RESUMO
The article presents an innovative approach for detecting defects in insulators used in high-voltage power transmission lines, employing an enhanced Detection Transformer (DETR) model, termed IF-DETR. The study addresses the significant challenges in traditional insulator defect detection methods, such as the loss of small defect features and confusion with background features. Firstly, we propose a multi-scale backbone network to better extract features of small objects. Secondly, as the contextual information surrounding objects plays a critical role in detecting small objects, we introduce a fusion module composed of ECA-Net and SAU to replace the original attention module for improved contextual information extraction. Lastly, we introduce the insulator defect (IDIoU) loss to optimize the instability in the matching process caused by small defects. Extensive experiments demonstrate the model's effectiveness, particularly in detecting small defects, marking a notable advancement in insulator defect detection technology. The IF-DETR achieved a 2.3% increase in AP compared to existing advanced methods. This method not only enhances the accuracy of defect detection, crucial for maintaining the reliability and safety of power transmission systems but also has broader implications for the maintenance and inspection of high-voltage power infrastructure.
Assuntos
Elementos Isolantes , Elementos Isolantes/genética , Reprodutibilidade dos Testes , Algoritmos , Fontes de Energia Elétrica , Modelos TeóricosRESUMO
A key question in regulatory genomics is whether cis-regulatory elements (CREs) are modular elements that can function anywhere in the genome, or whether they are adapted to certain genomic locations. To distinguish between these possibilities we develop MPIRE (Massively Parallel Integrated Regulatory Elements), a technology for recurrently assaying CREs at thousands of defined locations across the genome in parallel. MPIRE allows us to separate the intrinsic activity of CREs from the effects of their genomic environments. We apply MPIRE to assay three insulator sequences at thousands of genomic locations and find that each insulator functions in locations with distinguishable properties. All three insulators can block enhancers, but each insulator blocks specific enhancers at specific locations. However, only ALOXE3 appears to block heterochromatin silencing. We conclude that insulator function is highly context dependent and that MPIRE is a robust method for revealing the context dependencies of CREs.
Assuntos
Elementos Facilitadores Genéticos , Elementos Isolantes , Elementos Isolantes/genética , Elementos Facilitadores Genéticos/genética , Animais , Genoma/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Genômica/métodos , Camundongos , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Topologically associating domain (TAD) boundaries are the flanking edges of TADs, also known as insulated neighborhoods, within the 3D structure of genomes. A prominent feature of TAD boundaries in mammalian genomes is the enrichment of clustered CTCF sites often with mixed orientations, which can either block or facilitate enhancer-promoter (E-P) interactions within or across distinct TADs, respectively. We will discuss recent progress in the understanding of fundamental organizing principles of the clustered CTCF insulator codes at TAD boundaries. Specifically, both inward- and outward-oriented CTCF sites function as topological chromatin insulators by asymmetrically blocking improper TAD-boundary-crossing cohesin loop extrusion. In addition, boundary stacking and enhancer clustering facilitate long-distance E-P interactions across multiple TADs. Finally, we provide a unified mechanism for RNA-mediated TAD boundary function via R-loop formation for both insulation and facilitation. This mechanism of TAD boundary formation and insulation has interesting implications not only on how the 3D genome folds in the Euclidean nuclear space but also on how the specificity of E-P interactions is developmentally regulated.
Assuntos
Fator de Ligação a CCCTC , Cromatina , Elementos Isolantes , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Humanos , Elementos Isolantes/genética , Animais , Cromatina/genética , Cromatina/metabolismo , Genoma/genética , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Coesinas , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genéticaRESUMO
While the elements encoding enhancers and promoters have been relatively well studied, the full spectrum of insulator elements which bind the CCCTC binding factor (CTCF), is relatively poorly characterized. This is partly due to the genomic context of CTCF sites greatly influencing their roles and activity. Here we have developed an experimental system to determine the ability of minimal, consistently sized, individual CTCF elements to interpose between enhancers and promoters and thereby reduce gene expression during differentiation. Importantly, each element is tested in the identical location thereby minimising the effect of genomic context. We found no correlation between the ability of CTCF elements to block enhancer-promoter activity with the degree of evolutionary conservation; their resemblance to the consensus core sequences; or the number of CTCF core motifs harboured in the element. Nevertheless, we have shown that the strongest enhancer-promoter blockers include a previously described bound element lying upstream of the CTCF core motif. In addition, we found other uncharacterised DNaseI footprints located close to the core motif that may affect function. We have developed an assay of CTCF sequences which will enable researchers to sub-classify individual CTCF elements in a uniform and unbiased way.
