MIWI 2 targets RNAs transcribed from pi RNA ‐dependent regions to drive DNA methylation in mouse prospermatogonia
2018; Springer Nature; Volume: 37; Issue: 18 Linguagem: Inglês
10.15252/embj.201695329
ISSN1460-2075
AutoresToshiaki Watanabe, Xiekui Cui, Zhongyu Yuan, Hongying Qi, Haifan Lin,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoArticle14 August 2018free access Transparent process MIWI2 targets RNAs transcribed from piRNA-dependent regions to drive DNA methylation in mouse prospermatogonia Toshiaki Watanabe Corresponding Author [email protected] Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Xiekui Cui Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Zhongyu Yuan Zhiyuan College, Shanghai Jiaotong University, Shanghai, China Search for more papers by this author Hongying Qi Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Haifan Lin Corresponding Author [email protected] orcid.org/0000-0002-5374-9491 Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Toshiaki Watanabe Corresponding Author [email protected] Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Xiekui Cui Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Zhongyu Yuan Zhiyuan College, Shanghai Jiaotong University, Shanghai, China Search for more papers by this author Hongying Qi Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Haifan Lin Corresponding Author [email protected] orcid.org/0000-0002-5374-9491 Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Author Information Toshiaki Watanabe *,1, Xiekui Cui1, Zhongyu Yuan2, Hongying Qi1 and Haifan Lin *,1 1Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA 2Zhiyuan College, Shanghai Jiaotong University, Shanghai, China *Corresponding author. Tel: +81 70 4388 3690; E-mail: [email protected] *Corresponding author. Tel: +1 203 785 6239; E-mail: [email protected] EMBO J (2018)37:e95329https://doi.org/10.15252/embj.201695329 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Argonaute/Piwi proteins can regulate gene expression via RNA degradation and translational regulation using small RNAs as guides. They also promote the establishment of suppressive epigenetic marks on repeat sequences in diverse organisms. In mice, the nuclear Piwi protein MIWI2 and Piwi-interacting RNAs (piRNAs) are required for DNA methylation of retrotransposon sequences and some other sequences. However, its underlying molecular mechanisms remain unclear. Here, we show that piRNA-dependent regions are transcribed at the stage when piRNA-mediated DNA methylation takes place. MIWI2 specifically interacts with RNAs from these regions. In addition, we generated mice with deletion of a retrotransposon sequence either in a representative piRNA-dependent region or in a piRNA cluster. Both deleted regions were required for the establishment of DNA methylation of the piRNA-dependent region, indicating that piRNAs determine the target specificity of MIWI2-mediated DNA methylation. Our results indicate that MIWI2 affects the chromatin state through base-pairing between piRNAs and nascent RNAs, as observed in other organisms possessing small RNA-mediated epigenetic regulation. Synopsis The nuclear Piwi protein MIWI2 regulates DNA methylation of specific genomic regions in murine spermatogenic cells. Base-pairing between MIWI2-bound piRNAs and nascent RNA determines the target specificity of piRNA-dependent DNA methylation. piRNA-dependent regions are transcribed at the developmental stage when piRNA-mediated DNA methylation occurs. The MIWI2-piRNA complex specifically interacts with RNAs transcribed from piRNA-dependent regions. Removing a retrotransposon sequence either in a piRNA-dependent region or a piRNA cluster abolishes DNA methylation of the piRNA-dependent region. In Mili−/− prospermatogonia deficient in producing MIWI2-bound piRNAs, de novoDNA methylation is compromised in many MIWI2-dependent regions. Introduction Gene expression is regulated at multiple steps, including transcription, splicing, RNA export, RNA localization, translation, and RNA degradation, with multiple mechanisms acting together to determine which genes are expressed at