Chromatin-associatedRNA interference components contribute to transcriptional regulation in Drosophila

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 RNAimediated 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. Accumulating evidence indicates that RNAi components and small RNAs function in the nucleus to control heterochromatin formation, as well as repeat-induced gene silencing and transposable element mobilization. However, the global association of RNAi components with chromatin and their role in transcriptional regulation remains to be elucidated. To investigate a role for RNAi in the chromatin dynamics of a multicellular organism and, possibly, in transcriptional gene silencing, we first determined whether any of the key RNAi components are present in the nucleus of Drosophila cells. Cellular fractionation of embryonic tissue culture cells (S2 cells) showed that the microRNA-processing factors DCR1 and AGO1, as well the RNAi component AGO2, are equally distributed between the cytoplasm and the nucleus (Supplementary Fig. 1a). By contrast, the RNAi protein DCR2 is greatly enriched in the nuclear fraction (Supplementary Fig. 1a). To evaluate the association of RNAi components with the different nuclear compartments, we carried out chromatin fractionation experiments (Supplementary Fig. 1b, c). A substantial amount of DCR2 and AGO2 was detected in chromatin fractions, together with RNA polymerase II (RNA Pol II), Negative elongation factor E (NELFE), Polycomb (Pc) and histone H3 (Supplementary Fig. 1c). By contrast, most of the cellular DCR1 and AGO1 was found in the Triton X-100 soluble fraction (S1 fraction), together with tubulin, a marker for proteins that are not associated with chromatin (Supplementary Fig. 1c). Taken together, our data indicate that the DCR2–AGO2 complex is mainly associated with chromatin, whereas the DCR1–AGO1 complex is mostly cytoplasmic, in accordance with its cytoplasmic function (that is, post-transcriptional gene silencing). To determine whether RNAi components associate with chromatin in vivo, Drosophila polytene chromosomes were stained with AGO2and DCR2-specific monoclonal antibodies. Both AGO2 and DCR2 were detected at several hundred sites on polytene chromosomes from wild-type larvae (Supplementary Figs 1d, g and 2a, b). By contrast, little or no staining was detected in Ago2or Dcr2-null chromosomes (Supplementary Fig. 1f, i and Supplementary Table 1a). Strikingly, the majority of AGO2and DCR2-associated loci correspond to interbands, suggesting that AGO2 and DCR2 preferentially associate with euchromatic, transcriptionally active, loci (Supplementary Fig. 2a, b). In particular, the chromatin binding of DCR2 requires AGO2, but the converse is not true (Supplementary Fig. 3), suggesting that AGO2 also acts as the RNAi effector complex on chromatin. Interestingly, AGO2 and DCR2 are present at the 87A and 87C cytogenetic loci (Supplementary Fig. 1e, h). These cytogenetic loci contain copies of the heat-shock gene Hsp70, thus providing wellcharacterized inducible candidate genes with which to investigate the role of DCR2 and AGO2 in transcription and, in particular, in RNA Pol II pausing regulation. To determine whether DCR2 and AGO2 affect Hsp70 transcription, Hsp70 transcript levels were measured in control cells and in cells depleted of DCR2 or AGO2 (Supplementary Fig. 4). The knockdown of either RNAi component resulted in a significant increase in Hsp70 transcripts in non-heatshocked cells (Fig. 1a, b). Similar results were obtained for a second heat-shock gene, Hsp68 (Fig. 1a, b). The increased expression of Hsp70 and Hsp68 in cells depleted of DCR2 could be reversed by the expression of a Flag-tagged wild-type copy of DCR2 (DCR2–Flag) (Fig. 1c), indicating that the change in expression was not the result of an offtarget effect of the Dcr2 RNAi. Interestingly, the levels of the endogenous AGO2 protein increased together with the expression of wild-type DCR2–Flag (Supplementary Fig. 4a), suggesting that the DCR2–Flag protein is integrated into the RNAi pathway and that re-establishment of functional levels of DCR2 and AGO2 rescues the observed transcriptional defects in heat-shock genes. However, the depletion of DCR2 and AGO2 did not affect the expression of Hsp70 and Hsp68