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Context differences reveal insulator and activator functions of a Su(Hw) binding region.

ABSTRACT: 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.

SUBMITTER: Soshnev AA

PROVIDER: S-EPMC2493044 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Isha is a su(Hw) mRNA-binding protein required for gypsy insulator function.

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 pulldown coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif and RNA Polymerase II C-terminal domain-interacting domain (CID). We found that Isha physically interacts with total and elongating Polymerase 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 the core gypsy insulator component 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 Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.

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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.

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Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome.

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Isha is a su(Hw) mRNA-binding protein required for insulator function

Project description:This SuperSeries is composed of the SubSeries listed below.

2022-05-26 | GSE200995 | GEO

Isha is a su(Hw) mRNA-binding protein required for insulator function [RIP-seq]

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

Isha is a su(Hw) mRNA-binding protein required for insulator function [ChIP-seq]

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

SU(HW)-HB.BG3.SU(HW)_RNAi

Project description:modENCODE_submission_3718 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 | GSE32812 | GEO

The su(Hw) insulator can disrupt enhancer-promoter interactions when located more than 20 kilobases away from the Drosophila achaete-scute complex.

Project description:Here we report that the su(Hw) insulator may not necessarily separate promoters from enhancers to allow inhibition of transcription by the su(Hw) protein. For this purpose we used the strains of Drosophila melanogaster which carry inversion of the region containing the yellow gene and the achaete-scute complex (AS-C). Despite the reverse orientation of the region, the AS-C enhancers continue to activate achaete and scute gene expression. The su(Hw) insulator, located more than 20 kb away from the inversion, facilitates strong suppression of achaete and scute gene expression, although is does not separate the promoters from the AS-C enhancers.

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Role of Su(Hw) zinc finger 10 and interaction with CP190 and Mod(mdg4) proteins in recruiting the Su(Hw) complex to chromatin sites in Drosophila.

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.

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