The histone demethylase KDM3 prevents auto-immune piRNAs production in Drosophila

Abstract

In animals, genome integrity of the germ line is protected from transposable element (TE) activity by small, non-coding, dedicated RNAs acting as an immune system against TEs, and called PIWI-interacting RNAs (piRNAs). In Drosophila, the production of piRNAs is initiated from heterochromatic loci containing remnants of TEs and enriched in histone H3 trimethylated on lysine 9 (H3K9me3). These loci, called piRNA clusters, constitute a memory of past TE invasions. Little is known about how piRNA clusters are genetically defined. Using a genetic screen combined with a bimodal epigenetic state piRNA cluster (BX2), we identified the splicing factor Half pint (Hfp) and the histone demethylase KDM3 as being able to prevent BX2 piRNA production. Furthermore, we showed that Hfp is needed to splice Kdm3 transcripts. Germline expression of Kdm3 coding sequence (splicing-independent) rescued the hfp germline knock-down (GLKD) effect demonstrating that Kdm3 is sufficient to prevent BX2 piRNA production. Our data revealed that in the absence of Kdm3, dozens of gene-containing regions become bona fide germinal dual strand piRNA clusters. Indeed, they produce piRNAs originating from both DNA strands, become transcribed in a Moonshiner-dependent manner and enriched in di-and tri-methylation of lysine 9 of histone H3 (H3K9me2/3) and in Rhino, an HP1-like protein. Eggs laid by Kdm3 GLKD females do not hatch and show developmental defects phenocopying loss of function of genes included into the new piRNA clusters, suggesting an inheritance of functional ovarian "auto-immune" piRNAs. Our results demonstrate that some gene-containing regions are actively prevented for piRNA production by proteins that counteract piRNA cluster emergence. Hence, a non-piRNA-producing state is therefore not a "by default" state but rather a cellular lock that is actively controlled for some genomic loci.

ADMA-histones define genomic loading of Rhino at the initial step of piRNA cluster formation

“…*P < 0.05, **P < 0.01, and ***P < 0.001. HP1a KD: ***P = 8.49 × 10 -13 , DART1 KD: ***P = 1.83 × 10 -7 , DART4 KD: ***P = 2.83 × 10 -4 , DART8 KD: n.s. P = 0.655, CG17726 KD: n.s.…”

“…Kdm3 GLKD-dependent ectopic piRNA cluster ends were extracted using the following method. Among Rhino peaks listed in the MACS2 callpeak files “GSE203279_MACS2_narrowpeak_Rhino_Ctrl.txt” and “GSE203279_MACS2_ narrowpeak_Rhino_Kdm3GLKD.txt” in the GSE203279 dataset, we first extracted the Rhino peaks that appeared only in the latter 13 . The peaks closest to the 5′ and 3′ ends of the R1−R12 regions in Table S4 of the Casier et al (2023) were designated as the ends of ectopic piRNA clusters 13 .…”

“…Among Rhino peaks listed in the MACS2 callpeak files “GSE203279_MACS2_narrowpeak_Rhino_Ctrl.txt” and “GSE203279_MACS2_ narrowpeak_Rhino_Kdm3GLKD.txt” in the GSE203279 dataset, we first extracted the Rhino peaks that appeared only in the latter 13 . The peaks closest to the 5′ and 3′ ends of the R1−R12 regions in Table S4 of the Casier et al (2023) were designated as the ends of ectopic piRNA clusters 13 . (d) Genome browser snapshot of the indicated ChIP-seq data in representative ectopic piRNA clusters appearing in Kdm3 GLKD (R2_bi region and R4_jing region).…”

“…Like other HP1 family members, Rhino has a chromodomain through which it binds to H3K9me3 scattered on dual-strand piRNA clusters 6,8,9,12,13,14,15,16 . Deadlock and Cutoff then join Rhino, assembling the Rhino-Deadlock-Cutoff (RDC) complex, which further recruits the transcription factor IIA-L (TFIIA-L) paralogue Moonshiner, the transcription factor IIA-S (TFIIA-S), and the animal TATA-box-binding protein (TBP) paralog TRF2, initiating transcription of the clusters 6,7,10 .…”

“…Rhino has a propensity to spread across the genome. For example, depletion of the histone demethylase Kdm3 in the female germline causes Rhino to spread to adjacent chromatin 13 . Deletion of the region proximal to the transcription start site of the phospholipase D gene, which is in the region flanking piRNA cluster 42AB, also resulted in Rhino spreading toward the deleted genomic region 10 .…”