The Mechanism Of Gene Regulation By Nuclear ARGONAUTE 1 In Arabidopsis

Conventional RNA interference(RNAi)pathways suppress gene expression at the post-transcriptional or transcriptional level.The ARGONAUTE(AGO)family proteins are known as core effectors of RNAi pathways in eukaryotes.They function by associating with different classes of small RNAs(sRNAs)that are processed from double-stranded or stem-loop structured precursor RNAs by Dicer or Dicer-like(DCL)proteins.Arabidopsis thaliana encodes 10 AGOs.AGO1 is known to bind microRNAs(miRNAs)and post-transcriptionally repress the expression of target genes via mRNA cleavage and/or translational inhibition in the cytoplasm.Emerging evidence suggests that AGO proteins can also contribute to transcriptional regulation tFrrough mechanisms different from conventional RNAi.Previous studies have shown that AGO1 is also localized in the nucleus.However,the nuclear function of AGO1 in Arabidopsis remains elusive.We have previously found that AGO1 preferentially binds to the chromatin of genes through performing AGO1 chromatin immunoprecipitation followed by deep sequencing(ChIP-seq).In this study,to address whether chromatin-bound AGOl can regulate gene expression,we performed mRNA sequencing(RNA-seq)experiments in the wide-type(Col-0)as well as ago 1-36 mutant plants.Analyses of ChIP-seq and RNA-seq data revealed that AGOI-bound genes have higher expression levels compared to non-AGO1-bound genes.Importantly,the expression levels of AGO1-bound genes were generally decreased in agol-36.These results suggest that,different from cytoplasmic AGOl,chromatin-bound AGO1 positively regulates gene expression.Using nuclear run-on assays,which quantitatively measure gene transcription rates,we found that the agol-36 mutation caused a marked decrease in the transcription rates of AGO1-bound genes,indicating the regulation of the target genes by AGO1 occurs at the transcriptional level.Consistently,we found the enrichment of DNA-dependent RNA polymerase ?(Pol ?)at AGO1-bound genes was much lower in agol-36 compared to Col-0.Taken together,these results suggest that AGO1 promotes the transcription of AGO1-bound genes through assisting Pol ? recruitment.AGO proteins are usually guided to their targets by sRNAs through sequence complementarity.Deep sequencing analysis of nuclear AGO1-associated sRNAs revealed that sRNAs were detectable at 52%of AGOl-bound regions.Moreover,we found that AGOl binding to its target genes was significantly compromised in the dcl1-9 and dcl1/2/3/4 mutants but not in dcl2/3/4,suggesting that DCL1-dependent sRNAs are required for AGO1 binding to its targets.In agreement with these findings,compared to wide-type AGO1,AGO1 deficient in sRNA binding or slicer activity could not restore the expression of AGO1-bound genes to normal levels.We further identified SWI/SNF complexes as interacting partners with nuclear AGO1.Further experiments demonstrated that SWI/SNF complexes were required for both nuclear AGO1 targeting at target gene loci and gene expression.Taken together,we show that both sRNAs and SWI/SNF complexes are required for AGO1 binding to chromatin and its function in promoting gene transcription.Our findings reveal an unsuspected role for Arabidopsis AGO1 in facilitating gene transcription and provide novel insights into the molecular mechanisms of chromatin-bound AGO1 in promoting gene expression.