Argonaute2 Functions With LaminB to Mediate Transcriptional Silencing of Spermatogenesis Genes in Drosophila melanogaster

NAZER EZEQUIEL; LEI, ELISSA P

Simposio; CSHL meeting on Nuclear Organization and Function; 2016

Argonauteproteins 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 directlyon euchromatin to promote enhancer-promoter interaction at the homeotic Abd-B locus independently of the RNAinterference (RNAi) pathway. ChIP-seq analysis revealed that AGO2 bindsthousands of sites in the genome, raising the possibility that AGO2 couldmodulate global chromatin architecture.             To identify factors that canfunction with AGO2 to regulate transcription, we performed immunoaffinitypurification 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 inchromatin and nuclear matrix fractions. To directly assess the global role of bothproteins in transcription, we performed nascent RNA-Seq upon depletion of eitherAGO2 or LaminB in Kc167 cells. We found that both proteins co-repressed a highlysignificant number of genes, particularly those located at the borders ofLamin-associated domains (LADs).             In order to assess the physiologicalrole of AGO2 in transcriptional regulation, we performed mRNA-Seq in null versusRNA slicing catalytic activity mutant female larvae. Strikingly, we observedde-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 activatorof spermatogenesis gene expression. Null mutation of nht suppresses the up-regulation of spermatogenesis genes observedin AGO2 null mutants, suggesting thatAGO2 acts upstream of nht to silence thespermatogenesis gene program. Given that nhtis located within a LAD harboring flanking AGO2 chromatin binding sites, wehypothesized that AGO2 and LaminB could modulate chromatin topology to repress nht. Chromosomal conformation assays(3C) using the nht promoter as baitshowed a decrease in the frequency of interactions within the LAD upon AGO2 or LaminBknockdown. We conclude that both proteins may repress transcription at LADborders by regulating chromatin architecture.