Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity Josef P. Clark, Reazur Rahman, Nachen Yang, Linda H. Yang, Nelson C. Lau  Current Biology  Volume 27, Issue 17, Pages 2718-2726.e4 (September 2017) DOI: 10.1016/j.cub.2017.07.052 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Reporter Assay to Test Factors Impacting Initiation of PIWI-Directed Silencing (A) Left: design of the piRNA-targeted reporter assay in OSS cells. Right: a 2.4-kb piRNA-targeted region from the Flamenco (Flam) locus inserted in “sense” (same orientation as piRNAs) or “antisense” configurations into the 5′ intron or 3′ UTR of the Renilla luciferase plasmid. (B) Normalization procedure measures a factor’s impact on PIWI-directed silencing. Left: representative values of Renilla luciferase to firefly luciferase. Antisense reporter (dark blue) is repressed by PIWI/piRNAs in the siGFP-negative control but de-repressed upon PIWI knockdown (KD; by siPIWI siRNA). Right: full normalization of siPIWI KD changes against siGFP control. (C) Assay of known PIWI/piRNA pathway factors shows greater reporter de-repression when the Flam element is in the 5′ intron compared to the 3′ UTR. (D) Knockdown of chromatin-associated factors do not display reporter de-repression. (E) Assay of RNA-polymerase-II-associated factors shows loss of PAF1 and RTF1 enhances silencing of 5′ intron piRNA-targeted reporters. Standard deviation (SD) of biological triplicates with significant differences to siGFP control is shown; ∗p < 0.05; ∗∗p < 0.01; t test. See also Figures S1 and S2. Current Biology 2017 27, 2718-2726.e4DOI: (10.1016/j.cub.2017.07.052) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 PAF1 Modulation of piRNA-Targeted Reporters (A) Left: piRNA profiles for a Flam 600-bp element and a Tj-3′ UTR 300-bp element. Right: positions of sense and antisense piRNA-target segments inserted into 5′ UTR, 5′ intron, 3′ intron, or 3′ UTR reporter regions. (B and C) Assay results for Flam-600-bp element (B) and Tj-3′ UTR-300-bp element (C) during siPIWI and siPAF1 treatment. (D) Assay results of factors impacting transcription termination (CSTF64), elongation (DRE4/SPT16), and RNA Pol II pausing (CDK9/pTEFb). SD of biological triplicates with significant differences to siGFP control is shown; ∗p < 0.05; ∗∗p < 0.01; t test. Current Biology 2017 27, 2718-2726.e4DOI: (10.1016/j.cub.2017.07.052) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 OSS Cell Transcriptomes after PAF1 Knockdown Display Greater Repression of TE Transcripts (A) Boxplot of TE expression changes with minimum and maximum whiskers and ∗∗p < 0.01 and ∗∗∗p < 0.001; Wilcoxon rank sum test. (B) Coverage plots for nascent (top tracks) and mature RNA (bottom tracks) reads for TE consensus sequences, with small RNAs on the lowest track. Additional TE consensus tracks are in Figure S3B. (C) Boxplot of nascent and mature RNAs from expressed genes grouped by regulation and TE features, with the number of nascent and mature RNA genes in parentheses, respectively. Whiskers represent minimum and maximum values and ∗∗∗p < 0.001; Wilcoxon rank sum test. (D) Coverage plots for genes upregulated by siPIWI but downregulated by siPAF1, either at the nascent RNA (Crb), mature RNA (Sbb), or at both levels (CG34393 and CG6441). Full gallery is in Figure S3H. (E) Boxplot of TE-linked lncRNAs. Whiskers represent minimum and maximum values and ∗p < 0.05; Wilcoxon rank sum test. (F) Coverage plots of lncRNAs upregulated by siPIWI but downregulated by siPAF1. (G) Transcripts upregulated by siPIWI knockdown but cannot be called lncRNAs because of the same strand polarity in coverage across protein-coding genes. Dashed line brackets mark lncRNA and TE-linked long transcripts domains. See also Figure S3. Current Biology 2017 27, 2718-2726.e4DOI: (10.1016/j.cub.2017.07.052) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 PAF1 Antagonizes PIWI Silencing (A) ChIP/qPCR analyses of H3K4me3 and H3K9me3 on OSS cell loci and reporters after siRNA knockdown and reporter transfection. Left set of columns represent cells transfected with sense Flam reporters, and right set are cells with the antisense Flam reporters. SD of triplicate measurements from biological duplicates, with significant differences compared to siGFP control, is shown; ∗p < 0.05; t test. (B) Assay results for Flam reporter (top) and a mdg1-3′ end segment reporter (bottom). SD of biological triplicates with significant differences to the siGFP control is shown; ∗p < 0.05; ∗∗p < 0.01; t test. Additional bars mark tests between siPIWI and siPAF1 to siPIWI + siPAF1. (C) A model proposing that direct factors assist PIWI in targeting nascent and maturing transcripts, with silencing capacity influenced by proximity of the piRNA-targeted element to the promoter. PAF1/RTF1 reduction may stall nascent RNA release to enhance PIWI silencing. See also Figure S4. Current Biology 2017 27, 2718-2726.e4DOI: (10.1016/j.cub.2017.07.052) Copyright © 2017 Elsevier Ltd Terms and Conditions