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

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 Overall design: To investigate whether Effete and Cul4 directly affect transcription genome-wide, we performed neuRNA-seq to examine newly synthesized transcripts in control cells versus Effete or Cul4 deficient cells.