Life Science Alliance

{gamma}-secretase is a multi-subunit enzyme complex responsible for cleaving hundreds of substrates in diverse cellular contexts. Variation in subunit composition - including the use of alternate catalytic subunits Presenilin 1 (PSEN1) and Presenilin 2 (PSEN2) - results in diverse {gamma}-secretase complexes. Point mutations in PSEN1 and PSEN2 cause familial forms of Alzheimers disease, while loss-of-function mutations in the {gamma}-secretase subunits PSEN1, PSENEN and NCSTN cause acne inversa. To advance therapeutic strategies targeting {gamma}-secretase in Alzheimers disease, a better understanding of individual {gamma}-secretase complexes is required. In this study, we used CRISPR-Cas9 genome engineering to generate PSEN2-knockout iPSCs in order to compare the consequence of PSEN2 knockout versus PSEN1 knockout in iPSC-derived brain cells. In contrast to PSEN1-knockout, PSEN2-knockout did not alter APP cleavage or A{beta} generation in iPSC-neurons, nor did it disrupt Nicastrin maturation. Similarly, PSEN2-knockout had little impact on TREM2 processing in iPSC-microglia. Instead, our data indicate that loss of PSEN2 primarily impacts the endo-lysosomal system in iPSC-neurons, causing an accumulation of early endosome markers and a reduction in lysosomal markers - phenotypes not observed in PSEN1-knockout neurons. Taken together, these findings highlight distinct and non-redundant functions of PSEN1 and PSEN2 in human brain cells, reinforcing findings in animal models and subcellular localisation studies. This work advances our understanding of distinct {gamma}-secretase complex functions and provides insights that will support future therapeutic efforts to inhibit, modulate or stabilise {gamma}-secretase.

The multiple endocrine neoplasia type 1 (MEN1) gene encodes Menin, a nuclear scaffold protein and tumor suppressor that regulates transcription. It is frequently mutated in endocrine neoplasia. MEN1-gastrinomas are aggressive neuroendocrine tumors (NETs) that arise predominantly in the submucosal Brunners glands of the duodenum, an organelle rich in extracellular growth factors. Many duodenal NETs retain wild-type MEN1 allele and nuclear Menin, suggesting post-translational inactivation of its tumor-suppressor function. The Menin C-terminal domain (CTD) contains a conserved phosphorylation site at Ser487 within the first of three nuclear localization signals (NLS1-3). We hypothesized that extracellular signaling regulates Menin by phosphorylating the CTD at Ser487 blocking its nuclear localization. Using CTD deletion mapping, site-directed mutagenesis, and kinase activation in gastric cell lines, we show that loss of NLS1-3 reduces Menins nuclear localization, stability, and repression of GASTRIN. Cell stimulation by epiregulin, forskolin, or phorbol ester induced Menin Ser487 phosphorylation and its nuclear translocation, relieving repression of GASTRIN. The phospho-mimetic S487D mutant remained cytoplasmic and phenocopied CTD deletion of NLS1-3 sustaining de-repression of GASTRIN. These findings showed that Ser487 phosphorylation restricts nuclear accumulation of Menin and functionally links extracellular signaling to post-translational modification of Menin that ultimately contributes to transcriptional derepression and neuroendocrine tumorigenesis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=127 HEIGHT=200 SRC="FIGDIR/small/717082v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1fbc016org.highwire.dtl.DTLVardef@fffdfdorg.highwire.dtl.DTLVardef@7bf0a2org.highwire.dtl.DTLVardef@f32422_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intestinal epithelial cells are central players in mucosal barrier integrity and host-microbe interactions. Genetic studies have revealed that epithelial dysfunction is a key contributor to the pathogenesis of inflammatory bowel disease. Non-SMC condensin II complex subunit D3 (NCAPD3) is essential for chromatin organization and stability. NCAPD3 also promotes antimicrobial defense and autophagy responses in vitro. NCAPD3 expression is decreased in intestinal epithelial cells from patients with ulcerative colitis; however, it is not known whether loss of NCAPD3 expression drives intestinal barrier dysfunction or is a result of disease-associated inflammation. To investigate this relationship in vivo, a tissue-specific approach was required, as global constitutive knockout of NCAPD3 is embryonic lethal. Therefore, a transgenic mouse line with doxycycline-inducible expression of a short hairpin RNA targeting NCAPD3 restricted to villin-expressing cells was generated (NCAPD3KD mice) to enable the study of NCAPD3 function in the intestinal epithelium. Treatment of NCAPD3KD mice with 9-tert-butyl doxycycline resulted in [~]75% reduction of NCAPD3 protein in EpCAM intestinal cells. Short-term epithelial NCAPD3 knockdown did not induce spontaneous colitis but was associated with increased serum amyloid A and a trend towards increased intestinal permeability. Upon dextran sodium sulfate or Salmonella enterica serovar Typhimurium {Delta}AroA challenge, NCAPD3KD mice exhibited exacerbated weight loss, higher disease activity, increased histopathological damage, abnormal colonic cytokines and chemokines, and significantly increased intestinal permeability. These results indicate that NCAPD3 expression in the intestinal epithelium is required for optimal barrier maintenance and antimicrobial defense under chemical or microbial stress. These findings support prior in vitro observations and solidify NCAPD3 as a regulator of intestinal epithelial barrier function and mucosal host defense. Author SummaryNCAPD3 is a multifunctional protein with established roles in chromatin organization, genome stability, mitochondrial function, and antimicrobial defense. Dysregulated NCAPD3 is implicated in human diseases, such as inflammatory bowel disease (IBD) and microcephaly; however, due to its essential role in cellular division, determination of whether NCAPD3 loss drives these pathologies in vivo has been lacking. Using a new transgenic mouse model that selectively reduces NCAPD3 expression in intestinal epithelial cells, our study establishes NCAPD3 as an epithelial regulator of the mammalian intestine that enhances epithelial barrier resilience and antimicrobial defense during stress. Although dispensable for short-term basal homeostasis, NCAPD3 function becomes critical during epithelial injury and enteric infection. Reduced NCAPD3 expression may therefore lower the threshold for inflammatory disease by weakening barrier integrity, amplifying inflammatory cascades, and impairing antimicrobial defenses. These findings position NCAPD3 as a potential modulator of IBD susceptibility and highlight chromatin organization as an important, previously underappreciated layer of intestinal epithelial regulation.

