piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells

Abstract

Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments.

Keywords: H3K9me3; piRNA; transposon.

Figure 1.

Distribution of the H3K9me3 mark along the genome of mouse somatic and male germ cells. (A) Distribution of the H3K9me3 mark in liver cells of 10-dpp animals on four types of genomic partitions (TSSs, exons, introns, and intergenic space). Values of four ChIP-seq replicas were normalized to the respective input samples and averaged. Error bars show standard deviations. (B) Distribution of the H3K9me3 mark in liver cells of 10-dpp animals over five major TE classes (LINEs, SINEs, LTRs, DNA, and satellites). Values of four ChIP-seq replicas were normalized to the respective input samples and averaged. (C) Enrichment of the H3K9me3 mark over TE families within the LTR and the LINE classes in liver cells, testicular somatic cells, and FACS- or MACS-sorted male germ cells (spermatogonia) of 10-dpp animals. Only families with at least 5000 mapped reads in the library were considered. Families were sorted according to H3K9me3 levels in germ cells. For liver and testicular somatic cells, values of four and two ChIP-seq replicas were averaged, respectively. Three independent biological replicas were used to measure H3K9me3 signal in spermatogonia: One was obtained by FACS-sorting using GFP-Mili, and two were obtained by MACS-sorting with the EpCAM cell surface marker. (D) Distribution of the H3K9me3 mark on all families of LINE and LTR classes combined. For somatic testicular cells and liver cells, values from two and four ChIP-seq libraries were averaged, respectively. For MACS-sorted germ cells, two ChIP-seq libraries were averaged. The error bars show standard error.

Figure 2.

Profiles of the H3K9me3 mark along retrotransposon bodies and flanking sequences. (A) Distribution of normalized level of the H3K9me3 mark along an LTR IAPEz element consensus sequence in liver cells of 10-dpp animals. The Y-axis shows enrichment (log₂) of H3K9me3 signal in ChIP compared with input DNA. Signals above the red dashed line indicate at least twofold enrichment. (B) Metaplot of input-normalized H3K9me3 signal in regions flanking all IAPEz insertions in the genome of testicular somatic cells and spermatogonia. Only uniquely mapped reads were considered. Red and blue dashed lines show the distance at which the signal dropped twofold from the peak value. (C) Distribution of the normalized level of the H3K9me3 mark along a LINE1 element (L1-A) consensus sequence in liver cells of 10-dpp animals. The Y-axis shows enrichment (log₂) of H3K9me3 signal in ChIP compared with input. Signals above the red dashed line indicate at least twofold enrichment. (D) Distribution of the H3K9me3 mark on LTR and LINE families in liver cells, testicular somatic cells, and spermatogonia of 10-dpp animals. The black dots show enrichment on L1 regions corresponding to the first 500 bp of the consensus (the 5′ repeats). Only families that are enriched in the H3K9me3 mark are shown. (E) Metaplot of input-normalized H3K9me3 signal in genomic regions flanking all genomic L1-A insertions in testicular somatic cells and spermatogonia. Only uniquely mapped reads were considered. Red and blue dashed lines show the distance at which the signal dropped twofold from the peak value.

Figure 3.

