Abstract
Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions.
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Acknowledgements
We thank A. Leshinsky, R.F. Cook, and C.A. Whittaker from the Swanson Biotechnology Center at the Massachusetts Institute of Technology (Koch Institute for Integrative Cancer Research) for the sequencing of nascent transcript libraries, W. Gu (University of Massachusetts, Worcester) for providing siRNA sequencing data sets, and members of the Grishok, Maniatis and Hobert laboratories at Columbia University for discussions. Reagents were kindly provided by C. Mello (University of Massachusetts, Worcester) and A. Desai (University of California, San Diego). This work was supported by US National Institutes of Health grant 1DP2OD006412-01 (A.G.).
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G.C. designed and performed the experiments. G.C., S.H., S.O. and R.S. performed bioinformatic analyses of generated and published data. G.C., S.H., S.O., R.S. and A.G. analyzed the data, and G.C. and A.G. wrote the manuscript.
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Supplementary Figure 1 Histone abundance is not compromised in csr-1 hypomorph and drh-3(ne4253) mutant worms.
(a) Venn diagram showing an overlap between genes down-regulated at least 1.5 fold in csr-1(tm892) mutant (by tiling array15) and CSR-1 target genes15. A Fisher's exact test was used for calculating a P value for the significance of the overlap. (b) Western blotting showing that the levels of H2B (left panel) and H2A (center panel) in csr-1 hypomorph and the level of H2B in drh-3(ne4253) mutant (right panel) are not reduced compared to the control. Ponceau red staining is shown as a loading control (membrane). (c) Western blot showing the two isoforms of CSR-1 present in the cytoplasmic and nuclear fractions (top panel). In control western blots histone H3 is detected in the nuclear fraction (bottom panel) and actin is detected in the cytoplasmic fraction (middle panel). (d) Western blot showing reduced levels of CSR-1 in nuclear extracts from csr-1 hypomorph compared to the control. Ponceau red staining is shown as a loading control (membrane). (e) ChIP-qPCR results with histone H3 antibody detect no significant changes in the incorporation of histone proteins into chromatin at selected germline-specific CSR-1 targets and nontarget loci in csr-1 hypomorph and drh-3(ne4253) mutants in comparison to WT. Data are presented as the mean of the percentage of input in three independent experiments. Error bars, s.d. (n = 3 biological replicates).
Supplementary Figure 2 Transcription of WAGO target protein-coding genes is not globally affected in csr-1 hypomorph and drh-3(ne4253) mutants; GRO-seq and published microarray data show correlation.
(a) Cumulative distribution plots of normalized GRO-seq gene body reads (log2 of the csr-1/WT read ratio) showing the overall effect of csr-1 mutation on Pol II transcription of CSR-1 targets15 (orange line), WAGO protein-coding gene targets14 (purple line) and total genes (black line). (b) GRO-seq analysis performed as in a, considering drh-3(ne4253) and WT normalized GRO-seq gene body reads. (c) Venn diagrams showing significant overlaps between the groups of genes found up-regulated in csr-1 mutant by GRO-seq (this study) and tiling array15. A Fisher's exact test was used for calculating P values for the significance of the overlaps. (d) Venn diagrams as in (c) considering the down-regulated groups of genes identified by both approaches.
Supplementary Figure 3 CSR-1 affects Pol II transcription at promoters and gene ends and interacts with Pol II.
(a, b) Cumulative distribution plots of normalized GRO-seq promoter and gene end reads (log2 of the reads' ratios: drh-3(ne4253)/WT) showing the overall effect of drh-3 mutation on Pol II transcription of CSR-1 targets (orange line), and all genes (black line). (c) ChIP-qPCR results in L4- staged worms showing the reduction of Pol II binding at the promoters of CSR-1 target genes in drh-3(ne4253) versus WT. Error bars indicate the range (n = 2 biological replicates). (d) Coimmunopercipitation results with nuclear extracts from WT worms and csr-1 hypomorphic mutant strain show specific interaction between CSR-1 and the Pol II complex. The top panels show western blots with an antibody that recognizes the nonphosphorylated isoform of Pol IIand the bottom panels show western blots with an antibody recognizing the long and the short isoforms of CSR-1. Left panels show immunoprecipitation with CSR-1 antibody and right panels show immunoprecipitation with Pol II antibody. Long and short CSR-1 isoforms are indicated by the arrows.
