Close observation of chromatin states at piRNA-silenced genomic loci demonstrates the power of transposons to change native gene expression.
As reviewed in an earlier post, the Drosophila Piwi/piRNA transposon silencing pathway can be divided into two facets; a complex pathway operating in the germline centred on the Piwi-family argonautes Aubergine and AGO3 localised in peri-nuclear nuage, and a linear pathway operational in the somatic follicle cells. In this linear pathway, piRNAs derived from uni-directional piRNA clusters such as flamenco target Piwi to mediate silencing of a limited subset of retrotransposons. Unlike Aub and AGO3, Piwi is localised to the nucleus, leading to speculation that rather than silencing transposons post-transcriptionally by ‘slicing’ their transcripts, it may act at the transcriptional level. There are many precedents in other organisms for argonautes mediating transcriptional silencing via interactions with chromatin modification and DNA methylation pathways. However, whether one of these silencing modes is employed by Drosophila Piwi was unresolved. A new paper from the lab of Julius Brennecke, generally analysing the linear piRNA pathway active in a cell line derived from the somatic follicle cells surrounding the oocyte (OSC cells) includes important findings for a number of aspects of Piwi-mediated transposon silencing leading to insights on the wider genomic ecology of transposon insertions.
In the first section of the paper, Sienski et al. demonstrate that Maelstrom (Mael), a protein containing putative RNA and DNA binding domains, expressed in both cytoplasm and nuclei and previously implicated in a number of Piwi-pathway effects, acts downstream of Piwi to effect TE silencing. Silencing requires the nuclear localisation of both Piwi and Mael. Further, mutation of the residues necessary for ‘slicer’ activity in Piwi did not de-repress TEs, suggesting a different mechanism for Piwi-mediated silencing.
Sienski et al. go on to marshal three different high-throughput techniques to show that Piwi mediates gene silencing at the transcriptional level. Knocking down (KD) the expression of Piwi pathway factors (piwi, mael) in OSC cells they determined the set of repressed transposable elements (TEs) by comparing RNA levels (RNA-seq). Changes in the steady-state RNA levels were highly correlated with transcription rate as monitored by RNA polymerase II occupancy (ChIP-seq) and levels of nascent RNAs (GRO-seq). Judging by how closely correlated derepression of TEs was to transcription rate, it seems unlikely that the linear piRNA pathway active in follicle cells acts post-transcriptionally at all.
Reasoning that Piwi-mediated transcriptional gene silencing may involve chromatin modification, Sienski et al. profiled the distribution of the repressive histone mark H3K9me3 in OSCs after piwi or mael knockdown. H3K9me3 levels at transposable elements known to be repressed by the piRNA pathway were significantly reduced in the absence of Piwi (and to a lesser extent Mael). This data was from across the genome irrespective of whether the TE was inserted into heterochromatic or euchromatic regions. To negate general effects associated with heterochromatin, the authors looked more closely at TE insertions within euchromatic regions.
At a specific euchromatic insertion of the retrotransposon mdg1, they observed that upon either piwi KD or mael KD, transcription downstream of the insertion strongly increased. However, although this transcriptional bleeding into the surrounding area was similar upon TE derepression due to either piwi KD or mael KD, the pattern of H3K9me3 was very different. Normally this mdg1 insertion displays H3K9me3 in the surrounding 12kb, peaking at the insertion site. This was strongly reduced in piwi KD cells, but in mael KD, H3K9me3 was moderately reduced at the insertion site but had actually spread further downstream (see figure). Similar patterns were observed at nearly all euchromatic mdg1 insertions, as well as other TEs known to be targeted by the linear piRNA pathway active in OSC cells.
Strikingly, most euchromatic H3K9me3 peaks were sensitive to piwi knockdown, whilst 88% of H3K9me3 peaks were found within 5Kb of TE insertions. Piwi-mediated transposon silencing therefore seems to be the main trigger for H3K9 trimethylation in euchromatin.
This transposon silencing mechanism appears to have a major impact on native genes upon TE insertion in their vicinity. An insertion of the retrotransposon gypsy into the first intron of the expanded (ex) gene serves as paradigm for these effects. In OSC cells, the gypsy insertion triggered H3K9me3 spreading into the surrounding 10-12Kb. In control cells RNA polymerase II occupancy was observable at the ex transcription start site (TSS) but weak. Upon piwi or mael knockdown, transcription from the ex TSS was massively increased. As in the earlier mdg1 example, H3K9me3 levels were greatly reduced upon piwi KD but not in mael KD cells. Sienski et al. observed similar effects on the transcription of 28 more genes with nearby TE insertions in OSC cells.
This data has a number of ramifications speaking of a complex interplay between transcription, the establishment and maintenance of repressive chromatin states and the Piwi pathway. Firstly, H3K9me3 considered a transcriptionally repressive histone mark is compatible with transcription. In fact, based on it’s pattern in mael KD cells, the authors propose that downstream transcriptional bleeding leads to the spread of H3K9me3. Further, although H3K9me3 has an integral role in Piwi-mediated silencing, it is not the final silencing mark. H3K9 trimethylation is downstream of Piwi action, but is either upstream or acts in parallel to Mael, which mediates an unknown silencing step crucial to Piwi transcriptional gene silencing.
Importantly, this paper has demonstrated the impact that TE insertion and subsequent piRNA pathway transcriptional repression can have on native gene expression. There are two different modes in which the inactivation of Piwi-mediated TE silencing can lead to the transcriptional activation of these loci. Firstly, the spreading of repressive chromatin marks at transposons can suppress RNA polymerase II access to the genes promoter. Alleviation of TE repression hence leads to (re-)activation of gene expression. Conversely, as TEs (especially the long terminal repeats of some retrotransposons) can serve as promoters, the loss of their repressed chromatin state upon piRNA pathway loss, can activate transcription of downstream regions. Although both these modes lead to transcriptional activation after Piwi pathway loss, they demonstrate that transposon insertion can either activate or repress transcription within relatively extensive genomic surroundings. This underscores the scope for transposons to act as regulatory elements, or to produce new chimerical transcripts and hence potential new genes.
These experiments were mainly performed in one cell type that only partially reflects the activity of what is already a subset of piwi/piRNA action during Drosophila oogenesis. Piwi and Mael are also active in the nurse cells and oocyte, and this paper suggests that they have similar roles within the context of the expanded piRNA pathways active in the germline. It will be interesting to integrate this nuclear-localised transcriptional-silencing aspect of piRNA silencing into the context of ping-pong amplification and bi-directional piRNA cluster transcripts. Further, do these Piwi-mediated chromatin effects in the germline impact on the transcriptional status of TEs and genes later in somatic development? And if not, do other systems have equivalent activity?
This paper underlines again the importance of the arms race between mobile genetic elements and genomic immune systems such as the piRNA pathway on the wider genomic regulatory context. This contest is being observed to have shaped so many aspects of genome organisation throughout evolution that it sometimes becomes hard to differentiate parasitism from regulation. It is clear however, that to understand the evolutionary impact of mobile elements we must also understand the import of the various epigenetic mechanisms controlling their spread. The minutiae of these mechanisms with regard to their targets, plasticity, adaptability, heritability – often different from organism to organism – has major evolutionary significance. Evolution works differently depending on these mechanisms.
Sienski, G., Dönertas, D., & Brennecke, J. (2012). Transcriptional Silencing of Transposons by Piwi and Maelstrom and Its Impact on Chromatin State and Gene Expression Cell, 151 (5), 964-980 DOI: 10.1016/j.cell.2012.10.040