Assuntos
Fator de Ligação a CCCTC , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Sítios de Ligação/genética , Humanos , Animais , Camundongos , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Elementos Isolantes/genética , Ligação Proteica , Motivos de Nucleotídeos , Linhagem Celular , Regulação da Expressão Gênica , Diferenciação Celular/genéticaRESUMO
The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow some physical crosstalk across regulatory domains, which is at odds with the specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites cooperatively foster the robust insulation of regulatory domains. By genetically dissecting a couple of representative loci in mouse embryonic stem cells, we show that CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), while developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the insulation of regulatory domains, which in turn might help interpreting the pathological consequences of certain structural variants.
Assuntos
Fator de Ligação a CCCTC , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas , Regiões Promotoras Genéticas , Animais , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Camundongos , Regiões Promotoras Genéticas/genética , Elementos Facilitadores Genéticos/genética , Células-Tronco Embrionárias Murinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Elementos Isolantes/genéticaRESUMO
In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription.
Assuntos
Cromatina , Inativação Gênica , Retroelementos , Saccharomyces cerevisiae , Retroelementos/genética , Cromatina/genética , Cromatina/metabolismo , Saccharomyces cerevisiae/genética , Elementos Isolantes/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequências Repetidas Terminais/genética , Regulação Fúngica da Expressão Gênica , Transcrição Gênica , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismoRESUMO
Transvection is a phenomenon of interallelic communication in which enhancers can activate a specific promoter located on a homologous chromosome. Insulators play a significant role in ensuring functional interactions between enhancers and promoters. In the presented work, we created a model where two or three copies of the insulator are located next to enhancers and promoters localized on homologous chromosomes. Using the Su(Hw) insulator as a model, we showed that the functional interaction between a pair of insulators promotes enhancer-promoter trans-interactions. The interaction between the three insulators, on the contrary, can lead to the formation of chromatin loops that sterically hinder the full enhancer-promoter interaction. The results of the work suggest the participation of insulators in the regulation of homologous chromosome pairing and in communication between distant genomic loci.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Elementos Facilitadores Genéticos , Elementos Isolantes , Regiões Promotoras Genéticas , Animais , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Elementos Isolantes/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Cromatina/metabolismo , Cromatina/genéticaRESUMO
Chromatin insulators are DNA-protein complexes that promote specificity of enhancer-promoter interactions and maintain distinct transcriptional states through control of 3D genome organization. In this review, we highlight recent work visualizing how mammalian CCCTC-binding factor acts as a boundary to dynamic DNA loop extrusion mediated by cohesin. We also discuss new studies in both mammals and Drosophila that elucidate biological redundancy of chromatin insulator function and interplay with transcription with respect to topologically associating domain formation. Finally, we present novel concepts in spatiotemporal regulation of chromatin insulator function during differentiation and development and possible consequences of disrupted insulator activity on cellular proliferation.
