The comprehensive epigenome map of piRNA clusters

Abstract

PIWI-interacting RNA (piRNA) clusters act as anti-transposon/retrovirus centers. Integration of selfish jumping elements into piRNA clusters generates de novo piRNAs, which in turn exert trans-silencing activity against these elements in animal gonads. To date, neither genome-wide chromatin modification states of piRNA clusters nor a mode for piRNA precursor transcription have been well understood. Here, to understand the chromatin landscape of piRNA clusters and how piRNA precursors are generated, we analyzed the transcriptome, transcription start sites (TSSs) and the chromatin landscape of the BmN4 cell line, which harbors the germ-line piRNA pathway. Notably, our epigenomic map demonstrated the highly euchromatic nature of unique piRNA clusters. RNA polymerase II was enriched for TSSs that transcribe piRNA precursors. piRNA precursors possessed 5'-cap structures as well as 3'-poly A-tails. Collectively, we envision that the euchromatic, opened nature of unique piRNA clusters or piRNA cluster-associated TSSs allows piRNA clusters to capture new insertions efficiently.

Figure 1.

Histone code of the BmN4 cell genome. (A) Transcriptional activity in five histone modifications. Fold enrichments of the number of histone modification peaks overlapping with RNA-seq tags are shown. Every score was normalized to that in IgG-R (rabbit IgG) peaks. P-values were calculated using a Poisson distribution. *P < 0.01; NS, non-significant. (B) Heat map showing the overlaps among five histone modifications. Peaks not overlapping with IgG-R were considered. The statistical significance of each combination was evaluated using a Poisson distribution. NS, non-significant. Without NS, P < 0.01. (C) The chromatin landscape for whole-genome TSSs in the BmN4 genome. Every score was normalized to that in IgG-R peaks. Fold enrichment of RNA pol II peaks is also shown, where IgG-M (mouse IgG) peaks served as a control library. P-values were calculated using a Poisson distribution. *P < 0.01; NS, non-significant.

Figure 2.

The chromatin landscape of piRNA clusters. (A) The chromatin landscape of piRNA clusters producing >100 RPM piRNAs. Every score was normalized to those of IgG-R (rabbit IgG) peaks. The percent of piRNA clusters positive for RNA-seq tags is also shown. P-values were calculated using a Poisson distribution. *P < 0.01; NS, non-significant. (B) The chromatin landscape of Torimochi, a representative piRNA cluster in the BmN4 genome. The density of piRNAs in the Torimochi locus was visualized by using the ‘modified’ silkworm genome where we sequenced a part of unassembled region in the Torimochi locus (see ‘Materials and Methods’ section). The remaining unassembled region is shown as N. Mapping patterns of ChIP-seq and RNA-seq tags were visualized by using Genome studio (Illumina). The primer annealing sites for Figure 2C are indicated. (C) ChIP-PCR analyses for the Torimochi locus. In addition to five histone modifications, DNA samples immunoprecipitated with the two silkworm heterochromatin proteins were analyzed.

Figure 3.

The nature of piRNA precursor transcription. (A) 5′- and 3′-RACE for Torimochi piRNA precursor. The amplicons were cloned and sequenced to validate that 5′-end of the precursor was G-capped and that 3′-end of the precursor was poly-A-tailed. (B) The chromatin landscape of piRNA-cluster-associated TSSs. IgG-R peaks (rabbit IgG) served as a control for five histone modifications and IgG-M peaks (mouse IgG) served as a control for RNA pol II. P-values were calculated using a Poisson distribution. *P < 0.01; NS, non-significant. (C) Enrichment of RNA pol II in the Torimochi-associated TSSs. The arrows indicate TSSs in the Torimochi locus. Mapping patterns of ChIP-seq tags were visualized by using Genome studio (Illumina).