each step. Piwi-interacting RNAs (piRNAs) are 21–30 nt small RNAs that are mainly expressed in the germline (Watanabe & Lin, 2014). piRNAs act together with Piwi family proteins, a subfamily of Argonaute proteins. The Piwi-piRNA complex binds to target RNAs, including retrotransposon RNA, mRNA, and long non-coding RNA, via piRNA sequence complementarity (Bagijn et al, 2012; Sienski et al, 2012; Sytnikova et al, 2014; Wasik et al, 2015; Watanabe et al, 2015). The target RNAs are sometimes cleaved by the endonuclease (slicer) activity of Piwi proteins, which leads to degradation of the target RNAs. In addition to post-transcriptional regulation, nuclear Piwi proteins in mice and flies are essential for the establishment of repressive chromatin marks on some retrotransposon sequences, which is required for normal gametogenesis (Carmell et al, 2007; Klenov et al, 2011). Although the mechanisms of piRNA-mediated transcriptional regulation in the fly have been well studied (Sienski et al, 2012, 2015; Huang et al, 2013; Le Thomas et al, 2013; Rozhkov et al, 2013; Yu et al, 2015; Iwasaki et al, 2016; Peng et al, 2016), very little is known about these mechanisms in mammals. There are three Piwi proteins in mice: MIWI, MIWI2, and MILI. MIWI2 and MILI are expressed in prospermatogonia (Aravin et al, 2008; Kuramochi-Miyagawa et al, 2008), where the genome-wide establishment of DNA methylation takes place. MIWI2 is present in nuclei and is required for DNA methylation and histone H3 lysine 9 trimethylation (H3K9me3) of some retrotransposons, such as LINE1 (L1) and IAP (Aravin et al, 2007, 2008; Carmell et al, 2007; Kuramochi-Miyagawa et al, 2008; Shoji et al, 2009; Molaro et al, 2014; Pezic et al, 2014; Manakov et al, 2015; Nagamori et al, 2015; Kojima-Kita et al, 2016). Because piRNAs expressed at this stage are preferentially mapped to these retrotransposon sequences (Aravin et al, 2008; Kuramochi-Miyagawa et al, 2008), MIWI2-bound piRNAs likely function as sequence determinant for the transcriptional repression of these retrotransposons. However, there is little direct evidence that they play this role. Here, we show that MIWI2 affects chromatin states by targeting RNAs transcribed from piRNA-dependent regions using piRNAs as a guide. In addition, we report the salient features observed among the piRNA-dependent regions. Results Identification of hypomethylated regions in Mili knockout prospermatogonia Recent genome-wide studies have identified genomic targets of piRNA-mediated epigenetic regulation by comparing DNA methylation (or H3K9me3) pattern between Mili−/− (or Miwi2−/−) and control mice (Molaro et al, 2014; Pezic et al, 2014; Manakov et al, 2015; Nagamori et al, 2015). These studies used spermatocytes or spermatogonia from 10 days post-partum (dpp) testes for the analyses despite the occurrence of piRNA-mediated epigenetic regulation in prospermatogonia of ~16–19 days post-coitum (dpc) testes (Kato et al, 2007). Given that DNA methylation is dynamically regulated during spermatogonial development (Hammoud et al, 2015; Kubo et al, 2015), some genomic targets might acquire DNA methylation (or H3K9me3) in spermatogonia independently of piRNAs. Such target regions could not be identified using spermatogonium and spermatocyte data in those studies. To identify a more complete set of genomic targets of piRNA-mediated DNA methylation, we conducted whole-genome bisulfite sequencing analysis using DNA obtained from 0 to 2 dpp Mili−/− and Mili+/− prospermatogonia (Appendix Supplementary Results and Fig EV1), because genome-wide de novo DNA methylation has just completed at this stage (Kato et al, 2007). In Mili−/− prospermatogonia, not only MILI-bound piRNAs but also a large portion of MIWI2-bound piRNAs are lost, and therefore, de novo DNA methylation of many MIWI2-dependent regions is compromised (Aravin et al, 2008; Kuramochi-Miyagawa et al, 2008; Vasiliauskaite et al, 2017). Although global DNA methylation pattern was not changed (Fig EV2A), 6,541 genomic regions were identified to be hypomethylated in Mili−/− prospermatogonia by applying a stringent condition to the output of the Bisulfighter program (Saito et al, 2014). 