The Integrity of piRNA Clusters is Abolished by Insulators in the Drosophila Germline.

Elizaveta Radion,Alla Kalmykova,Pavel Komarov,Olesya Sokolova,Yuri Abramov,Sergei Ryazansky

doi:10.3390/genes10030209

Elizaveta Radion, Alla Kalmykova + Show 4 more

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https://doi.org/10.3390/genes10030209

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Abstract

Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, we studied the effects of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. We show that transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.

Highlights

The Piwi-interacting RNA pathway is an essential mechanism that protects genome integrity by suppressing transposable element (TE) activity in animal gonads [1]
We show here that transgenes bearing Su(Hw) recognition sites embedded in endogenous pericentromeric and telomeric Piwi-interacting RNAs (piRNAs) clusters interfere with the local transcription of piRNA precursors, production of small RNAs and formation of specific chromatin structure
We suggested that Su(Hw) binding could impede piRNA production and performed ovarian small RNA sequencing of transgenic strains bearing su(Hw)V /su(Hw)f mutations

Summary

Introduction

The Piwi-interacting RNA (piRNA) pathway is an essential mechanism that protects genome integrity by suppressing transposable element (TE) activity in animal gonads [1]. In Drosophila, piRNA precursors are derived from distinct genomic regions termed piRNA clusters, which are enriched in TE fragments [2]. The specific chromatin structure of piRNA clusters ensures the recruitment of the noncanonical transcriptional machinery that drives piRNA precursor expression [3,4,5,6]. The chromatin of piRNA clusters is enriched in a common heterochromatic histone mark, trimethylated lysine 9 of histone H3 (H3K9me3) and by two chromodomain-containing proteins, heterochromatic protein. The protein Maelstrom represses canonical transcription from TEs and neighbouring gene promoters in dual-strand piRNA clusters [10]. Noncanonical convergent transcription from both genomic strands initiated at multiple

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