Biology subjects

Eastwood, E. L.

Publications and source records attributed to Eastwood, E. L..

5 recordsLinked to original sources

bioRxiv2024

A dual histone code specifies the binding of heterochromatin protein Rhino to a subset of piRNA source loci

Animal germ cells deploy a specialized small RNA-based silencing system, called the PIWI-interacting RNA (piRNA) pathway, to prevent unwanted expression of transposable elements and maintain genome integrity. In Drosophila germ cells, the majority of piRNA populations originate from dual-strand piRNA clusters, genomic regions highly enriched in transposable element (TE) fragments, via an elaborate protein machinery centred on the heterochromatin protein 1 homolog, Rhino. Although Rhino binds to peptides carrying trimethylated H3K9 in vitro, it is not fully understood why in vivo only a fraction of H3K9me3-decorated heterochromatin is occupied by Rhino. Recent work revealed that Rhino is recruited to a subset of piRNA clusters by the zinc finger protein Kipferl. Here we identify a Kipferl-independent mode of Rhino targeting that, in addition to the previously established role of H3K9me3, also depends on the histone H3 lysine 27 methyltransferase Enhancer of Zeste. At Kipferl-independent sites, we find that Rhino, through its chromodomain, specifically binds to loci marked by both H3K9me3 and H3K27me3. Although the exact mechanism of how Rhino binding is influenced by dual histone modifications remains unclear from a structural and biochemical perspective, our work suggests that combinatorial modifications can play a crucial role in influencing the specificity of chromatin-binding protein interactions. These findings provide an enhanced understanding of the multifaceted mechanisms by which Rhino targets piRNA source loci highlighting the sophisticated epigenetic landscape governing TE silencing in Drosophila germ cells. Our work further reveals a role for dual histone modifications defining the binding specificity of a key chromatin protein.

molecular biology↗

bioRxiv2021

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

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.

molecular biology↗

bioRxiv2019

Specialization of the Drosophila nuclear export family protein, Nxf3, for piRNA precursor export

The piRNA pathway is a conserved, small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilisation. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use non-canonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by Nuclear export factor 1. Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila Nuclear export factor family protein, Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We find that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.

molecular biology↗

bioRxiv2019

piRNA-guided co-transcriptional silencing coopts nuclear export factors

The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. Here we show that Nuclear Export Factor 2 (Nxf2) and its cofactor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. We mapped the protein domains crucial for the Nxf2/Panx complex formation and show that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing.

molecular biology↗

bioRxiv2019

Daedalus and Gasz recruit Armitage to mitochondria, bringing piRNA precursors to the biogenesis machinery

The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialised machinery that converts long single-stranded precursors into small RNAs of ~25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb-body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and for the correct localisation of the indispensable piRNA biogenesis factor, Armitage (Armi). We find that Gasz and Daed interact with each other and likely provide a mitochondrial \"anchoring platform\" to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb-bodies in a manner that depends upon Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing, namely how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.

molecular biology↗