Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

Abstract

A persistent question in biology is how cis-acting sequence elements influence trans-acting factors and the local chromatin environment to modulate gene expression. We reported previously that the DNA transposon 1360 can enhance silencing of a reporter in a heterochromatic domain of Drosophila melanogaster. We have now generated a collection of variegating phiC31 landingpad insertion lines containing 1360 and a heat-shock protein 70 (hsp70)-driven white reporter to explore the mechanism of 1360-sensitive silencing. Many 1360-sensitive sites were identified, some in apparently euchromatic domains, although all are close to heterochromatic masses. One such site (line 1198; insertion near the base of chromosome arm 2L) has been investigated in detail. ChIP analysis shows 1360-dependent Heterochromatin Protein 1a (HP1a) accumulation at this otherwise euchromatic site. The phiC31 landing pad system allows different 1360 constructs to be swapped with the full-length element at the same genomic site to identify the sequences that mediate 1360-sensitive silencing. Short deletions over sites with homology to PIWI-interacting RNAs (piRNAs) are sufficient to compromise 1360-sensitive silencing. Similar results were obtained on replacing 1360 with Invader4 (a retrotransposon), suggesting that this phenomenon likely applies to a broader set of transposable elements. Our results suggest a model in which piRNA sequence elements behave as cis-acting targets for heterochromatin assembly, likely in the early embryo, where piRNA pathway components are abundant, with the heterochromatic state subsequently propagated by chromatin modifiers present in somatic tissue.epigenetics | position-effect variegation T ransposable elements (TEs) are major structural features of nearly all eukaryotic genomes, and can play a role as cisacting regulatory features with profound influence over the gene regulatory networks of their host (1). However, if left unchecked, the deleterious effects of TE mobilization can become insurmountable for the system, damaging genome integrity. Thus, silencing mechanisms that prevent TE mobilization are fundamental to the faithful propagation of genetic information. Position-effect variegation (PEV), a mosaic expression pattern resulting from silencing in some cells that normally express a gene, has frequently been used as an indicator of heterochromatic environments (2, 3). We reported previously that the DNA transposon 1360 can enhance reporter silencing in repeat-rich heterochromatic regions (4). To gain insight into the mechanisms involved, we sought to identify sequence elements present in 1360 that contribute to 1360-sensitive PEV, using an adjacent heat-shock protein 70 (hsp70)-driven white gene as the reporter.We used the phiC31 landing pad system, where phiC31 integrase mediates recombination between a landing pad (inserted in the genome) containing phage attachment (attP) sites and a donor construct containing bacterial attachment (attB) sites (5). This system allows us to maintain a constant genomic cont...

Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors

“…Similarly, in Drosophila, two distinct RNAi-like processes for silencing Tns have been elucidated. In the germline, the Piwi-interacting RNA (piRNA) pathway generates small RNAs that suppress Tns by inducing heterochromatin formation (Vagin et al 2006;Aravin et al 2007;Brennecke et al 2007;Sentmanat and Elgin 2012;Le Thomas et al 2013). In somatic cells, esiRNAs silence retroTns via a Dicer 2 (Dcr-2)/Argonaute 2 (Ago2)-dependent mechanism (Chung et al 2008;Czech et al 2008;Ghildiyal et al 2008;Saito and Siomi 2010;Xie et al 2013).…”

Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes

“…Most piRNA sequences in the Drosophila genome map to heterochromatic regions (Saito et al 2006), and several lines of investigation have demonstrated that Piwi and HP1a can physically interact (Brower-Toland et al 2007;Mendez et al 2011). Moreover, transcriptional silencing of retrotransposons by the Piwi-piRNA system involves recruitment of HP1a to transposable elements (Wang and Elgin 2011;Sentmanat and Elgin 2012;Huang et al 2013). Piwi also participates in activities that are not directly related to transposon silencing.…”

Targeting of P-Element Reporters to Heterochromatic Domains by Transposable Element 1360 in Drosophila melanogaster

“…A careful examination of this domain revealed a correlation between the presence of the repetitive element 1360 and the variegating phenotype (Sun et al 2004). Follow-up studies found that incorporating 1360 sequences into the hsp70-white transgene construct could increase the gene silencing observed at a subset of insertion sites (Haynes et al 2006;Sentmanat and Elgin 2012). However, these studies also showed that the presence of 1360 was not sufficient to induce PEV-and heterochromatin formation-in general, as full expression, rather than variegation, is observed for insertions of the 1360-hsp70-white transgene at most sites in the euchromatic chromosome arms (Haynes et al 2006;Sentmanat and Elgin 2012).…”

“…Follow-up studies found that incorporating 1360 sequences into the hsp70-white transgene construct could increase the gene silencing observed at a subset of insertion sites (Haynes et al 2006;Sentmanat and Elgin 2012). However, these studies also showed that the presence of 1360 was not sufficient to induce PEV-and heterochromatin formation-in general, as full expression, rather than variegation, is observed for insertions of the 1360-hsp70-white transgene at most sites in the euchromatic chromosome arms (Haynes et al 2006;Sentmanat and Elgin 2012).The available data suggest that PEV is caused by spreading of adjacent heterochromatin to encompass the reporter gene (Weiler and Wakimoto 1995;Vogel et al 2009). The effectiveness of this spreading is thought to be stochastic, thus leading to the variegating phenotype.…”

“…The effectiveness of this spreading is thought to be stochastic, thus leading to the variegating phenotype. As noted above, proximity to high repeat density regions is key, even if a repeated element such as 1360 is included in the transgene (Wallrath and Elgin 1995;Sun et al 2000;Haynes et al 2006;Sentmanat and Elgin 2012). While the importance of a high density of repetitive elements is well supported, genetic evidence indicates that there are several different mechanisms operating in the various heterochromatic domains of the Drosophila genome (Phalke et al 2009).…”