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Role of Su(Hw) in DNA amplification in Drosophila ovaries

ABSTRACT: Suppressor of Hairy wing (Su(Hw)) is an insulator protein that participates in regulating chromatin architecture and gene repression in Drosophila. In previous studies we have shown that Su(Hw) is also required for pre-replication complex (pre-RC) recruitment on Su(Hw)-bound sites (SBSs) in Drosophila S2 cells and pupa. Here, we describe the effect of Su(Hw) on developmentally regulated amplification of 66D and 7F Drosophila amplicons in follicle cells (DAFCs), widely used as models in replication studies. We show Su(Hw) binding co-localizes with all known DAFCs in Drosophila ovaries, whereas disruption of Su(Hw) binding to 66D and 7F DAFCs causes a two-fold decrease in the amplification of these loci. The complete loss of Su(Hw) binding to chromatin violates pre-RC recruitment to all amplification regulatory regions of 66D and 7F loci at early oogenesis (prior to DAFCs amplification). These changes coincide with a considerable Su(Hw)-dependent condensation of chromatin at 66D and 7F loci. Although we observed the Brm, ISWI, Mi-2, and CHD1 chromatin remodelers at SBSs genome wide, their remodeler activity does not appear to be responsible for chromatin decondensation at the 66D and 7F amplification regulatory regions. We have discovered that, in addition to the CBP/Nejire and Chameau histone acetyltransferases, the Gcn5 acetyltransferase binds to 66D and 7F DAFCs at SBSs and is dependent on Su(Hw). We propose that the main function of Su(Hw) in developmental amplification of 66D and 7F DAFCs is to establish a chromatin structure that is permissive to pre-RC recruitment.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE168894 | GEO | 2021/08/22

REPOSITORIES: GEO

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NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization

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.

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Expression analyses in Drosophila ovaries

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Gene expression analyses from 3-day testes of D. melanogaster

Project description:Suppressor of Hairy-wing [Su(Hw)] is a globally expressed twelve-zinc finger transcription factor in Drosophila. Su(Hw) is well-known for its role in establishing the gypsy insulator within the gypsy retrotransposon. A direct role of Su(Hw) in gene activation and repression was uncovered through the study in the Drosophila oogenesis, revealing that Su(Hw)-dependent repression of neuronal genes is essential for egg production. Motivated from the spatially restricted expression of Su(Hw) in the testis, we investigated the role of Su(Hw) in spermatogenesis. We discovered that Su(Hw) is required for sperm development and male fertility. Data presented here examined the transcriptional role of Su(Hw) in the Drosophila testis. These studies identified ~400 genes with altered expression in the absence of Su(Hw). Coupled with whole-genome Su(Hw) binding profiles, we identified 145 direct targets of Su(Hw) regulation, of which ~80% show increased transcription upon Su(Hw) loss. Upregulated Su(Hw) targets are enriched for neuronal expression, emphasizing that Su(Hw) represents a functional homolog of mammalian REST that represses neuronal gene expression in non-neuronal tissues.

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Expression analyses in Drosophila ovaries

Project description:Suppressor of Hairy-wing [Su(Hw)] is a multi-zinc finger DNA binding factor required for gypsy insulator function and female germline development in Drosophila. The enhancer-blocking and barrier functions of the gypsy retrotransposon involve Su(Hw) binding to twelve clustered Su(Hw) binding sites (SBSs) and recruitment of the Centrosomal Protein of 190 kD (CP190) and Modifier of mdg4 67.2 kD isoform (Mod67.2) insulator proteins. In contrast, the Su(Hw) germline function involves binding to non-clustered genomic SBSs and does not require CP190 or Mod67.2. Here, we use genome-wide expression analyses in the ovary to identify the first Su(Hw) regulated target genes. Ovaries for RNA isolation were dissected from 4-6 hour old virgin females of wild type (Canton S and BL15598) and su(Hw) null sterile mutants (su(Hw)A2663/v and su(Hw)Pb/2) Drosophila melanogaster. At this stage of development, ovaries only contain egg chamber stages 1-8. Loss of Su(Hw) causes apoptosis at stage 9. Thus, the experimental design compares transcriptional changes in the ovary prior to induction nof apoptosis in su(Hw) mutants.

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in vivo binding of su(Hw) in the Drosophila genome

Project description:There is considerable evidence that insulator elements are likely to play a key role in the organisation of the regulatory architecture of the genome. In Drosophila, one of the best studied insulator elements is the gypsy insulator in the gypsy retrotransposon whose function is dependent on the Su(Hw) Zn-finger DNA binding protein. Although there are several hundred Su(Hw) sites in the genome which are proposed to act as endogenous insulator elements, analysis of the role of the Su(Hw) protein has focussed on the gypsy insulator and few endogenous sites have yet been identified. We have used chromatin immunopurification coupled to genomic microarray analysis to identify Su(Hw) binding sites within a representative region of the Drosophila genome; the 3MB Adh region on chromosome 2L. We have located about 60 Su(Hw) binding sites across this region and this has enabled us to construct a robust new Su(Hw) binding site consensus based on these in vivo sites. In contrast to the gypsy insulator which contains 12 Su(Hw) binding sites within 340bp, the endogenous sites are not present in clusters. We identify two key features of these endogenous Su(Hw) sites. Firstly, in contrast to most analyses of DNA binding protein specificity, we find that strong matches to the binding consensus are good predictors of binding site occupancy. Secondly, examination of Su(Hw) binding site occupancy in 0-20hr embryos, 3rd larval instar brains or 3rd larval imaginal discs reveals a constant pattern of Su(Hw) binding indicating that most , if not all Su(Hw) sites are constitutively occupied. These two features support a constant genomic architectural role for the Su(Hw) protein. Keywords: ChIP-chip

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Genome-wide analyses of Su(Hw) binding in Drosophila ovary

Project description:The twelve zinc finger Suppressor of Hairy-wing [Su(Hw)] protein binds thousands of sites in Drosophila genome and is essential for the function of the gypsy insulator. Loss of the globally expressed Su(Hw) protein causes female sterility due to tissue-specific defect limited to female germline. Using chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq), we determine the extent of tissue-specific binding of Su(Hw) in Drosophila ovary. We demonstrate that Su(Hw) binding sites (SBSs) are largely constitutively occupied in the germline and soma. Our analyses indicate that SBSs fall into several non-uniform classes, as determined by the partner protein distribution and DNA sequence conservation. Further, we show that only a subset of SBSs is required for the female fertility. These sites are maintained in the su(Hw)f zinc finger 10 mutant background, which is fertile but does not support gypsy insulator function. Together, our data are consistent with the model where Su(Hw) serves multiple regulatory roles in the genome, and contribute to understanding of how loss of a single zinc finger affects chromosome localization of a DNA binding protein. Examination of Su(Hw) localization in the ovaries of less than 6 hour old wild type and su(Hw)f mutant Drosophila females

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Project description:Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, we studied the effects of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. We show that transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.

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Role of Su(Hw) in DNA amplification in Drosophila ovaries

Project description:Role of Su(Hw) in DNA amplification in Drosophila ovaries

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