RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

Abstract

A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, we performed genomewide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.

Argonaute2 promotes tumor metastasis by way of up-regulating focal adhesion kinase expression in hepatocellular carcinoma

“…37 Mutation of Ago2 led to a reduction of chromosomal looping interactions, with altering gene expression. 38 In humans, Ago2 can target the intragenic LINE-1 transcription complexes that repress cancer-associated genes through epigenetic mechanism. 39 Nuclear Ago2 can bind to a set of regulatory genes, including Ago2 itself, Oct4, Sox2, Nanog, GATA, STAT3, and b-catenin, which play fundamental functions in stem cells.…”

Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration

“…Large-scale loss-of-function analysis by RNA interference (RNAi) using small hairpin (shRNA) or small interfering RNA (siRNA) -mediated knockdown of genes is a powerful screening approach in mammalian cells (2)(3)(4)(5)(6)(7). ShRNAs provide the opportunity for long-term silencing by stable infection of cells maintained under selection pressure.…”

The RNA-binding protein Rumpelstiltskin antagonizes gypsy chromatin insulator function in a tissue-specific manner

“…For immunoprecipitation, Rump 5G4 tissue culture supernatant was used. For ChIP, 5G4 and 10C3 tissue culture supernatants against Rump (Jain and Gavis, 2008), guinea pig serum against Mod(mdg4)2.2, rabbit serum against Mod(mdg4)2.2 (Van Bortle et al, 2012), rabbit serum against Su(Hw) (Bushey et al, 2009), rabbit serum against CP190 (Moshkovich et al, 2011), rabbit serum against Shep (Matzat et al, 2012) and guinea pig serum against Shep (Matzat et al, 2012) were used.…”

“…Imaginal discs and brains were dissected from at least six larvae of each genotype and subjected to whole-mount staining, as previously described (Moshkovich et al, 2011), using ProLong Gold (Life Technologies, Carlsbad, CA) mounting media. For insulator body localization experiments, six out of seven independent experiments achieved similar results.…”

“…In both Drosophila and mammals, genome-wide binding profiles of insulator proteins are largely invariant across cell types (Bushey et al, 2009;Kim et al, 2007), suggesting that the particular higher-order chromatin configurations mediated by insulator proteins might be regulated in different cell types. Recent work has shown that RNA and RNA-binding proteins can modulate the activities of the gypsy insulator (Lei and Corces, 2006;Matzat et al, 2013;Matzat et al, 2012), the Drosophila CTCF insulator complex (Lim et al, 2013;Moshkovich et al, 2011), and mammalian CTCF and its partner cohesin (Sun et al, 2013;Yao et al, 2010). In the case of the RNA-binding protein Alan Shepard (Shep), gypsy insulator activity and insulator body localization is antagonized in a tissue-specific manner (Matzat et al, 2012).…”