Grainyhead-like 2 (GRHL2) is an epithelial transcription factor with context-dependent regulatory roles, yet the sequence rules governing its DNA recognition remain incompletely defined. In this study, a high-density genomic Specificity and Affinity for Protein (SNAP) DNA-binding array containing 772,732 tiled probes derived from GRHL2 ChIP-seq regions was used to resolve GRHL2 binding specificity at 6 base pair resolution across genomic sequences. From high-affinity probes, de novo motif analysis recovered the canonical 5-AACCGGTT-3 motif. Sequence specificity landscapes revealed a stepwise reduction in binding as mismatches were introduced, with the strongest effects at the C (position 3) and G (position 6) within the motif, greater tolerance at the central CG dinucleotide, and intermediate tolerance at the A/T bases at the motif edges. This analysis also demonstrated the influence of nearby flanking sequences. Extended motif and spacing analyses indicated dimeric binding at paired motifs, with periodic helical spacing consistent with interactions on the same face of the DNA helix. Integration of SNAP array binding with ChIP-seq data distinguished direct, motif-encoded GRHL2 occupancy from indirect, cofactor-mediated recruitment at genomic sites. These results define the sequence specificity of GRHL2 interactions with variations in the DNA consensus motif and flanking sequences within an endogenous genomic context. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/719077v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1a28904org.highwire.dtl.DTLVardef@1d197aforg.highwire.dtl.DTLVardef@13d9e97org.highwire.dtl.DTLVardef@76d55f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID are complex chronic conditions that often follow infectious triggers with overlapping clinical features but poorly defined pathophysiological relationships. This study aimed to identify disease-specific immune signatures through multiparameter immunophenotyping of monocytes, dendritic cells, and T-cell subsets. A total of 207 participants were included (ME/CFS: n = 103; long COVID: n = 63; healthy controls: n = 41). Peripheral blood mononuclear cells were analyzed using multiparameter flow cytometry. Statistical analyses included non-parametric testing, age-adjusted ANCOVA, correlation network analysis, and principal component analysis (PCA). Long COVID was characterized by increased M2-like monocyte polarization, elevated CD80 expression across monocyte subsets, expansion of dendritic cells, and reduced expression of activation markers, indicating persistent immune activation with features of immune exhaustion. In contrast, ME/CFS exhibited reduced costimulatory molecule expression, impaired CCR7-mediated immune cell trafficking, and less coordinated activation patterns, consistent with a state of immune suppression. Correlation network analysis revealed more extensive and integrated immune interactions in long COVID, while PCA identified distinct immunophenotypic components and enabled moderate discrimination between the two conditions. These findings demonstrate that ME/CFS and long COVID are characterized by distinct immune profiles, supporting the concept of divergent immunopathological mechanisms. The identified signatures may contribute to biomarker development and guide targeted therapeutic approaches.

