Author response: Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila

Abstract

In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex. Introduction Large portions of eukaryotic genomes are occupied by repetitive sequences and mobile genetic elements (Cosby et al., 2019). The de novo establishment of heterochromatin at these regions prevents transposon mobilisation and aberrant recombination events, and the ability of small RNAs to provide sequence specificity to this mechanism has emerged as a fundamental element of eukaryotic genome defence systems (Martienssen and Moazed, 2015). Specifically, nuclear Argonaute proteins bound to small RNA partners recognise nascent RNA transcripts and trigger a molecular cascade that leads to recruitment of epigenetic modifiers to the target region (Czech et al., 2018; Martienssen and Moazed, 2015; Ozata et al., 2019). This co-transcriptional process ultimately results in the formation of a repressive chromatin environment at the target locus, with most of our understanding originating from studies in yeast and plants (Martienssen and Moazed, 2015). In animal gonads, the piRNA pathway represses mobile element activity, in part, through the cotranscriptional gene silencing (TGS) of transposon insertions (Czech et al., 2018; Ozata et al., 2019). In Drosophila melanogaster, where this pathway has been extensively studied, piRNA-guided TGS depends on the PIWI-clade Argonaute protein Piwi (Klenov et al., 2014; Le Thomas et al., 2013; Rozhkov et al., 2013; Sienski et al., 2012; Wang and Elgin, 2011). Loci targeted by Piwi are associated with repressive chromatin states marked by the absence of di-methylated H3K4 (H3K4me2) and the presence of diand tri-methylated H3K9 (H3K9me2/3). These regions are also coated with Heterochromatin Protein 1a (HP1a/Su(var)205) (Wang and Elgin, 2011). Loss of Piwi results in marked de-repression of transposon mRNA levels which is coupled with loss of repressive Eastwood et al. eLife 2021;10:e65557. DOI: https://doi.org/10.7554/eLife.65557 1 of 29 RESEARCH ARTICLE histone modifications and increased occupancy of RNA polymerase II at transposon insertions (Klenov et al., 2014; Le Thomas et al., 2013; Rozhkov et al., 2013; Sienski et al., 2012; Wang and Elgin, 2011). Transposon control at the transcriptional level through nuclear PIWI proteins is not unique to Drosophila. In mouse embryonic testes, the nuclear PIWI protein MIWI2 directs TGS to evolutionarily young transposon insertions, which are silenced in a process that alters both the histone modification and DNA methylation landscapes (Aravin et al., 2008; Carmell et al., 2007; Molaro et al., 2014; Pezic et al., 2014). While the mechanism of TGS in mammals is not fully understood, recent work linked two nuclear partners of MIWI2, TEX15 and SPOCD1, to this process (Schöpp et al., 2020; Zoch et al., 2020). The molecular mechanism linking target recognition by Piwi with transcriptional repression is yet to be fully resolved. Removal of H3K4 methylation marks depends on the lysine demethylase Lsd1/ Su(var)3–3, assisted by CoRest, while H3K9me3 is deposited by the histone methyltransferase dSetDB1/Eggless (Egg) and its cofactor Windei (Wde) (Czech et al., 2013; Handler et al., 2013; Koch et al., 2009; Muerdter et al., 2013; Osumi et al., 2019; Rangan et al., 2011; Sienski et al., 2015; Yu et al., 2015). The chromatin-associated protein Ovaries absent (Ova), which bridges HP1a and Lsd1, the SUMO ligase Su(var)2–10, the nucleosome remodeller Mi-2 and the histone deacetylase Rpd3 additionally contribute to the heterochromatin formation process (Mugat et al., 2020; Ninova et al., 2020; Yang et al., 2019). Loss of any of these factors in ovaries impairs TGS and transposon repression, yet how they are recruited to transposon loci and cooperate in target repression remains elusive (Czech et al., 2013; Handler et al., 2013; Muerdter et al., 2013; Mugat et al., 2020; Ninova et al., 2020; Sienski et al., 2015; Yang et al., 2019; Yu et al., 2015). Recent work has uncovered a complex consisting of Panoramix (Panx), Nuclear export factor 2 (Nxf2) and NTF2-related export protein 1 (Nxt1) termed PICTS (also known as SFiNX) that functions downstream of Piwi and upstream of the chromatin modifying machinery to effect TGS (Batki et al., 2019; Fabry et al., 2019; Murano et al., 2019; Sienski et al., 2015; Yu et al., 2015; Zhao et al., 2019). While both Panx and Nxf2 can induce transcriptional repression when artificially recruited to reporters, the link to epigenetic modifiers and the transcriptional silencing activity of the PICTS complex seems to reside within Panx itself (Batki et al., 2019; Fabry et al., 2019). Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is a critical TGS factor and an integral part of the PICTS complex. Ctp is essential for transposon repression in both the somatic and germline compartments of Drosophila ovaries and, like Panx and Nxf2, can induce transcriptional silencing when artificially tethered to a reporter locus. In contrast to Panx and Nxf2, whose functions are specific to transposon control (Batki et al., 2019; Fabry et al., 2019; Murano et al., 2019; Sienski et al., 2015; Yu et al., 2015; Zhao et al., 2019), Ctp is a ubiquitously expressed and essential protein with wide-ranging cellular functions (Barbar, 2008; Jespersen and Barbar, 2020; Rapali et al., 2011b). While first identified as a component of the dynein motor complex (King and Patel-King, 1995), Ctp, which itself forms a homodimer, has since been shown to function as a dimerisation hub protein, promoting the assembly and stabilisation of many dyneinindependent protein complexes (Barbar and Nyarko, 2014; Jespersen and Barbar, 2020). We show that Ctp promotes dimerisation of the PICTS complex through its interaction with two conserved motifs in Panx. In the absence of Ctp, Panx fails to self-associate and is unable to support transposon silencing. Considered together, our data reveal that Ctp-mediated higher order PICTS complex assembly is essential for heterochromatin formation and silencing. Results Ctp associates with the PICTS complex and is required for transposon control Previously, we and others showed that Panx, Nxf2, and Nxt1 form a complex that acts downstream of Piwi target engagement to silence transposons at the transcriptional level (Batki et al., 2019; Fabry et al., 2019; Murano et al., 2019; Zhao et al., 2019). As part of this study, we immunoprecipitated GFP-tagged Panx and Nxf2 from ovary lysates and identified the associated proteins by mass spectrometry (Fabry et al., 2019). As well as known components of the PICTS complex, the protein Cut-up (Ctp) was significantly enriched in both experiments (Figure 1A). This was an unexpected result, since Ctp is a member of the LC8 family of dynein light chains and a component of Eastwood et al. eLife 2021;10:e65557. DOI: https://doi.org/10.7554/eLife.65557 2 of 29 Research article Chromosomes and Gene Expression