88.1% of HMRs were 200–3,000 nt in length (Fig EV2B). Most HMRs identified in prospermatogonia were also hypomethylated in Mili−/− spermatocytes (Fig EV2C), although, overall, the extent of hypomethylation of these HMRs seemed to be more severe in prospermatogonia than in spermatocytes (Fig EV2D and E). Conversely, most of the 1,033 hypomethylated regions identified in Mili−/− spermatocytes were also hypomethylated in Mili−/− prospermatogonia (Fig EV2F). Of the 1,033 spermatocyte hypomethylated regions, 828 were overlapped with prospermatogonium HMRs. These results suggest almost the same loci are hypomethylated in Mili−/− prospermatogonia and spermatocytes. Click here to expand this figure. Figure EV1. Statistics for bisulfite sequencing Statistics for the CpG methylation rate. Pearson correlation and mean Euclidean distance between CpG methylation rates of two biological replicates are shown. Only CpG sites covered with more than 10 mapped reads in both replicates were considered for the analyses. Of 12406548 (Mili+/−) and 8228713 (Mili−/−) CpG sites that were considered for the analyses, 7,650,754 CpG sites were common between Mili+/− and Mili−/−. Genomic coverage of bisulfite sequencing. Total read numbers of both biological replicates were used for this analysis. For mean coverage, only CpGs covered with at least one unique sequence read were considered. DNA methylation pattern of maternally and paternally methylated loci. Hypomethylated region identified in the Rasgrf1 differentially methylated region (DMR) is indicated by green bar. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Features of hypomethylated regions identified in Mili−/− prospermatogonia (HMRs) DNA methylation pattern is not changed in Mili−/− prospermatogonia at chromosome levels. Average CpG DNA methylation levels (2-kb window) are plotted on each chromosome. Length distribution of hypomethylated regions in Mili−/− prospermatogonia (HMRs). The numbers in parentheses represent the numbers of regions examined. Most HMRs are kept hypomethylated in Mili−/− spermatocytes. Circles in the charts show 6,541 HMRs. X-axis (Y-axis) represents average DNA methylation level in Mili−/− (control) mice. The results in prospermatogonia (top) and spermatocytes (bottom) are shown. DNA methylation of HMRs seems to be more severely affected in prospermatogonia compared with spermatocytes. X-axis (Y-axis) represents ratio of DNA methylation level between Mili KO and control mice in prospermatogonia (spermatocytes). A representative genomic locus that shows clear difference in DNA methylation level between Mili mutant and control mice in prospermatogonia compared with spermatocytes. Most hypomethylated regions in Mili KO spermatocyte are also hypomethylated in prospermatogonia. See (D) for information about the chart. Download figure Download PowerPoint Consistent with studies analyzing epigenetic patterns in spermatogonia and spermatocytes of piRNA pathway mutants (Molaro et al, 2014; Pezic et al, 2014; Manakov et al, 2015; Nagamori et al, 2015), 80.9% of the 6,541 HMRs were located in the intergenic regions (Appendix Fig S1A), and the HMRs frequently included LINE and LTR retrotransposons, with a predominance of young retrotransposons (e.g., L1Md_T, L1Md_A, RLTR10, ETn, IAP, L1Md_Gf, MMERVK10C, and MMERGLN; Appendix Fig S1B–D). Notably, approximately two-thirds (4,299/6,541) of the HMRs included young L1 retrotransposon sequences that showed > 90% identity with the consensus sequence. Within L1 sequences, the 5′ regions were more frequently associated with the HMRs than internal or 3′ regions (Appendix Fig S1E). This result is in accordance with the studies showing that the 5′ regions, but not internal or 3′ regions, of L1s, are specifically affected in piRNA-mutant mice (Molaro et al, 2014; Manakov et al, 2015). For subsequent analyses, HMRs were divided into two classes according to the presence (HMR_+L1s, 4,299 regions) or absence (HMR_−L1s, 2,242 regions) of young L1s (Fig EV2A, Appendix Fig S1A, B and F) to allow effective analysis of L1-free