A Major Epigenetic Programming Mechanism Guided by piRNAs Xiao A. Huang, Hang Yin, Sarah Sweeney, Debasish Raha, Michael Snyder, Haifan Lin  Developmental Cell  Volume 24, Issue 5, Pages 502-516 (March 2013) DOI: 10.1016/j.devcel.2013.01.023 Copyright © 2013 Elsevier Inc. Terms and Conditions

Developmental Cell 2013 24, 502-516DOI: (10.1016/j.devcel.2013.01.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Distribution of ChIP-Seq (U+M) Scores over Genomic Features Distribution of ChIP-seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, introns, transposons, repetitive sequences, and intergenic regions within the whole genome (X, 2L, 2R, 3L, 3R, 4, XHet, 2LHet, 2RHet, 3LHet, 3RHet, YHet, U, Uextra). The top and bottom rows show the distributions in wild-type and piwi1/piwi2 flies, respectively. See also Figure S1 and Table S1. Developmental Cell 2013 24, 502-516DOI: (10.1016/j.devcel.2013.01.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Genome-wide Colocalization of Piwi and Piwi-Associated piRNAs (A) Genome-wide localization of Piwi and Piwi-associated piRNAs are shown for X, 2L, 2R, 3L, 3R, 4, and unassembled Contig U. The horizontal line shows the length of each chromosome arm proportionally. The red peak above the chromosomal line represents Piwi ChIP scores [sum of ChIP-seq (U+M) scores per 10 kb window; both unique-mapping and multiple-mapping reads were considered], and the green peak below the chromosomal line represents the abundance of Piwi-associated piRNAs (numbers of piRNAs per 10 kb window). Gray ovals indicate centromeres. Asterisks denote enrichment of Piwi in telomere regions. Boxed regions labeled as B, C, and D are 260 kb region containing the 42AB piRNA cluster, a 150 kb region of sporadic transposons, and a 75 kb region containing a gene CG32377, respectively. See also Figure S2. (B–D) Zoomed-in views of localization of Piwi and Piwi-associated piRNAs at the 42AB piRNA cluster (B), a sporadic transposon region (C), and CG32377 (D). Developmental Cell 2013 24, 502-516DOI: (10.1016/j.devcel.2013.01.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Ectopic piRNA Target Sequences Recruit Piwi and HP1a De Novo (A) A diagram depicts the experimental design of the ectopic recruitment assay for Piwi-piRNA complexes. (B and C) Two piRNA target sequences (originally located in 2L and 3R) were inserted to the same genomic location (cytoband 68A4 in 3L) via Phi31C recombination-based site-specific transgenic system. The resulting transgenic strains were referred to as line 1 and line 2, respectively. (D) An exogenous LacI sequence of similar length or same tandem-arrayed copies of scrambled sequences for line 1 or 2 was also inserted to the same ectopic site as negative controls. The resulting transgenic strains were referred as line 3, 1S, and 2S, respectively. (E–J) Piwi is ectopically recruited to piRNA target insertion site. Piwi is not present in the ectopic site before the insertion (E) but is recruited to ectopic piRNA target insertion sites at a comparable level of enrichment as in the original sites in line 1 (F) and line 2 (G). Negative control lines (H-J) did not show any Piwi recruitment. IP: ChIP using antibody against Piwi. IgG: ChIP using nonspecific IgG. (K–P) HP1a is not enriched in the ectopic site before the insertion (K), but is also recruited to ectopic piRNA target sequence insertion sites in both line 1 (L) and line 2 (M), but not in negative control lines (N–P). IP: ChIP using antibody against HP1a. IgG: ChIP using nonspecific IgG. Error bars in (E)–(P) denote SD. Developmental Cell 2013 24, 502-516DOI: (10.1016/j.devcel.2013.01.