Suppressor of hairy-wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi.
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ABSTRACT:
SUBMITTER: Carballar-Lejarazu R
PROVIDER: S-EPMC4935592 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Malaria parasites are transmitted to humans by mosquitoes of the genus Anopheles, and these insects are the targets of innovative vector control programs. Proposed approaches include the use of genetic strategies based on transgenic mosquitoes to suppress or modify vector populations. Although substantial advances have been made in engineering resistant mosquito strains, limited efforts have been made in refining mosquito transgene expression, in particular attenuating the effects of insertions sites, which can result in variations in phenotypes and impacts on fitness due to the random integration of transposon constructs. A promising strategy to mitigate position effects is the identification of insulator or boundary DNA elements that could be used to isolate transgenes from the effects of their genomic environment. We applied quantitative approaches that show that exogenous insulator-like DNA derived from the Drosophila melanogaster gypsy retrotransposon can increase and stabilize transgene expression in transposon-mediated random insertions and recombinase-catalyzed, site-specific integrations in the malaria vector mosquito, Anopheles stephensi. These sequences can contribute to precise expression of transgenes in mosquitoes engineered for both basic and applied goals.
| S-EPMC3645527 | biostudies-literature
Project description:The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. Here we investigate the function of Su(Hw) in the ovary. We show that Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, we demonstrate that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190kDa (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions.
| S-EPMC3143288 | biostudies-literature
Project description:Suppressor of Hairy-wing [Su(Hw)] is a DNA-binding factor required for gypsy insulator function and female germline development in Drosophila. The insulator function of the gypsy retrotransposon depends on Su(Hw) binding to clustered Su(Hw) binding sites (SBSs) and recruitment of the insulator proteins Centrosomal Protein 190 kD (CP190) and Modifier of mdg4 67.2 kD (Mod67.2). By contrast, the Su(Hw) germline function involves binding to non-clustered SBSs and does not require CP190 or Mod67.2. Here, we identify Su(Hw) target genes, using genome-wide analyses in the ovary to uncover genes with an ovary-bound SBS that are misregulated upon Su(Hw) loss. Most Su(Hw) target genes demonstrate enriched expression in the wild-type CNS. Loss of Su(Hw) leads to increased expression of these CNS-enriched target genes in the ovary and other tissues, suggesting that Su(Hw) is a repressor of neural genes in non-neural tissues. Among the Su(Hw) target genes is RNA-binding protein 9 (Rbp9), a member of the ELAV/Hu gene family. Su(Hw) regulation of Rbp9 appears to be insulator independent, as Rbp9 expression is unchanged in a genetic background that compromises the functions of the CP190 and Mod67.2 insulator proteins, even though both localize to Rbp9 SBSs. Rbp9 misregulation is central to su(Hw)(-/-) sterility, as Rbp9(+/-), su(Hw)(-/-) females are fertile. Eggs produced by Rbp9(+/-), su(Hw)(-/-) females show patterning defects, revealing a somatic requirement for Su(Hw) in the ovary. Our studies demonstrate that Su(Hw) is a versatile transcriptional regulatory protein with an essential developmental function involving transcriptional repression.
| S-EPMC3742144 | biostudies-literature
Project description:Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.
| S-EPMC4981481 | biostudies-literature
Project description:For the compact Drosophila genome, several factors mediating insulator function, such as su(Hw) and dCTCF, have been identified. Recent analyses showed that both these insulator-binding factors are functionally dependent on the same cofactor, CP190. Here we analysed genome-wide binding of CP190 and dCTCF. CP190 binding was detected at CTCF, su(Hw) and GAF sites and unexpectedly at the transcriptional start sites of actively transcribed genes. Both insulator and transcription start site CP190-binding elements are strictly marked by a depletion of histone H3 and, therefore, a loss of nucleosome occupancy. In addition, CP190/dCTCF double occupancy was seen at the borders of many H3K27me3 'islands'. As before, these sites were also depleted of H3. Loss of either dCTCF or CP190 causes an increase of H3 and H3K27 trimethylation at these sites. Thus, for both types of cis-regulatory elements, domain borders and promoters, the chromatin structure is dependent on CP190.
| S-EPMC2670862 | biostudies-literature
Project description:Chromatin insulators or boundaries are proposed to structure the chromatin fiber into functionally independent domains by promoting the formation of chromatin loops. These elements can block the communication between an enhancer and a gene when placed between them. Interestingly, it has been previously observed that two tandem copies of the Drosophila Su(Hw) insulator abolish this enhancer-blocking activity, presumably through pairing. This bypass effect has not been described with other insulators, however. In this report, we show that the insertion of binding sites for the GAGA factor (GAF) between an enhancer and the Su(Hw) insulator allows bypassing of the insulator. This bypass relies on the activity of both the GAF protein and the Mod(mdg4)-67.2 protein, a factor required for Su(Hw) insulator activity. We show that these two proteins interact in vitro and in vivo, providing molecular evidence of pairing between the GAF sites and the Su(Hw) insulator. Finally, we show that placing the Mcp boundary together with the Su(Hw) insulator between an enhancer and a promoter leads to bypass, again in a GAF- and Mod(mdg4)-dependent manner. Our data provide direct evidence that heterologous insulators can be bypassed by distal enhancers and identify the interaction between GAF and Mod(mdg4) as a possible means to regulate insulator activity.
| S-EPMC522021 | biostudies-literature
Project description: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 their normal localization is correlated with proper 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) are also required for recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy binding site DNA, 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.
2022-10-14 | PXD032165 | Pride
Project description:Insulators define chromosomal domains such that an enhancer in one domain cannot activate a promoter in a different domain. We show that the Drosophila gypsy insulator behaves as a cis-stimulatory element in the larval fat body. Transcriptional stimulation by the insulator is distance dependent, as expected for a promoter element as opposed to an enhancer. Stimulation of a test alcohol dehydrogenase promoter requires a binding site for a GATA transcription factor, suggesting that the insulator may be facilitating access of this DNA binding protein to the promoter. Short-range stimulation requires both the Suppressor of Hairy-wing protein and the Mod(mdg4)-62.7 protein encoded by the trithorax group gene mod(mdg4). In the absence of interaction with Mod(mdg4)-62.7, the insulator is converted into a short-range transcriptional repressor but retains some cis-stimulatory activity over longer distances. These results indicate that insulator and promoter sequences share important characteristics and are not entirely distinct. We propose that the gypsy insulator can function as a promoter element and may be analogous to promoter-proximal regulatory modules that integrate input from multiple distal enhancer sequences.
| S-EPMC99942 | biostudies-literature
Project description: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 their normal localization is correlated with proper 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) are also required for recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy binding site DNA, 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.
2022-06-28 | GSE198361 | GEO
Project description:Chromatin insulators are DNA-protein complexes that establish independent higher-order DNA domains to influence transcription. Insulators are functionally defined by two properties: they can block communication between an enhancer and a promoter, and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy insulator complex contains three core components; Su(Hw), CP190 and Mod(mdg4)67.2. Here, we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When clamp is knocked down, gypsy-dependent enhancer-blocking and barrier activities are strongly reduced. CLAMP associates physically with the core gypsy insulator complex, and ChIP-seq analysis reveals extensive overlap, particularly with promoter-bound CP190 on chromatin. Depletion of CLAMP disrupts CP190 binding at a minority of shared sites, whereas depletion of CP190 results in extensive loss of CLAMP chromatin association. Finally, reduction of CLAMP disrupts CP190 localization within the nucleus. Our results support a positive functional relationship between CLAMP and CP190 to promote gypsy chromatin insulator activity.
| S-EPMC6432716 | biostudies-literature