Project description:Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression. In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown. Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator. AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions. Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome. ChIP-seq of AGO2 in two Drosophila cell types (S2 and S3)
Project description:Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals that endogenous small interfering RNAs (siRNAs) are produced via bidirectional transcription. >100 cis-NATs with overlapping 3' exons generate 21-nt, Dicer-2 (Dcr-2)dependent, 3'-end modified siRNAs. To determine whether any co-expressed cisNATs are denied entry into the RNAi pathway, we analyzed the gene expression profile of S2 cells. The analysis suggested that the processing of cis-NATs by RNAi are actively restricted, and the selected loci are enriched for nucleic acidbased functions and include Argonaute-2 (AGO2) itself. Keywords: Gene expression
