This year, the virtual EMBO Workshop: “the Mobile Genome: Genetic and Physiological Impacts of Transposable Elements” centered around the broad impacts of TEs on organismal biology. It turned out to be a diverse meeting with interesting cross-disciplinary discussions, assembling experts from diverse fields including genomics, epigenetics, structural biology, developmental biology, immunology, cancer biology and neurobiology.
Over 90 posters were available on the virtual platform and three presenters stood out with their poster flash talks. An extraordinary accomplishment in the virtual format. With no further ado, we are introducing the winners and their research!
The repressive SCAN zinc-finger protein family targets transposable elements
Presenter: Wayo Matsushima
Transposable elements (TEs) contribute to genome innovations through insertions of coding and non-coding elements. KRAB zinc-finger proteins (KZFPs) function as sequence-specific repressors by recruiting KAP1/TRIM28, a scaffold protein of a heterochromatin-inducing complex, to TE-derived sequences. An ancestral KZFP gave rise to another family of transcription factors through the exonisation of the gag gene from the Gmr1-like transposon family as the SCAN domain. This SCAN zinc-finger protein (SZFP) family experienced multiple rounds of segmental duplication events, resulting in the presence of ~300 family members in diverse amniote genomes. Despite its abundance and evolutionary proximity to KZFPs, the functions of the SZFP family are still not well understood.
To analyse evolutionary conservation of SZFPs, we compared the DNA-contacting zinc-finger amino acid sequences of the human SZFPs to those of 65 other amniote species. This revealed that the zinc finger signatures of SZFPs are under rapid lineage-specific selection, and that several human SZFPs harbour primate-specific zinc finger sequences.
To characterise the genomic targets of all of the 55 human SZFPs, we performed ChIP-seq on 293T cell lines, each expressing one of the SZFPs with an HA-tag. We found that the binding sites of a number of SZFPs significantly overlap with specific TE subfamilies, and further observed that the evolutionary ages of SZFPs and their TE targets often matched, suggesting the TE-driven positive selection of this transcription factor family.
We next performed luciferase assays to study the regulatory function of SZFPs. We discovered that SZFPs act as transcriptional repressors owing to their SCAN domain with its conserved C-flanking 15 amino acid residues. Similar levels of repression were obtained in KAP1/TRIM28 knockout cells, demonstrating that, in contrast to KZFPs, SZFPs act independently of this master corepressor.
Together, this study identifies SZFPs as putative controllers of the regulatory potential of TEs, through their lineage-specific zinc finger repertoire and the repressive domain derived from the co-opted retroviral gene. Future work will explore the potential impact of SZFPs and their TE targets on species-specific gene regulatory networks.
The poster contains unpublished data and can therefore not be published. Follow Wayo Matsushima on Twitter for more information on his projects.
A natural transposon affects gene regulation and fitness related traits depending on the developmental stage and environmental conditions in D. melanogaster
Presenter: Miriam Merenciano
TEs have been considered a genome-wide source of regulatory elements capable of regulating nearby gene expression. In Drosophila melanogaster, the FBti0019985 natural TE insertion has been previously reported to add a transcription start site to the Lime transcription factor. In this work, we performed invivo enhancer assays and gene expression analysis with CRISPR/Cas9 mutants and natural populations to explore the effects of FBti0019985 on Lime expression under different stress conditions and different developmental stages. We found that this insertion acts as an enhancer in the adult stage under immune-stress conditions. Indeed, the deletion of predicted immune-related binding sites in the TE significantly reduces its enhancer activity in infected conditions, confirming that it harbors functional cis-regulatory elements. We also found that the TE upregulates Lime in embryos, however, in this case we could not pinpoint the molecular mechanism. Finally, we found that TE-induced Lime upregulation was associated with tolerance to bacterial infection and with increased egg-to-adult viability. Our results suggest that different developmental stages and environmental conditions should be tested in order to fully characterize the molecular and functional effects of a genetic variant.
An ATPase filament bridge: How a transposon and CRISPR stick together
Presenter: Irma Querques
Since the discovery of bacterial adaptive immunity, CRISPR-Cas systems have been mainly regarded as a mechanism to counteract horizontal transfer of mobile genetic elements including transposons in prokaryotic genomes. Conversely, a distinct family of Tn7-like elements co-opted CRISPR-Cas RNA-guided machineries to direct transposon insertion into specific target sites. In type V CRISPR-associated transposons, RNA-directed transposition relies on the cross-talk between the pseudonuclease Cas12k, the transposase TnsB, the zinc-finger protein TniQ and the ATPase TnsC. Yet, the molecular mechanisms underpinning this interplay have remained unknown. Here we present a cryo-electron microscopy structure of DNA-associated TnsC in its ATP-bound state. The structure reveals that the AAA+ ATPase forms an ATP-dependent helical filament that encloses and remodels the underlying target DNA. One strand only of the duplex is tracked by consecutive TnsC protomers with an unexpected two-nucleotide periodicity, resulting in a DNA helix with 12 base pairs per turn. Biochemical studies show that TnsC polymerization is a critical aspect of the system that enables the coupling of RNA-guided target recognition by Cas12k with the downstream recruitment of TnsB by direct protein interactions. In turn, TnsB triggers filament disassembly upon ATP hydrolysis, establishing target immunity. We also show that TniQ directly interacts with TnsC, restricting its polymerization. A crystal structure of TniQ reveals structural diversity within the TniQ protein superfamily, further suggesting a role in TnsC regulation rather than DNA targeting. Together, our data point to a mechanistic model whereby TnsC oligomers bridge between the RNA-guided target selector Cas12k and the transpososome, promoting target DNA remodeling and ultimately transposon integration. This work discloses first mechanistic insights into targeting and regulation of type V CRISPR-associated elements and will guide the rational design of these systems as programmable, site-specific gene insertion tools.
The EMBO Workshop: “The Mobile Genome: Genetic and Physiological Impacts of Transposable Elements” took place from 29 August – 1 September 2021.