multicopy BE28 element maps to pericentric regions of the D. melanogaster 2L, 2R and X chromosome arms to which D1 has been shown to localize. In yeast, BEs that mark the periphery of silenced chromosomal domains have recently been shown to block the spreading of heterochromatin assembly. We propose that the BE28 repeat clusters could fulfill a similar function, acting as a local boundary between hetero- and euchromatin in a process involving interactions between the BEAF and D1 proteins.