Assuntos
Cromatina , Genoma , Elementos Isolantes , Animais , Cromatina/genética , Cromatina/metabolismo , Elementos Isolantes/genética , Genoma/genética , Humanos , Coesinas , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas , Drosophila/genética , Drosophila/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genéticaRESUMO
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
In Drosophila, a group of zinc finger architectural proteins recruits the CP190 protein to the chromatin, an interaction that is essential for the functional activity of promoters and insulators. In this study, we describe a new architectural C2H2 protein called Madf and Zinc-Finger Protein 1 (Mzfp1) that interacts with CP190. Mzfp1 has an unusual structure that includes six C2H2 domains organized in a C-terminal cluster and two tandem MADF domains. Mzfp1 predominantly binds to housekeeping gene promoters located in both euchromatin and heterochromatin genome regions. In vivo mutagenesis studies showed that Mzfp1 is an essential protein, and both MADF domains and the CP190 interaction region are required for its functional activity. The C2H2 cluster is sufficient for the specific binding of Mzfp1 to regulatory elements, while the second MADF domain is required for Mzfp1 recruitment to heterochromatin. Mzfp1 binds to the proximal part of the Fub boundary that separates regulatory domains of the Ubx and abd-A genes in the Bithorax complex. Mzfp1 participates in Fub functions in cooperation with the architectural proteins Pita and Su(Hw). Thus, Mzfp1 is a new architectural C2H2 protein involved in the organization of active promoters and insulators in Drosophila.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Elementos Isolantes , Proteínas Nucleares , Regiões Promotoras Genéticas , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Elementos Isolantes/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Genes Essenciais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ligação Proteica , Regulação da Expressão Gênica , Eucromatina/metabolismo , Eucromatina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Associadas aos MicrotúbulosRESUMO
Although promoters and their enhancers are frequently contained within a topologically associating domain (TAD), some developmentally important genes have their promoter and enhancers within different TADs. Hypotheses about molecular mechanisms enabling cross-TAD interactions remain to be assessed. To test these hypotheses, we used optical reconstruction of chromatin architecture to characterize the conformations of the Pitx1 locus on single chromosomes in developing mouse limbs. Our data support a model in which neighboring boundaries are stacked as a result of loop extrusion, bringing boundary-proximal cis-elements into contact. This stacking interaction also contributes to the appearance of architectural stripes in the population average maps. Through molecular dynamics simulations, we found that increasing boundary strengths facilitates the formation of the stacked boundary conformation, counter-intuitively facilitating border bypass. This work provides a revised view of the TAD borders' function, both facilitating and preventing cis-regulatory interactions, and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Animais , Camundongos , Elementos Facilitadores Genéticos/genética , Cromatina/genética , Cromossomos , Regiões Promotoras Genéticas/genética , Elementos IsolantesRESUMO
During the past decades, tremendous advances have occurred in manufacturing recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. Nevertheless, the production of stable high-producing cell lines has remained a major obstacle in the development process of the CHO cell line. It has been shown that genomic regulatory elements can promote cell line development efficiency by improving transgenes' productivity and stability. Such elements include insulators, ubiquitous chromatin opening elements, scaffold/matrix attachment regions, and antirepressors. In addition, tDNA elements are shown to act as insulators in mammalian cells. This study examines the effect of the tDNA insulator on stable expression of a vascular endothelial growth factor receptor-Fc fusion protein.
Assuntos
Elementos Isolantes , Fator A de Crescimento do Endotélio Vascular , Animais , Cricetinae , Células CHO , Cricetulus , Anticorpos Monoclonais , Receptores de Fatores de Crescimento do Endotélio VascularRESUMO
CP190 protein is one of the key components of Drosophila insulator complexes, and its study is important for understanding the mechanisms of gene regulation during cell differentiation. However, Cp190 mutants die before reaching adulthood, which significantly complicates the study of its functions in imago. To overcome this problem and to investigate the regulatory effects of CP190 in adult tissues development, we have designed a conditional rescue system for Cp190 mutants. Using Cre/loxP-mediated recombination, the rescue construct containing Cp190 coding sequence is effectively eliminated specifically in spermatocytes, allowing us to study the effect of the mutation in male germ cells. Using high-throughput transcriptome analysis we determined the function of CP190 on gene expression in germline cells. Cp190 mutation was found to have opposite effects on tissue-specific genes, which expression is repressed by CP190, and housekeeping genes, that require CP190 for activation. Mutation of Cp190 also promoted expression of a set of spermatocyte differentiation genes that are regulated by tMAC transcriptional complex. Our results indicate that the main function of CP190 in the process of spermatogenesis is the coordination of interactions between differentiation genes and their specific transcriptional activators.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Espermatócitos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/genética , Proteínas Associadas aos Microtúbulos/genética , Drosophila/genética , Diferenciação Celular/genética , Elementos IsolantesRESUMO
