Argonaute2 Cooperates with LaminB to Repress Transcription at Lamin-associated Domains in Drosophila melanogaster

NAZER EZEQUIEL; CHINEN, MADOKA; DALE, RYAN K.; LEI, ELISSA P

Chicago

Congreso; Annual meeting of the American Society for Biochemistry and Molecular Biology; 2017

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. Circular chromosome conformation capture assays (4C-Seq) 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.