Argonaute2 Functions With LaminB to Mediate Transcriptional Silencing of Spermatogenesis Genes Argonaute proteins are commonly known as core components of RNA silencing pathways. However, Argonaute proteins have also been shown to possess nuclear functions, such as regulation of transcription, splicing and chromatin architecture. Previous work showed that Drosophila AGO2 functions directly on euchromatin to promote enhancer-promoter interaction at the homeotic Abd-B locus independently of the RNA interference (RNAi) pathway. ChIP-seq analysis revealed that AGO2 binds thousands of sites in the genome, raising the possibility that AGO2 could modulate global chromatin architecture. To identify factors that can function with AGO2 to regulate transcription, we performed immunoaffinity purification of AGO2 from nuclear extracts followed by mass spec analysis. Interestingly, we found that LaminB is enriched among the top AGO2-associated proteins. Reciprocal co-immunoprecitation validated the specificity of this interaction, and biochemical fractionation assays confirmed that both proteins reside in chromatin and nuclear matrix fractions. To directly assess the global role of both proteins in transcription, we performed nascent RNA-Seq upon depletion of either AGO2 or LaminB in Kc167 cells. We found that both proteins co-repressed a highly significant number of genes, particularly those located at the borders of Lamin-associated domains (LADs). In order to assess the physiological role of AGO2 in transcriptional regulation, we performed mRNA-Seq in null versus RNA slicing catalytic activity mutant female larvae. Strikingly, we observed de-repressed transcription of spermatogenesis genes in the absence of AGO2, independent of its catalytic activity. One of the de-repressed genes is nht, which encodes a key upstream activator of spermatogenesis gene expression. Null mutation of nht suppresses the up-regulation of spermatogenesis genes observed in AGO2 null mutants, suggesting that AGO2 acts upstream of nht to silence the spermatogenesis gene program. Given that nht is located within a LAD harboring flanking AGO2 chromatin binding sites, we hypothesized that AGO2 and LaminB could modulate chromatin topology to repress nht. Chromosomal conformation assays (3C and 4C) using the nht promoter as bait showed a decrease in the frequency of interactions within the LAD upon AGO2 or LaminB knockdown. We conclude that both proteins may repress transcription at LAD borders by regulating chromatin architecture. Currently, a set of experimental approaches, including DamID and DNA FISH, are underway in order to provide additional molecular insights into this novel mechanism of transcriptional regulation. Structure/function analysis of AGO2 with respect to CTCF/CP190 insulator activity In order to gain mechanistic insight into how AGO2 contributes to CTCF/CP190 chromatin insulator activity, we constructed a series of AGO2 conserved domain truncations that will allow us to identify domains responsible for interaction and Fab-8 insulator activity. Crystal structures of both eukaryotic and prokaryotic Argonautes are available, and AGO2 is well studied biochemically with respect to RNAi. Although we found that RNAi catalytic activity is not required for AGO2 insulator function, it is possible that its RNA-binding ability is needed. To this end, we generated point mutants Y897E in the PIWI RNase H-like domain or added alanine to the C-terminus (+A) in order to block the RNA-binding pocket without disrupting protein folding. These constructs are under control of the endogenous AGO2 promoter and contain an N-terminal Myc epitope tag. These constructs were verified to express at similar levels when transfected into cell lines, and transgenic rescue lines in the null AGO2 genetic background have also been generated. Using an established in vivo assay, the wildtype line is functional for RNAi while the mutants are defective. We are currently testing whether these constructs are functional for Fab-8 and additional insulator activities using transgenic reporter assays. These constructs were examined for nuclear localization in cells by immunofluorescence and biochemical fractionation. Constructs will be tested for the ability to rescue chromatin looping at Fab-8 within the Abd-B locus using directed 3C and 4C. We will also examine the chromatin topology at the nht locus in these mutants. Preliminary nascent RNA-seq analysis of RNA-binding mutants compared to wildtype suggests that AGO2-dependent transcription requires its RNA-binding capacity.

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Caravaca, Juan Manuel; Lei, Elissa P (2016) Maintenance of a Drosophila melanogaster Population Cage. J Vis Exp :
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