RNAi‐independent role for Argonaute2 in CTCF/CP190 insulator function

Chromatin insulators are DNA‐protein complexes that control nuclear organization to effect changes in gene expression. RNA silencing pathways have been proposed to modulate long‐range chromosomal interactions; however, the underlying mechanisms are unknown. We performed genome‐wide localization of the Drosophila small interfering RNA (siRNA) pathway effector protein Argonaute2 (AGO2) by chromatin immunoprecipitation and sequencing (ChIP‐seq). This analysis reveals extensive overlap of AGO2 with insulator sites throughout the genome. Unexpectedly, AGO2 chromatin binding does not correspond to regions of the genome that produce siRNA. Comprehensive genetic analysis reveals that AGO2 but not other RNA silencing factors is required for the activity of the CTCF/CP190 dependent Fab‐8 insulator, and catalytic activity of AGO2 is not required for insulator function. Furthermore, AGO2 and CP190 interact physically, and depletion of either CTCF or CP190 results in loss of AGO2 chromatin association with insulator as well as non‐insulator sites. This general dependence suggests that AGO2 chromatin association may be achieved in part by CTCF/CP190‐mediated looping interactions between cis ‐regulatory elements. Collectively, our findings suggest Dicer‐independent recruitment of AGO2 to chromatin by insulator proteins promote the definition of transcriptional domains throughout the genome.