HMRs so that they will not be "buried" by the abundant presence of L1s. RNA expression in piRNA-dependently methylated regions If MIWI2 targets the chromatin of piRNA-dependent regions via nascent RNAs, RNAs with piRNA-complementary sequences would be transcribed in these regions. Consistent with this, piRNA-complementary sequences were specifically observed in HMRs (Appendix Supplementary Results, Fig EV3). A study of DNA methylation analysis in Mili−/− spermatogonia (Molaro et al, 2014) has shown positive correlation between the extent of demethylation in Mili−/− spermatogonia and RNA expression level at the stage of piRNA-mediated DNA methylation among individual L1 sequences, supporting the view that piRNA-mediated epigenetic regulation requires RNA expression. However, such correlation has not been observed for non-L1 retrotransposons, such as IAP, despite their demethylation in Mili−/− spermatogonia (Molaro et al, 2014). This raises a question as to whether HMR_−L1s are transcriptionally active in prospermatogonia. Click here to expand this figure. Figure EV3. HMRs frequently include target sites of MIWI2-bound piRNAs A. Box plots showing the number of piRNAs mapped to 1-kb regions spanning the centers of HMRs and randomly selected sites. "–LINE" (–LTR and –SINE): piRNAs derived from LINE (LTR and SINE) were removed before mapping. The numbers in parentheses represent the numbers of regions examined. B–D. Heatmap showing the number of piRNAs mapped to HMRs with ("HMR_+L1s"; B and C) or without ("HMR_−L1s"; D) young LINE1 retrotransposons. In (B and D), "–LINE" and "–LTR" on the top of the charts show that piRNAs derived from LINE and LTR sequences, respectively, were removed before mapping. In (C), HMR_+L1s containing sense- and antisense-oriented L1s are shown as separate maps. HMR_+L1s containing both sense- and antisense-orientated L1s were not analyzed. Download figure Download PowerPoint To examine this, we carried out RNA-seq analysis using RNA isolated from 16.5 dpc prospermatogonia. Heatmap and metaplot analyses revealed significantly higher levels of RNA expression in HMR_−L1s compared with the surrounding regions despite the high proportions of repeat sequences in HMR_−L1s (Fig 1A and B), which leads to underestimation of the expression level. Although unique RNA-seq reads were not observed in 10.5% of HMR_−L1s (Appendix Fig S2A), these HMR_−L1s were enriched in repetitive sequences (Appendix Fig S2B). Hence, it appears that more HMR_−L1s actually express RNAs than those where the unique RNA-seq reads are derived. RNAs expressed from HMR_−L1s seemed to be polyadenylated, because all five individual HMR_−L1-derived RNAs examined were enriched in oligo (dT) bead-selected RNAs as compared to non-polyadenylated RNAs (histone H3.1 mRNA, 18S rRNA, and 7SK RNA; Appendix Fig S2C). Thus, RNA expression at the stage of genome-wide de novo DNA methylation seems to be a common feature shared among HMRs. Figure 1. Hypomethylated regions express RNAs potentially targeted by MIWI2-bound piRNAs A, B. HMRs express RNAs. Heatmaps (A) and metaplots (B) show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses. Positions (1)–(4) in (A and B) (green arrows) indicate the same positions (±2 kb from the centers). The lower chart in (B) represents mappability defined as the percentage of unique sequences in all possible 50-nt sequences (see Appendix Fig S2B). TSS isoforms are included in 55,798 TSSs. Low mappability at the centers of HMR_−L1s is due to high occurrence of repeat (retrotransposon) sequences. RPM, reads per million mapped reads. Download figure Download PowerPoint Hypomethylated regions show specific epigenetic features The presence of L1 promoter regions in the HMR_+L1s (Fig EV4C, Appendix Fig S1E) and preferential demethylation of promoter regions within retrotransposon sequences in Mili−/− spermatocytes (Molaro et al, 2014) suggested that HMR_−L1s might also harbor promoters that are active at the stage of de novo DNA methylation. Indeed, heatmap and metaplot analyses of the RNA-seq data of 16.5 dpc prospermatogonia showed a peak of RNA expression at