Distribution of insulator proteins CP190, dCTCF, Su(Hw) and Mod(mdg4)2.2 in Drosophila S2 cells in the presence or absence of dPARP inhibitor 3AB
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE41354 | GEO | 2013/09/15
SECONDARY ACCESSION(S): PRJNA176855
REPOSITORIES: GEO
Similar Datasets
Project description:We mapped the localization of insulator proteins in Drosophila S2 cells in the presence or absence of 12mM of 3AB. We found that inhibition of PARylation affects DNA binding of insulator proteins only at a small subsets of genomic sites. Examination of genomic occupancy for insulator proteins in S2 cells with or without 3AB inhibition.
2013-09-15 | E-GEOD-41354 | biostudies-arrayexpress
Project description: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.
| S-EPMC5825117 | biostudies-literature
Project description:Chromatin insulators are DNA-protein complexes that establish higher order independent DNA domains to influence transcriptional regulation. Insulators are defined by two different functions: 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 depleted by RNAi, gypsy-dependent enhancer blocking activity decreases and barrier activity is reduced in all tissues. Furthermore, Clamp RNAi knockdowns and mutation result in disorganized insulator complex localization in the nucleus. Co-immunoprecipitation experiments showed that CLAMP physically associates with core gypsy-insulator proteins. Co-localization of CLAMP with gypsy components on polytene chromosomes and ChIP-seq analysis demonstrates co-localization of CLAMP with a subset of insulator sites across the genome. Thus, our findings suggest a ubiquitous, genome-wide role for CLAMP in promoting gypsy-dependent chromatin insulator activity.
2019-01-28 | GSE103601 | GEO
Project description: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 (Hipp?1) 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.
| S-EPMC6911044 | biostudies-literature
Project description: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.
| S-EPMC5666082 | biostudies-literature
Project description:Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here we utilized RNA-affinity pull down coupled with mass spectrometry to identify a novel RNA-binding protein, Isha, that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif (RRM) and RNA Polymerase II CTD-interacting domain (CID). We found that Isha physically interacts with total and elongating RNA Pol II and associates with chromatin at the 5’ end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with gypsy factor CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.
2022-05-26 | GSE200994 | GEO
Project description:Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here we utilized RNA-affinity pull down coupled with mass spectrometry to identify a novel RNA-binding protein, Isha, that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif (RRM) and RNA Polymerase II CTD-interacting domain (CID). We found that Isha physically interacts with total and elongating RNA Pol II and associates with chromatin at the 5’ end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with gypsy factor CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.
2022-05-26 | GSE200993 | GEO
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:modENCODE_submission_3670 This submission comes from a modENCODE project of Gary Karpen. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: The goal of these experiments are to validate and confirm the locations of 125 chromosomal proteins across the Drosophila melanogaster genome. To do this, we are using RNAi to deplete individual non-histone chromosomal proteins in Drosophila BG3 and S2 tissue culture cells, and then using antibodies to perform Chromatin ImmunoPrecipitation (ChIP) using genomic tiling arrays. Comparison of a protein factor's binding profiles before and after depletion will increase the confidence of our predictions. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
2011-10-13 | GSE32780 | GEO
Project description:Insulators are DNA elements that divide chromosomes into independent transcriptional domains. The Drosophila genome contains hundreds of binding sites for the Suppressor of Hairy-wing [Su(Hw)] insulator protein, corresponding to locations of the retroviral gypsy insulator and non-gypsy binding regions (BRs). The first non-gypsy BR identified, 1A-2, resides in cytological region 1A. Using a quantitative transgene system, we show that 1A-2 is a composite insulator containing enhancer blocking and facilitator elements. We discovered that 1A-2 separates the yellow (y) gene from a previously unannotated, non-coding RNA gene, named yar for y-achaete (ac) intergenic RNA. The role of 1A-2 was elucidated using homologous recombination to excise these sequences from the natural location, representing the first deletion of any Su(Hw) BR in the genome. Loss of 1A-2 reduced yar RNA accumulation, without affecting mRNA levels from the neighboring y and ac genes. These data indicate that within the 1A region, 1A-2 acts an activator of yar transcription. Taken together, these studies reveal that the properties of 1A-2 are context-dependent, as this element has both insulator and enhancer activities. These findings imply that the function of non-gypsy Su(Hw) BRs depends on the genomic environment, predicting that Su(Hw) BRs represent a diverse collection of genomic regulatory elements.
| S-EPMC2493044 | biostudies-literature