Benjamin Czech

Glioblastoma (GBM) remains a devastating disease with few meaningful therapeutic advances over the past three decades. Dendritic cell (DC) vaccination is a promising immunotherapeutic strategy for GBM, but its efficacy is limited by the clinical use of dexamethasone to control cerebral oedema and associated symptoms. Here we show that steroid signalling is a central regulator of DC dysfunction in GBM. Through targeted metabolomics of primary GBM samples, we identified a steroid-rich tumour microenvironment in which dexamethasone is present at high levels. Across bulk and single-cell transcriptomic and epigenomic datasets, NR3C1 emerged as the dominant steroid receptor in GBM immune cells and was negatively associated with activated DC states. In patient-derived DCs, dexamethasone altered NR3C1 chromatin occupancy, induced broad transcriptional and chromatin remodelling, and suppressed co-stimulatory antigen-presentation and cytokine programmes. DC-specific deletion of Nr3c1 restricted syngeneic glioblastoma growth, enhanced DC activation, promoted cytotoxic CD8+ T cell responses and remodelled myeloid states in vivo. In GBM patient-derived DCs, pharmacological or non-viral CRISPR-mediated disruption of NR3C1 restored inflammatory, antigen presentation and T cell-stimulatory programmes, enhanced antigen-specific CD8+ T cell priming, and improved tumour lysate-loaded DC vaccination. Together, these findings identify glucocorticoid signalling as a key barrier to DC immunotherapy in GBM and establish NR3C1-targeted, steroid-resistant DCs as a potential therapeutic strategy.

Transposable elements are a highly diverse group of selfish genomic elements, prevalent across the tree of life, whose uncontrolled propagation poses a threat to genome stability. Recent studies have explored the evolution of Drosophila melanogaster transposable elements, their co-evolution with the host genome, and mechanisms that regulate their activity. However, little is known about their cross-species evolutionary patterns. Long terminal repeat (LTR) retrotransposons are the most active group of transposable elements in Drosophila . They are broadly separated into retroelements, which are active in the germline, and insect endogenous retroviruses that are active in the soma. Somatic elements are hypothesised to infect the germline through their acquisition of virus-derived proteins such as Envelope and sORF2, thus multiplying through successive generations. In this study, we curated the sequences of LTR retrotransposons in 249 drosophilid genomes, allowing us to study their evolution across these species and highlight their varying degrees of conservation. Furthermore, we reveal multiple instances of Envelope protein loss or inactivation that suggest shifts in the expression pattern of these transposons, likely accompanied by adopting different transcriptional control mechanisms. We contrast this with the evolutionary history of sORF2, which we found to be much more stable. Lastly, we examined variations in transposon LTR regions responsible for transcriptional regulation and use predictive modelling to suggest six transcription factors likely involved in their tissue-specific expression. Altogether, we reveal complex, interspecies evolutionary patterns of Gypsy -family LTR retrotransposons and highlight examples of their co-evolution with their host genome.

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 (TEs) and maintain genome integrity. In Drosophila melanogaster germ cells, the majority of piRNA populations originate from dual-strand piRNA clusters, genomic regions highly enriched in TE fragments, via an elaborate machinery centered on the Heterochromatin Protein 1 homolog, Rhino. Although Rhino binds to peptides carrying tri-methylated 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 Kipferl. Here we identify a Kipferl-independent mode of Rhino recruitment 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 specifically binds to loci marked by both H3K9me3 and H3K27me3 via its chromodomain. 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 may regulate the specificity of chromatin-binding protein interactions. These findings provide an enhanced understanding of how Rhino targets piRNA source loci, highlighting the sophisticated epigenetic landscape governing TE silencing in Drosophila germ cells.

The gene-regulatory mechanisms controlling the expression of the germline PIWI-interacting RNA (piRNA) pathway components within the gonads of metazoan species remain largely unexplored. In contrast to the male germline piRNA pathway, which in mice is known to be activated by the testis-specific transcription factor A-MYB, the nature of the ovary-specific gene-regulatory network driving the female germline piRNA pathway remains a mystery. Here, usingDrosophilaas a model, we combined multiple genomics approaches to reveal the transcription factor Ovo as regulator of the germline piRNA pathway in ovarian germ cells. Ectopic expression of Ovo in ovarian somatic cells activates germline piRNA pathway components, including the ping-pong factors Aubergine, Argonaute-3, and Vasa, leading to assembly of perinuclear cellular structures resembling nuage bodies of germ cells. We found that in ovarian somatic cells, transcription ofovois repressed byl(3)mbt, thus preventing expression of germline piRNA pathway genes in the soma. Cross-species ChIP-seq and motif analyses demonstrate that Ovo is binding to genomic CCGTTA motifs within the promoters of germline piRNA pathway genes, suggesting a regulation by Ovo in ovaries analogous to that of A-MYB in testes. Our results also show consistent engagement of the Ovo transcription factor family at ovarian piRNA clusters across metazoan species, reflecting a deep evolutionary conservation of this regulatory paradigm from insects to humans.