Effect of Miwi2 mutation on the H3K9me3 mark and TE expression in spermatogonia. (A) Differences in H3K9me3 levels over LINE and LTR families in spermatogonia of 10-dpp Miwi2 knockout (KO) animals relative to those in their heterozygous littermates. Levels of H3K9me3 on 5′ repeats of selected LINEs are displayed as black dots. Heat map shows H3K9me3 levels in LINE and LTR families in germ cells from Miwi2 heterozygous animals; the scale bar at the bottom shows the log₂ (ChIP/input) values of these levels. Two L1 families, L1-A and L1-T, analyzed by independent ChIP-qPCR experiments shown on B, are marked in red. (B) ChIP-qPCR on L1-T and L1-A elements confirms a decrease in the H3K9me3 mark in spermatogonia of Miwi2 knockout animals. Spermatogonia were sorted from testes of 10-dpp animals by MACS using the EpCAM cell surface marker. The signal was normalized to respective ChIP input and internally normalized to signal over IAPLTR1a. Two independent ChIP experiments were performed, and error bars show standard deviation. P-values were calculated using t-test based on two sets of qPCR triplicates for each ChIP. (C) Distribution of the H3K9me3 mark along the consensus sequence of L1-A in spermatogonia of Miwi2 knockout and control animals. The Y-axis shows enrichment (log₂) of H3K9me3 signal in ChIP-seq compared with input. Signals above the red dashed line indicate at least twofold enrichment. (D) Metaplot of the input-normalized level of the H3K9me3 mark in genomic regions flanking all L1-A insertions in spermatogonia of Miwi2 knockout and control animals. Only uniquely mapped reads were considered. Tandem L1-A genomic insertions were excluded from the analysis. (E) Differential expression of genes and TEs in testes of 10-dpp Miwi2 knockout and control animals as measured by RNA-seq. The fold change in expression upon Miwi2 knockout (Y-axis) is plotted against the average expression level (X-axis). Each dot corresponds to a gene or TE family. Genes (red) and TEs (blue) that passed the multiple testing-adjusted P-value < 0.01 threshold are shown. (F) Correlation between the changes in H3K9me3 and expression levels of TE families upon Miwi2 knockout. The X-axis shows the fold difference of H3K9me3 signal in TE families in spermatogonia of Miwi2 knockout and control animals (negative values indicate the loss of H3K9me3 signal in Miwi2 knockout animals). The Y-axis shows the fold difference in abundance of TE transcripts in Miwi2 knockout compared with control mice. LINE and LTR families with at least 5000 mapped reads were considered.

Figure 4.

Distribution of the H3K9me3 mark on individual L1 copies. (A) Correlation between the length of L1-A insertions and their derepression in testes of Miwi2 knockout (KO) mice. All genomic copies of L1-A were binned in groups based on their length. The number of loci in each category is indicated above the boxes. The Y-axis shows fold change in the transcript abundance in testes of 10-dpp Miwi2 knockout animals relative to their heterozygous littermates measured by RNA-seq. Boxes correspond to the 25th and 75th percentiles, and the lines inside the boxes are the medians. The whiskers spread to either 1.5 of IQR (interquartile range) or the farthest outlier if the outlier was within the 1.5 IQR distance. (B) Metaplot of the input-normalized level of the H3K9me3 mark in genomic regions flanking L1-A insertions in spermatogonia of 10-dpp Miwi2 knockout and control mice. All genomic L1-A copies were separated into full-length (with the preserved 500 bp of the 5′ end) and truncated copies. Only reads uniquely aligned to flanks of stand-alone L1-A insertions were considered for this analysis. (C) Distribution of the H3K9me3 mark in 1-kb flanks upstream of full-length (>5 kb) and truncated (<2 kb) L1-A insertions in spermatogonia of Miwi2 knockout and control animals. Only uniquely mapped ChIP-seq reads were considered. Boxes and whiskers show percentiles and IQR. (D) Scatter plot representation of the results shown in C. The plot shows the input-normalized level of the H3K9me3 mark in the 1-kb upstream flanks of individual L1-A copies (Y-axis) in relation to the length of each insertion (X-axis) in spermatogonia of Miwi2 knockout and control mice. The dots correspond to individual L1 copies that had at least one read mapped to their flanks in both ChIP and input libraries (9855 insertions in control and 10,072 in knockout). (E) ChIP-qPCR analysis of H3K9me3 and H3K4me2/3 levels on eight individual full-length L1-A insertions in Miwi2 knockout and control spermatogonia. The four loci in group I had a strong decrease in the H3K9me3 mark upon Miwi2 deficiency according to ChIP-seq shown on D, while the four loci in group II showed mild or no decrease in ChIP-seq. Germ cells were sorted by MACS from 10-dpp animals. H3K9me3 and H3K4me2/3 ChIPs were performed on the same material. H3K9me3 signal was normalized to input and a control region with high H3K9me3 (RMER1B). H3K4me2/3 signal was normalized to input and H3K4me2/3 level on the promoter of the gene encoding RNA polymerase II (Pol II). Error bars show standard deviation. Genomic loci and P-values for analyzed L1-A loci are listed in Supplemental Table S5. P-values were calculated using the t-test. (F) The correlation between the number of piRNAs that are able to target individual full-length L1-A loci and their repression as measured by the drop in H3K9me3 signal in Miwi2 knockout animals compared with the control. The X-axis shows the fold difference of H3K9me3 signal in 1-kb flanks of individual L1-A insertions in spermatogonia of Miwi2 knockout and control animals (negative values indicate the loss of H3K9me3 signal in Miwi2 knockout animals). The Y-axis shows the abundance of MIWI2-associated piRNAs that target the corresponding L1-A loci in wild-type embryonic (embryonic day 16.5 [E16.5]) testes. The number of piRNAs was normalized to lengths of L1-A loci (reads per kilobase of sequence per million mapped reads [RPKM]). An unlimited number of perfect (i.e., no mismatches) genomic alignments per piRNA read was allowed for this analysis.

