Biology subjects

van Lopik, J.

Publications and source records attributed to van Lopik, J..

4 recordsLinked to original sources

bioRxiv2024

FlaHMM: unistrand flamenco-like piRNA cluster prediction in Drosophila species using hidden Markov models

PIWI-interacting RNAs are a class of small non-coding RNAs that are essential for transposon control in animal gonads. In Drosophila ovarian somatic cells, piRNAs are transcribed from large genomic regions called piRNA clusters, which are enriched for transposon fragments and acts as a memory of past invasions. Despite being widely present across Drosophila species, somatic piRNA clusters are notoriously difficult to identify and study due to their lack of sequence conservation and limited synteny. Current identification methods rely either on extensive manual curation or availability of high-throughput small RNA-seq data, limiting large-scale comparative studies. We now present FlaHMM, a hidden Markov model developed to automate genomic annotation of flamenco-like unistrand piRNA clusters in Drosophila species without the need of experimental data beyond a genome assembly. FlaHMM uses transposable element content across 5 or 10 kb bins calculated from genome sequence alone and is thus able to detect candidate piRNA clusters without the need to obtain flies and experimentally perform small RNA sequencing. We show that FlaHMM performs on par with piRNA-guided or manual methods, and thus provides a scalable and efficient approach to piRNA cluster annotation in new genome assemblies. FlaHMM is freely available at https://github.com/Hannon-lab/FlaHMM under an MIT licence.

bioinformatics↗

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↗

bioRxiv2023

Unistrand piRNA clusters are an evolutionary conserved mechanism to suppress endogenous retroviruses across the Drosophila genus

The PIWI-interacting RNA (piRNA) pathway prevents endogenous genomic parasites, transposable elements, from damaging the genetic material of animal gonadal cells. Specific regions in the genome, called piRNA clusters, are thought to define each species piRNA repertoire and therefore its capacity to recognize and silence specific transposon families. The unistrand cluster flamenco (flam) is essential in the somatic compartment of the Drosophila ovary to restrict Gypsy-family transposons from infecting the neighbouring germ cells. Disruption of flam results in transposon derepression and sterility, yet it remains unknown whether this silencing mechanism is present more widely. Here, we systematically characterised 119 Drosophila species and identify five additional flam-like clusters separated by up to 45 million years. Small RNA-sequencing validated these as bona-fide unistrand piRNA clusters expressed in somatic cells of the ovary, where they selectively target transposons of the Gypsy family. Together, our study provides compelling evidence of a widely conserved transposon silencing mechanism that co-evolved with virus-like Gypsy-family transposons.

molecular biology↗

bioRxiv2021

Single Cell Variability of CRISPR-Cas Interference and Adaptation

CRISPR-Cas defence is a combination of adaptation to new invaders by spacer acquisition, and interference by targeted nuclease activity. While these processes have been studied on a population level, the individual cellular variability has remained unknown. Here, using a microfluidic device combined with time-lapse microscopy, we monitor invader clearance in a population of Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we show large cell-to-cell variability of clearance times, which originates from primed CRISPR adaptation. Faster growth and cell division, as well as higher levels of Cascade, increase the chance of clearance by interference. In contrast, faster growth is associated with decreased chances of clearance by priming. A mathematical model explains the experimental findings, and identifies Cascade binding to the mutated invader DNA, rather than spacer integration, as the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond to invading threats in a timely manner. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.

microbiology↗