The PIWI-interacting RNA (piRNA) pathway is essential for transposable element (TE) silencing in animal gonads. While the transcriptional regulation of piRNA pathway components in germ cells has been documented in mice and flies, their control in somatic cells of Drosophila ovaries remains unresolved. Here, we demonstrate that Traffic jam (Tj), the Drosophila orthologue of large Maf transcription factors in mammals, is a master regulator of the somatic piRNA pathway. Tj binds to regulatory regions of somatic piRNA factors and the major piRNA cluster flamenco , which carries a Tj-bound enhancer downstream of its promoter. Depletion of Tj in somatic follicle cells causes downregulation of piRNA factors, loss of flam expression and de-repression of gypsy -family TEs. We propose that the arms race between the host and TEs led to the co-evolution of promoters in piRNA pathway genes as well as TE regulatory regions that both rely on a shared transcription factor. Highlights: - Traffic jam (Tj) acts as a master regulator of the somatic piRNA pathway in Drosophila . - Tj regulates a network of piRNA pathway genes, mirroring the gene-regulatory mechanism of A-MYB in the mouse testis and Ovo in fly ovaries. - Cis -regulatory elements with Tj motifs are present at the promoters of somatic piRNA pathway genes. - The expression of the flamenco piRNA cluster is directly controlled by Tj.

PIWI-interacting RNAs (piRNAs) 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 act as a memory of past invasions. Despite being widely present across Drosophila species, somatic piRNA clusters are difficult to identify and study due to their lack of sequence conservation and limited synteny. Current identification methods rely on either extensive manual curation or availability of high-throughput small RNA sequencing 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, requiring only a genome assembly and transposon annotations. FlaHMM uses transposable element content across 5- or 10-kb bins, which can be 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.

Mapping the molecular identities and functions of cells within their spatial context is key to understanding the complex interplay within and between tissue neighbourhoods. A wide range of methods have recently enabled spatial profiling of cellular anatomical contexts, some offering single-cell resolution. These use different barcoding schemes to encode either the location or the identity of target molecules. However, all these technologies face a trade-off between spatial resolution, depth of profiling, and scalability. Here, we present B arcoding by A ctivated L inkage of Indexes (BALI), a method that uses light to write combinatorial spatial molecular barcodes directly onto target molecules in situ, enabling multi-omic profiling by next generation sequencing. A unique feature of BALI is that the user can define the number, size, and shape, and resolution of the spatial locations to be interrogated, with the potential to profile millions of distinct regions with subcellular precision. As a proof of concept, we used BALI to capture the transcriptome, chromatin accessibility, or both simultaneously, from distinct areas of the mouse brain in single tissue sections, demonstrating strong concordance with publicly available datasets. BALI therefore combines high spatial resolution, high throughput, histological compatibility, and workflow accessibility to enable powerful spatial multi-omic profiling.

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.

The gene-regulatory mechanisms controlling the expression of the germline PIWI- interacting RNA (piRNA) pathway components within the gonads of metazoan species remain largely unexplored. In contrast to the male germline piRNA pathway, which in mice is known to be activated by the testis-specific transcription factor A-MYB, the nature of the ovary-specific gene-regulatory network driving the female germline piRNA pathway remains a mystery. Here, using Drosophila as a model, we combine multiple genomics approaches to reveal the transcription factor Ovo as the master regulator of the germline piRNA pathway in ovaries. The enforced expression of Ovo in somatic cells activates germline piRNA pathway components, including the ping-pong factors Aubergine, Argonaute-3, and Vasa, leading to assembly of peri-nuclear cellular structures resembling nuage bodies of germ cells. Cross-species ChIP-seq and motif analyses demonstrate Ovo binding to genomic CCGTTA motifs within the promoters of germline piRNA pathway genes, suggesting a regulation by Ovo in ovaries analogous to that of A-MYB in testes. Our results also show consistent engagement of the Ovo transcription factor family at ovarian piRNA clusters across metazoan species, reflecting a deep evolutionary conservation of this regulatory paradigm from flies to humans.