Drosophila insulators were the first DNA elements found to regulate gene expression by delimiting chromatin contacts. We still do not know how many of them exist and what impact they have on the Drosophila genome folding. Contrary to vertebrates, there is no evidence that fly insulators block cohesin-mediated chromatin loop extrusion. Therefore, their mechanism of action remains uncertain. To bridge these gaps, we mapped chromatin contacts in Drosophila cells lacking the key insulator proteins CTCF and Cp190. With this approach, we found hundreds of insulator elements. Their study indicates that Drosophila insulators play a minor role in the overall genome folding but affect chromatin contacts locally at many loci. Our observations argue that Cp190 promotes cobinding of other insulator proteins and that the model, where Drosophila insulators block chromatin contacts by forming loops, needs revision. Our insulator catalog provides an important resource to study mechanisms of genome folding.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos Isolantes/genética , Proteínas Nucleares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismoRESUMO
Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). Here, we demonstrate an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components su(Hw) and Cp190 results in a significant reduction in γH2Av levels in chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av, but not H2Av, is a component of insulator bodies, which are protein condensates that form during osmotic stress. Phosphatase activity is required for insulator body dissolution after stress recovery. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Cromatina/genética , Cromatina/metabolismo , DNA/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Histonas/genética , Histonas/metabolismo , Elementos Isolantes/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Monoéster Fosfórico Hidrolases/genética , Cromossomos Politênicos/genéticaRESUMO
Insulators in vertebrates play a role in genome architecture and orchestrate temporo-spatial enhancer-promoter interactions. In plants, insulators and their associated binding factors have not been documented as of yet, largely as a result of a lack of characterized insulators. In this study, we took a comprehensive strategy to identify and validate the enhancer-blocking insulator CW198. We show that a 1.08-kb CW198 fragment from Arabidopsis can, when interposed between an enhancer and a promoter, efficiently abrogate the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and tobacco plants. In plants, both transcriptional crosstalk and spreading of histone modifications were rarely detectable across CW198, which resembles the insulation property observed across the CTCF insulator in the mammalian genome. Taken together, our findings support that CW198 acts as an enhancer-blocking insulator in both Arabidopsis and tobacco. The significance of the present findings and their relevance to the mitigation of mutual interference between enhancers and promoters, as well as multiple promoters in transgenes, is discussed.
Assuntos
Arabidopsis , Elementos Isolantes , Animais , Elementos Isolantes/genética , Elementos Facilitadores Genéticos/genética , Arabidopsis/genética , Regiões Promotoras Genéticas/genética , Transgenes/genética , Nicotiana/genética , Mamíferos/genéticaRESUMO
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.
Assuntos
Proteínas de Drosophila , Elementos Isolantes , Animais , Montagem e Desmontagem da Cromatina , Nucleossomos/genética , Nucleossomos/metabolismo , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Drosophila/genética , Drosophila/metabolismo , Cromatina/genética , Cromatina/metabolismoRESUMO
CTCF is the most thoroughly studied chromatin architectural protein and it is found in both Drosophila and mammals. CTCF preferentially binds to promoters and insulators and is thought to facilitate formation of chromatin loops. In a subset of sites, CTCF binding depends on the epigenetic status of the surrounding chromatin. One such variable CTCF site (vCTCF) was found in the intron of the Ubx gene, in close proximity to the BRE and abx enhancers. CTCF binds to the variable site in tissues where Ubx gene is active, suggesting that the vCTCF site plays a role in facilitating contacts between the Ubx promoter and its enhancers. Using CRISPR/Cas9 and attP/attB site-specific integration methods, we investigated the functional role of vCTCF and showed that it is not required for normal Drosophila development. Furthermore, a 2161-bp fragment containing vCTCF does not function as an effective insulator when substituted for the Fab-7 boundary in the Bithorax complex. Our results suggest that vCTCF function is redundant in the regulation of Ubx.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Cromatina/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/metabolismo , Elementos Isolantes/genética , Mamíferos/genética , Mamíferos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.