Structure-function analysis of Argonaute2 in chromatin insulator activity. Madoka Chinen, Elissa Lei. Laboratory of Cellular and Developmental Biology, NIDDK, Bethesda, MD.

   Chromatin insulators are DNA-protein complexes distributed throughout the genome that can act as barriers to prevent spreading of repressive chromatin and interfere with enhancer-promoter interaction by promoting alternative chromatin loop formation. We described a role for Argonaute2 (AGO2), a canonical member of the siRNA pathway, in CTCF/CP190-dependent Fab-8 insulator activity, which prevents inappropriate enhancer interactions with the Hox gene Abd-B promoter. AGO2 is important for promoting or stabilizing chromatin loop formation at the Abd-B locus. Genome-wide localization analysis demonstrated that AGO2 localizes extensively throughout euchromatin including the Fab-8 element as well as many promoters. Interestingly, an AGO2 RNAi-catalytic mutant does not show defects in insulator activity, indicating that insulator-related activity of AGO2 is independent of catalytic activity. Since the AGO2 RNAi-catalytic mutant can bind to RNA, it is unclear whether RNA is involved in AGO2-related insulator function. Drosophila AGO2 contains 4 domains including the GRR, DUF, PAZ, and PIWI domains, the latter 3 of which are conserved to humans. The PAZ domain binds nucleic acid, and the PIWI domain has RNase-H like nuclease activity. The function of the GRR and DUF domains are not well understood; the GRR is specific to Drosophila and is not required for RNAi activity. Here, we seek to define which domains are important for AGO2 nuclear localization and insulator function. As a first step, we generated transgenic AGO2 point mutants, RNA-binding mutant and truncation mutants, which are expressed under control of the genomic AGO2 promoter. We are currently examining whether these mutants are functional for enhancer blocking activity at Fab-8 in the AGO2 null mutant background. The minimal AGO2 domain required for chromatin binding will be determined by chromatin immunoprecipitation and immunostaining salivary gland polytene chromosomes expressing AGO2 truncation mutants. Current progress of our AGO2 mutant analysis will be reported.