Supplementary Figure 4 Increased antisense Pol II transcription in CSR-1 pathway mutants.
(a, b) Cumulative distribution plots of normalized GRO-seq reads showing the global increase in antisense transcription (red line) compared to the sense transcription (black line) in csr-1 and drh-3 mutants.
Supplementary Figure 5 Categories of genes affected by CSR-1 pathway mutants.
(a) Box plot showing changes in Pol II transcription in csr-1 mutant versus WT among different categories of genes defined by their expression profiles. The number of genes in each category is shown in parentheses. Each box shows the median (black line), the 25th-75th percentile (box) ± 1.5 interquartile range (whiskers). The boxes in each gene category show significant changes compared to all genes, based on Wilcoxon rank-sum test with P value < 0.0001. (b) Expected (grey bars) and observed (blue bars) numbers of CSR-1 target genes among different expression categories. Asterisks indicate significance as more or less genes than expected by chance calculated using hypergeometric test, P < 0.0001. (c) Expected (grey bars) and observed (colored bars) numbers of CSR-1 target genes among the top 20% highly-transcribed genes and the bottom 20%. Asterisks indicate significance as more or less genes than expected by chance calculated using hypergeometric test, P < 0.0001. (d) Cumulative distribution plots of normalized GRO-seq gene body reads showing the effect of drh-3(ne4253) on the top 20% highly-transcribed genes (blue line), the bottom 20% genes (red line) and all genes (black line). Only reads from the gene body were considered for this analysis. (e) The average of the log2 ratios between the drh-3(ne4253) mutant and WT normalized gene body reads quantified for genes grouped by quartiles of expression based on GRO-seq.
Supplementary Figure 6 An example of a genomic locus containing an active CSR-1 target genes next to silent non-target genes.
Top, an example of a genomic locus that shows a decrease in transcriptionally-engaged Pol II at the germline-specific gene (mes-4) in the csr-1 hypomorphic mutant versus WT. Track listing from top to bottom: GRO-seq normalized reads aligned to the negative strand (red) from WT larvae, GRO-seq normalized reads aligned to the negative strand (red) from the csr-1 hypomorphic larvae, GRO-seq normalized reads aligned to the positive strand (blue) from WT larvae (blue), GRO-seq normalized reads aligned to the positive strand (blue) from the csr-1 hypomorphic larvae. Bottom, a rescaled view of GRO-seq normalized reads to appreciate the increased transcription of non-target silenced genes next to the active CSR-1 target gene in the csr-1 hypomorphic mutant compared to WT. CSR-1 IP 22G-RNA track shows reads of CSR-1 immunoprecipitated (IP) endo-siRNAs from wild type young adult worms and reported in ref. 15. Gene models are based on UCSC Genome Browser (ce6).
Supplementary Figure 7 CSR-1 regulates transcription independently of MES-4.
(a) ChIP-qPCR results with L4-staged worms showing the enrichment of MES-4 at CSR-1 target regions compared to the non-target regions. There are no differences in MES-4 binding in drh-3(ne4253) mutant compared to WT. The enrichment has been calculated relative to the non-target gene ZK381.2. Error bars indicate the range (n = 2 biological replicates). (b) ChIP-qPCR results with L4-staged worms, as in (a), showing the levels of H3K36me3 normalized to the total levels of histone H3 at CSR-1 target regions compared to the non-target regions. There are no differences in the levels of H3K36me3 in drh-3(ne4253) mutant compared to WT. Error bars indicate the range (n = 2 biological replicates).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8 (PDF 3932 kb)
Supplementary Table 1
Gene sets used in this study (XLSX 516 kb)
Supplementary Table 2
Gene set designations and references (XLSX 11 kb)
Supplementary Table 3
Primer sequences (XLSX 10 kb)
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Cecere, G., Hoersch, S., O'Keeffe, S. et al. Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape. Nat Struct Mol Biol 21, 358–365 (2014). https://doi.org/10.1038/nsmb.2801
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DOI: https://doi.org/10.1038/nsmb.2801
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