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 The Ectopic Recruitment of Piwi and HP1a Is Sensitive to RNase Treatment and Is Achieved by piRNA from the Original Site (A) Piwi ChIP in the presence or absence of RNase A digestion. In both line 1 and line 2, Piwi binding to the original site and the ectopic site is abolished by RNase treatment. (B) HP1a ChIP in the presence or absence of RNase A. Similar to (A), HP1a binding to original site and the ectopic site is impaired in both line 1 and line 2 by RNase treatment. (C) H3K9me3 ChIP in the presence or absence of RNase A. Unlike (A) and (B), H3K9me3 signal in the original site and the ectopic site is not affected in both line 1 and line 2 by RNase treatment. (D) The impact of mismatches (MM) on the efficacy of piRNA binding to its target sequence. S, scrambled sequence. See also Figure S3. (E and F) The ectopic sites in line 1 (C) and line 2 (D) produce negligible levels of RNA transcripts comparing to their original piRNA-generating sites. O: original site; L and R: sequences spanning the left and right insertion junctions at the ectopic site. Error bars in (A)–(F) denote SD. Developmental Cell 2013 24, 502-516DOI: (10.1016/j.devcel.2013.01.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 The Change of Chromatin State at the Ectopic Site upon Recruitment of Piwi ChIP-qPCR using antibodies specific to the histone marks (A–H) and RNA Pol II (I) indicates that the repressive histone marks H3K9me2/3 but not active histone marks are enriched at the ectopic sites. In addition, RNA Pol II binding is reduced upon ectopic Piwi recruitment. Error bars denote SD. Developmental Cell 2013 24, 502-516DOI: (10.1016/j.devcel.2013.01.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Distinct Colocalization Patterns of Piwi and Piwi-Associated piRNAs in Euchromatin and Heterochromatin Suggest Two Modes of Piwi-piRNA Guidance Mechanism (A and B) Relative positions of Piwi, Piwi-associated piRNA, and transposons within the euchromatic genome (X, 2L, 2R, 3L, 3R, 4) and the heterochromatic genome (XHet, 2LHet, 2RHet, 3LHet, 3RHet, YHet, U, and Uextra). Piwi-associated piRNAs were aligned at their 5′ ends in the same direction. Gray dash lines indicate positions of piRNAs. Piwi ChIP-seq scores within upstream and downstream regions surrounding piRNA-transcribing regions (±3 kb) were separately plotted for euchromatic genome (orange) and heterochromatic genome (blue), together with the transposon density (TE density; green). See also Figure S4. (C and D) Relative positions of Piwi with piRNAs derived from piRNA clusters (green) and other sporadic piRNAs (orange). (E) Heatmaps depict Piwi ChIP-seq scores over various types/classes of transposons within genome. Average Piwi ChIP-seq scores of all same types of transposons within genome (total) or only within piRNA clusters (cluster) were separately calculated. (F) Heatmaps depict levels of chromatin-associated RNA Pol II over various types/classes of transposons within wild-type flies (left) and piwi1/piwi2 mutants (right). Average RNA Pol II ChIP-seq scores were separately calculated for all same types of transposons on chromosomal arms (X, 2L, 2R, 3L, 3R, 4), on contigs (U, Uextra) as well as for all same types of transposon within genome (genome average) or within piRNA clusters (cluster average). Developmental Cell 2013 24, 502-516DOI: (10.1016/j.devcel.2013.01.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Chromosome-wide Changes of Chromatin States in Piwi Mutants (A) Distribution of various epigenetic regulators/marks over the entire chromosome arm 2L [ChIP-seq (U+M) scores; both unique-mapping and repetitive sequences were considered] in wild-type and piwi1/piwi2 mutants. cen, the centromeric end of 2L; tel, the telomeric end of 2L. ChIP-seq scores were binned and averaged for every 20 kb window on the plots. See also Figures S5 and S6 and Tables S2 and S3. (B) Distribution of various epigenetic regulators/marks over the entire contig Uextra (ChIP-seq [U+M] scores; both unique-mapping and repetitive sequences were considered) in wild-type and piwi1/piwi2 mutants. See also Figures S5 and S6 and Tables S2 and S3. Developmental Cell 2013 24, 502-516DOI: (10.1016/j.devcel.2013.01.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 8 The Piwi-piRNA Epigenetic Guidance Model The steps of epigenetic factor recruitment by the Piwi-piRNA complex. For details, see text. Developmental Cell 2013 24, 502-516DOI: (10.1016/j.devcel.2013.01.023) Copyright © 2013 Elsevier Inc. Terms and Conditions