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Effete and Cullin 4 affect nuclear organization of the gypsy chromatin insulator [RNA-Seq]

ABSTRACT: Chromatin insulators demarcate the genome into distinct transcriptional domains and contribute to higher-order genome organization. InDrosophila, Su(Hw), CP190, and Mod(mdg4)67.2 are core components ofgypsyinsulator complex. Insulator bodies are polymerization of insulator proteins in cell nuclei, and their proper localization is correlated with insulator function. Post-modification of insulator proteins is required to fulfill insulator function, but few proteins in this process have been revealed. Here, we identify Effete(Eff), a ubiquitin-conjugating enzyme, and its interactor Cul4 as novel regulators of insulator bodies localization through a high-content RNAi screen. Effete and Cul4 physically associate with gypsy insulator proteins and promote gypsy-dependent insulator barrier activity. Moreover, Effete enriches in Satellite DNA and gypsy sites shared by Su(Hw). Cul4 overlaps with CP190 and localizes to gene promoters. Effete and Cul4 have little overlap, but they mutually impact the chromatin binding of one another. Effete and Cul4 are required for the binding of Su(Hw) in repetitive sequences and regulate the transcription of genes near TAD borders. Depletion of Effete alters the chromatin organization and position of gypsy insulator in nuclei. Overall, our findings reveal novel regulation roles of ubiquitin enzymes in chromatin insulator activity, 3D genome organization, and gene expression

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE243451 | GEO | 2026/02/27

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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Effete and Cullin 4 affect nuclear organization of the gypsy chromatin insulator [ChIP-Seq]

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