the centers of HMR_−L1s, as observed in transcription start sites (TSSs) of coding genes (Fig 1A and B). Furthermore, plus-strand RNAs were more highly expressed in the downstream regions of HMR_−L1s [(2) in Fig 1A and B] than in their upstream regions [(1) in Fig 1A and B]; the opposite trend was observed for minus-strand RNAs [(3) and (4) in Fig 1A and B)]. Again, a similar pattern was observed for TSSs of coding genes (Fig 1A and B). Click here to expand this figure. Figure EV4. A unique epigenetic signature in hypomethylated regions and LINE1 promoters Enrichment of histone marks associated with promoters against input or histone H3 in two representative HMR_−L1s. Y-axis represents reads per million uniquely mapped reads (RPM). The numbers in parentheses represent the coverage for a whole genome. Only the results of one of the two replicates are shown when there are biological replicates (Appendix Table S1), as the results of the two replicates were similar. The ChIP-seq data with biological replicates are indicated as "rep1" or "rep2" in the panel. HMR_−L1 shown in the left panel is located in an intergenic region on chromosome 1. HMR_−L1 shown in the right panel is located within the Rasgrf1 differentially methylated region (DMR). L1 promoters are active in 15.5 dpc prospermatogonia. ChIP-seq signals normalized by H3 or input signals are shown. For the analyses of L1Md_Tf and L1Md_A, all the reads were mapped to their consensus sequences. For transcription start sites (TSSs), unique reads were used for mapping, and the average signals across all coding genes are shown. Download figure Download PowerPoint To investigate the presence of promoters in HMR_−L1s, several histone modifications associated with active promoters (H3K4me1, H3K4me3, H3K9ac, H3K27ac, and H2A.Zac) were examined in 15.5 dpc prospermatogonia using chromatin immunoprecipitation and sequencing (ChIP-seq) analysis. The results of two representative HMRs are shown in Fig EV4A. Metaplot and heatmap analyses showed clear peaks at the centers of the HMR_−L1s for all of these histone modifications (Fig 2A), suggesting the presence of promoters in many HMR_−L1s. However, the analyses also revealed some differences in the pattern of histone modification between HMR_−L1s and coding gene promoters (Fig 2A). First, HMR_−L1s showed much stronger H2A.Zac signals than coding gene promoters. Second, although the H3K4me3 signal in HMR_−L1s was higher than that in coding gene promoters, the opposite pattern was observed for the H3K4me1 signal. Similar trends (high H2A.Zac and H3K4me3 signals and low H3K4me1 signal as compared to coding gene promoters) were observed with promoters of L1s that are often found in HMR_+L1s (Fig EV4B). Thus, HMRs appear to carry unique epigenetic signatures. Figure 2. Hypomethylated regions show features of active promoters Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons (HMR_−L1s). Metaplots show the average ChIP-seq signals across HMR_−L1s, transcription start sites (TSSs), and randomly selected sites. The signals were normalized by either H3 or input signal. The results shown in heatmaps were not normalized by H3 or input. The initiation form of Pol2 with an unphosphorylated carboxy-terminal domain (Pol2 null-P CTD) is highly enriched in HMR_−L1s. Download figure Download PowerPoint To obtain additional evidence for the presence of active promoters in HMRs, Pol2 ChIP were carried out using 15.5 dpc prospermatogonia. After the initiation of transcription, Pol2 pauses at the promoter-proximal region, and active elongation starts after the serine residue at position 2 of the carboxy-terminal domain (CTD) of Pol2 is phosphorylated (Adelman & Lis, 2012; Liu et al, 2015). Therefore, CTD phosphorylation on the serine at position 2 (S2P CTD) is observed only in the elongated form of Pol2, whereas unphosphorylated CTD (null-P CTD) is mainly observed in the initiation form of Pol2 (Nojima et al, 2015). Consistent with the pattern of promoter-associated histone modifications (Fig 2A), the null-P CTD signal peaked at the centers of HMR_−L1s, while the S2P CTD signal increased