Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila

Abstract

RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21-30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.

Figure 1. Pol II promoter-proximal pausing on hsp70 is reduced in Dcr2 RNAi cells

a-c) Quantitative RT-PCR analysis of heat shock gene transcripts. RNA from S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA (a), or AGO2 dsRNA (b) were analyzed with primers specific for the indicated heat shock genes. c) Induction of Dcr2-flag transgene is able to revert the phenotype induced by Dcr2-depletion. S2 cells stably transformed with a Dcr2-flag transgene were treated with EGFP dsRNA (control) or Dcr2 dsRNA. The Dcr2-flag expression is induced only in the Dcr2 RNAi sample by the addition of copper. The samples were analyzed before and after 72h induction of the transgene. The transcript levels are shown with respect to the EGFP control (experiment/control ratio). n=3, bars represent the mean±standard deviation. d) hsp70 DNA-FISH on polytene chromosomes from wild type (WT), homozygous Dcr2^L811fsX or homozygous Ago2⁴¹⁴ mutant larvae; lower pictures show DNA staining; upper pictures show the merge of DNA (blue) and FISH (green) signals. e) Schematic representation of the hsp70 transcription unit with the position of the PCR amplicons used in this study. The numbers indicate the middle of each amplicon with respect to the transcription start site. f-h) ChIP analysis of the hsp70 heat shock gene. Chromatin from S2 cells or S2 cells after exposure to heat shock (HS) was immunoprecipitated with anti-Pol II 4H8 (recognises the Ser5-phosphorylated CTD domain), anti-Dcr2 or anti-AGO2 antibodies. n=3, bars represent the mean± standard deviation. i-j) ChIP analysis of the hsp70 heat shock gene. Chromatin from S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA was immunoprecipitated with anti-Pol II 4H8 or anti-NELF-E antibodies. The resulting DNA has been analyzed by quantitative PCR. Protein binding is expressed as a percentage of input subtracted by the background signal. n=3, bars represent the mean± standard deviation. Differences in Pol II ChIP values in 2f and 2i are due to different batches of antibody used in these assays. k) Permanganate mapping of open transcription bubbles on hsp70. Permanganate reacts with single-stranded thymine residues, like those in a open transcription bubble, revealing a transcriptionally engaged RNA polymerase. The autoradiograph includes a G/A ladder, used to determine the position of the bands, and the permanganate reactivity of thymine residues observed in S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA. The hyper-reactive thymines +22 and +30 are labeled. Two independent biological samples have been analyzed. Shown is a representative picture l) Quantification of the autoradiograph.

Figure 2. Chromatin localization of Pol II and AGO2 after heat shock

Co-immunolocalization of AGO2 (red) and elongating Pol II (green) in WT (a) and homozygous Ago2⁴¹⁴ mutant chromosomes (b) after heat shock (HS). DNA is stained in blue. Single signals are shown in black and white. The bottom row shows a higher magnification of the boxed area. (c) Immunolocalization of elongating Pol II (green) in WT (c), homozygous Ago2^V966M (d), homozygous Dcr2^L188F (e), homozygous Dcr2^P1496L (f), chromosomes shows that missense mutations in Dcr2 or AGO2 influence the behaviour of elongating Pol II. Chromosomes on the lower panel have been exposed to heat shock (HS). DNA is stained in blue.

Figure 3. Dcr2 and the RNAi effector protein AGO2 associate with Pol II and NELF

a) Nuclear extracts from Drosophila S2 cells were immunoprecipitated with the indicated antibodies, and the immunoprecipitated protein complexes were analyzed by western blot for the presence of Pol II, Dcr2, AGO2 and NELF-E proteins. b) Nuclear extracts from S2 cells were immunoprecipitated with the indicated antibodies (TBP: TATA-binding protein) and analyzed by western blot for the presence of Pol II.

Figure 4. Features of AGO2-associated small RNAs

a) Top 20 short RNAs most-enriched promoters in heat shock vs. no heat shock conditions. Comparison of the short RNA fold enrichment in the AGO2-IP libraries across the normal and heat shock condition for all promoters. y-axis is shown in log scale. b) Relative enrichment calculated as fold change (y-axis) for the sum of all sense or antisense tags across different conditions. Gene definitions and conditions labeled below the chart. Sense tag enrichment colored in brown (little to no enrichment), antisense tags enrichment colored in green (strongest enrichment in heat shock conditions).