Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

Abstract

Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression.

Argonaute2 attenuates active transcription by limiting RNA Polymerase II elongation in Drosophila melanogaster

“…First, we transfected NELF-E and CDK9 HA-tagged constructs into Kc cells and performed immunoprecipitation from nuclear extracts followed by Western blotting against AGO2. In concordance with our previous AGO2 mass spec analysis of nuclear interacting factors 3 , we found that NELF-E but not CDK9 associates with AGO2 (Fig. 4A ).…”

“…Previously, we showed that AGO2 forms a nuclear complex with Pol II, and chromatin association profiles of both factors correlate positively across chromatin states throughout the genome 3 . These findings prompted us to test whether AGO2 influences Pol II occupancy genome-wide.…”

“…Furthermore, AGO2 is known to be required for proper chromatin looping between the enhancer and promoter of the homeotic gene Abd-B , thus affecting its transcription 6 . Importantly, the function of AGO2 in transcription and chromatin topology has been shown to occur independently of the siRNA pathway 3 , 6 , 7 .…”

“…Genome-wide transcriptional analyses by nascent euRNA-seq (neuRNA-seq) and steady-state RNA-seq revealed that depletion of AGO2 primarily causes up-regulation of certain active promoters 3 . Moreover, it has been shown that LaminB, a key component of the nuclear lamina, also modulates AGO2-dependent genes likely by controlling chromatin topology 3 . Comparisons with several chromatin features determined that such affected promoters are bound by AGO2 and associated with RED chromatin.…”

“…In addition, RED chromatin is associated with genes that are susceptible to modulation during development as well as external signals 8 . In this vein, AGO2 was found to interact physically with the Pol II complex 1 , 3 and the Negative Elongation Factor (NELF) 1 , a factor implicated in transcriptional pausing 9 , 10 . Although NELF has been characterized to inhibit transcription elongation in vitro 1 , 9 , 10 , it can also play a positive role in transcription by keeping promoters in a primed, open state to allow for regulated activation 11 .…”