from the centers toward the sides of HMR_−L1s (Fig 2B). However, compared with coding genes, HMR_−L1s displayed a considerably higher null-P CTD signal and lower S2P CTD signal (Fig 2B). Similarly, the enrichment of null-P CTD was observed in L1 promoters that are frequently found in HMR_+L1s (Fig EV4B). MIWI2 interacts with RNAs transcribed from hypomethylated regions To investigate whether the RNAs derived from HMR_−L1s are indeed targeted by piRNAs, we examined the RNAs bound to EGFP-tagged MIWI2 (EGFP-MIWI2) using BAC transgenic mice expressing EGFP-MIWI2 from the Miwi2 promoter (Aravin et al, 2007). EGFP-MIWI2 was immunoprecipitated with an EGFP antibody from lysates extracted from the FACS-purified 16.5 dpc prospermatogonia. RNAs bound to EGFP in 16.5 dpc prospermatogonia from knock-in mice expressing EGFP from the Oct4 locus serve as negative controls (Lengner et al, 2007). HMR_−L1-derived RNAs, but not prospermatogonium-expressed mRNAs, were significantly enriched in the RNAs bound to EGFP-MIWI2 (Fig 3A left), whereas no such enrichment was observed in the RNAs bound to EGFP (Fig 3A right). Furthermore, this specific interaction between EGFP-MIWI2- and HMR_−L1-derived RNAs was not observed in testes from Mili−/− neonatal mice, in which binding of EGFP-MIWI2 proteins to piRNAs was largely impaired (Fig 3B and Appendix Fig S3A). These results are consistent with our hypothesis that MIWI2 interacts with HMR_−L1-derived RNAs in a manner dependent on piRNAs. Figure 3. MIWI2 and piRNAs target RNAs from hypomethylated regions RNA immunoprecipitation (RIP) analyses of EGFP-MIWI2 and EGFP. EGFP-MIWI2 and EGFP were immunoprecipitated from 16.5 dpc prospermatogonia, and long RNAs bound to these proteins and input RNAs were sequenced. RNAs expressed from HMR_−L1s and piRNA clusters, but not Ensembl and prospermatogonium-expressed mRNAs, are overall enriched by immunoprecipitation (IP) of EGFP-MIWI2 (left). This enrichment is not observed when EGFP is immunoprecipitated (right). The numbers of mRNAs, piRNA clusters, and HMR_−L1s analyzed were shown in parentheses. Prospermatogonium-expressed mRNAs are those that are highly and specifically expressed in prospermatogonia. The bottom and top of boxes represent 25% percentile and 75% percentile, respectively. The line in the box represents the median. Circles represent outliers, and the bottom (top) of whiskers represents the minimum (maximum) values except for outliers. RIP analyses of EGFP-MIWI2 in newborn testes from WT and Mili KO mice. RNA bound to EGFP-MIWI2 and input RNA was sequenced. All RNAs derived from each class was counted for this analysis. HMR_−L1s preferentially generate piRNAs. X- and Y-axes represent expression levels of long RNAs (16.5 dpc prospermatogonia) as determined by RNA-seq and MIWI2-bound piRNAs (18.5 dpc testes) as determined by small RNA-seq, respectively. Circles in the charts represent individual mRNAs, piRNA clusters, and HMR_−L1s. More than half of the HMR_−L1s (right) are located in the area of y > x (upper part of the diagonal), whereas most mRNAs (left) are located in the area of y < x (lower part of the diagonal). RPKM, reads per kilobase per million mapped reads. Frequent 10-nt overlap between sense and antisense piRNAs derived from HMR_−L1s. Top 100 HMR_−L1s that generate the largest number of unique piRNAs were analyzed. Frequencies of 1- to 18-nt overlaps were analyzed for each HMR_−L1. piRNAs derived from HMR_−L1s are enriched in adenine at their 10th nucleotide. HMR_−L1s generating more than 40 unique piRNAs were analyzed (678 HMR_−L1s). The proportions of piRNAs with adenine (left) and uridine (right) at the indicated positions were analyzed for each HMR_−L1. See (A) for the explanation of box plots. Download figure Download PowerPoint We found that piRNA precursors were also enriched in the RNAs bound to EGFP-MIWI2 (Fig 3A). Therefore, the interaction between MIWI2- and HMR_−L1-derived RNAs might reflect the generation of piRNAs from HMR_−L1-derived RNAs rather than the direct targeting of these RNAs by MIWI2. Consistent with this, 5′ ends of HMR-derived RNAs bound to