Figure 5.

The influence of L1 on the expression of adjacent genes. (A) Relationship between the gene expression level in testis and the distance between the gene promoter and the neighboring L1 insertion. Genes with TSSs within 25 kb of full-length (≥6000 bp) L1-A, L1-T, and L1-Gf elements were considered. The Y-axis shows normalized expression as determined by RNA-seq averaged between testes of Miwi2 knockout (KO) and heterozygous 10-dpp animals. Boxes and whiskers show percentiles and IQR as in Figure 3E. (B) RT-qPCR analysis of expression of 15 genes with TSSs <25 kb from a full-length L1 insertion in testes of Miwi2 knockout and heterozygous 10-dpp animals. Results of two biological replicates were averaged; error bars correspond to standard deviations. (C) Genomic environment of the L1-T insertion in the intron of the Clca4 gene (chr3: 144,796,386–144,849,612). Shown are RNA-seq (polyA-selected total RNA) tracks from 10-dpp Miwi2 heterozygous (Het) and knockout testis. Only unique mappers are shown. Arrows designate TSSs and direction of the transcription for Clca4 and L1-T. A portion of the Mili gene is shown for comparison with the Clca4 locus. The Y-axis shows the number of reads. Note that the majority of the RNA-seq reads detected in Miwi2 knockout mapped to intronic sequences of the Clca4 locus, while almost all reads from the Mili gene correspond to exons. (D) ChIP-qPCR analysis of H3K4me2/3 enrichment on the promoters of Clca4 and Pkhd1 in MACS-sorted spermatogonia from 10-dpp Miwi2 knockout and control littermates. Signal was normalized to input and to the H3K4me2/3 level on the promoter of the gene encoding RNA Pol II. Error bars indicate standard deviation. (E) RT-qPCR analysis of the L1-T/Clca4 chimeric transcript in testes of 10-dpp Miwi2 knockout and control animals. Forward primer is in the L1-T insertion, and the reverse primer is in the intronic region of Clca4. RNA was isolated from total testis of two independent sets of Miwi2 heterozygous and knockout littermates. RNA levels were normalized to actin mRNA levels. Error bars indicate standard deviation.

Figure 6.

H3K9me3 and DNA methylation marks on individual L1 loci. (A) Level of the H3K9me3 mark on three individual L1 loci in spermatogonia of 10-dpp Miwi2 knockout (KO) and control animals. Spermatogonia were purified by MACS, and the H3K9me3 signal was measured by ChIP-qPCR. Means for two qPCRs on ChIP samples from two Miwi2 heterozygous animals and one knockout animal are shown. Error bars show standard deviation. An L1-F insertion served as a control for ChIP efficiency. All ChIP signals are normalized to input and a negative control region. (B) DNA methylation analysis of the same three loci shown in A and an additional L1-T locus. The plot shows the percentage of methylated CpGs on each locus. The actual sequenced clones are shown below the graph. (C) Analysis of H3K9me3 and DNA methylation on the L1-A locus on chr3 shown in A and B performed on the same starting material.

Figure 7.

Model for piRNA-induced establishment of the H3K9me3 mark on L1 elements. Transcripts from a full-length LINE in the nucleus of embryonic prospermatogonia are recognized by a MIWI2–piRNA complex, which recruits a histone methyltransferase (HMTase). This results in deposition of the H3K9me3 mark on LINE 5′ repeats and in the adjacent upstream region. The piRNA associates only with transcripts from actively transcribed copies of TEs. Truncated copies, which are not transcribed, are not targeted by piRNAs.