EGFP-MIWI2 were preferentially aligned with 5′ portions of piRNAs from the same HMRs (Appendix Fig S3B and see below for piRNA generation from HMRs). However, given that target RNAs of piRNAs are processed into piRNAs in some cases and piRNAs are preferentially generated from 5′ ends of RNAs sliced by Piwi-piRNA complexes (Brennecke et al, 2007; Gunawardane et al, 2007; De Fazio et al, 2011; Han et al, 2015; Homolka et al, 2015; Mohn et al, 2015; Yang et al, 2016), these results are nevertheless consistent with the notion that RNAs transcribed from HMRs are targeted by piRNAs. RNAs transcribed from hypomethylated regions are targeted by piRNAs To further examine whether transcripts from HMRs are targeted by piRNAs, we analyzed piRNAs biogenesis with regard to these transcripts. In prospermatogonia, after the cleavage of target RNAs by MILI, secondary piRNAs are generated from the 5′ ends of the 3′ cleavage products (Aravin et al, 2007; Kuramochi-Miyagawa et al, 2008; Shoji et al, 2009; De Fazio et al, 2011). These secondary piRNAs are incorporated into MILI and MIWI2. Because MILI and MIWI2 bind to similar sets of piRNAs in prospermatogonia (Appendix Fig S3C) (Aravin et al, 2008), we reasoned that if HMR_−L1-derived RNAs are targeted by MIWI2 in nuclei, they would also be targeted by MILI when exported to the cytoplasm, resulting in the generation of secondary piRNAs. Consistent with this, the HMR_−L1s produced a much larger number of piRNAs compared with coding genes showing the same expression level (Fig 3C). In fact, 22 HMR_−L1s were found within the top 95 piRNA clusters that generated the largest number of piRNAs in prospermatogonia (Kuramochi-Miyagawa et al, 2008), further suggesting that HMR_−L1s preferentially generate piRNAs. We observed piRNA generation in 86% (1,924/2,242) of the HMR_−L1s. However, given that the remaining 14% of HMR_−L1s tended to be shorter in length and were more enriched in repetitive sequences (Appendix Fig S3D and E), it appears that most, if not all, HMR_−L1s produce piRNAs. These results further support targeting of HMR_−L1-derived RNAs by piRNAs. We found many HMR_−L1s expressing piRNAs and their precursor RNAs from both strands (Fig EV5A–C). In fact, piRNAs were expressed bidirectionally in all of the top 100 HMR_−L1s showing the highest piRNA expression levels. These observations raised the possibility of a ping-pong cycle between cis-natural sense and antisense transcripts (i.e., repeated generation of secondary piRNAs from both strands; Brennecke et al, 2007; Gunawardane et al, 2007). Secondary piRNAs have two specific features: (i) Because cleavage by Piwi proteins occurs between the 10th and 11th position of piRNAs, the 5′ ends of the piRNAs and the newly generated secondary piRNAs overlap by 10 nt; (ii) because the first nucleotide of the majority of primary piRNAs is uridine, the 10th nucleotide of secondary piRNAs is enriched in adenine. These features can be used to reveal the presence of secondary piRNAs. In support of the generation of secondary piRNAs from HMR_−L1s, a frequent 10-nt overlap between sense and antisense piRNAs and enrichment of adenine at their 10th position were observed among piRNAs from HMR_−L1s (Figs 3D and E, and EV5D). Thus, traces of RNA targeting by piRNAs are found in many HMR_−L1s. Click here to expand this figure. Figure EV5. Ping-pong cycle within each hypomethylated region Bidirectional piRNA expression from HMR_−L1s. Direction index is defined as indicated. When half (all) of piRNAs are derived from the same strand, direction index is 0.5 (1). Only unique piRNA reads were used for this analysis. HMR_−L1s and mRNAs that generate appreciable level of unique piRNAs (≥ 10 unique piRNAs) were selected for this analysis. We divided them into two groups according to the number of generating unique piRNAs (10–99 and ≥ 100, top and bottom panels) in order to rule out the possibility that the difference is arisen from the number of piRNAs analyzed. The numbers of HMR_−L1s and mRNAs analyzed are shown in parentheses. Bidirectional RNA expression
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