Accurate chromosome segregation relies on proper centromere and kinetochore formation and phospho-regulation. We previously demonstrated that a pluripotent state confers a low fidelity of chromosome segregation, however it is unknown how a pluripotent state impacts centromere and kinetochore function. Here, we demonstrate that both centromere and kinetochore structural organization and phosphorylation in mitosis are developmentally regulated. CENP-A, CENP-C, and HEC1 protein abundance is reduced at mitotic centromeres and kinetochores of human pluripotent stem cells (hPSCs) compared to isogenic somatic cells; however, elevating their levels does not improve chromosome segregation fidelity. Rather, we find that reduced phosphorylation of kinetochores is responsible for their low fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs compared to isogenic somatic cells at Cyclin B/Cdk1 and Aurora kinase phospho-sites. Inhibiting PP2A phosphatase activity or differentiation increases HEC1 phosphorylation at hPSC kinetochores decreasing chromosome segregation errors. Thus, mitotic fidelity in non-transformed human cells depends on the developmental regulation of the kinase and phosphatase networks controlling kinetochore phosphorylation. SummaryGalaviz Sarmiento et al show that the developmental regulation of kinetochore phosphorylation governs mitotic fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs during mitosis contributing to their high rate of chromosome segregation errors. While differentiation increases HEC1 phosphorylation improving chromosome segregation fidelity.

Intracellular calcium signaling plays a vital role in regulating various cellular processes including gene regulation, motility, metabolism and cell death. Inositol 1,4,5-trisphosphate receptors (IP3R) on the Endoplasmic Reticulum (ER) are a major cation channel that regulates stimulus-induced calcium release from the ER. While several molecular players regulate activity of IP3R, its regulation by actin filaments were uncharacterized. Here we show that actin filaments polymerized by a specific actin nucleator INF2 facilitates agonist-induced IP3R activity. Our results demonstrate that INF2-mediated actin filaments regulate formation and/or stability of IP3R clusters on the ER that have been previously shown to be hotspots of ER calcium release. Using cell-biological and biochemical techniques we further show that INF2 physically interacts with IP3R isoforms, often at IP3R clusters. While INF2-IP3R interaction is independent of INF2-activity, the ability of INF2 to mediate IP3R clusters is dependent on its actin polymerization activity. Finally, we demonstrate that in addition to its calcium mobilization activity, INF2 on ER specifically regulates IP3R cluster positioning to mediate ER-mitochondrial contacts and facilitate ER to mitochondrial calcium transfer. Overall, these results reveal an actin-dependent step in regulation of IP3R activity both in terms of ER calcium release and modulation of ER-mitochondrial contacts. HighlightsO_LIINF2-mediated actin filaments potentiate agonist-induced IP3R-mediated ER calcium release without affecting the ER calcium stores per se. C_LIO_LIER-localization of INF2 is dispensable for its role on IP3R activity. Moreover INF2-mediated actin filaments affect the activity of all IP3R isoforms. C_LIO_LIINF2 interacts with IP3R in an activity and actin filament independent manner through its C-terminal region. C_LIO_LIINF2 regulates IP3R cluster formation in actin-filament dependent manner and thereby regulates IP3R activity. C_LIO_LIFurther we show that ER-localized INF2 specifically regulate IP3R cluster positioning thereby promoting ER to mitochondrial contact and calcium transfer. C_LI

Common sex chromosome aneuploidies (SCAs) often present with cognitive and cardiovascular dysfunction in humans. To address SCA effects on gene expression and DNA methylation in relevant cell types, we differentiated neural precursor cells (NPCs) and cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) with different numbers of sex chromosomes, including isogenic and independent lines. As expected, the expression of genes that escape X inactivation (escapees) mostly increases with the number of inactive X chromosomes (Xi). However, allelic analysis shows dampening of escapees specifically on the Xi in XXY compared to XX in both NPCs and CMs, revealing a novel type of dosage compensation in SCA. In contrast, Y-linked gene expression is higher in XXY versus XY NPCs, but the opposite is observed in CMs. This may explain the greater number of differentially expressed autosomal genes in NPCs versus CMs with an added Y chromosome, while effects of added X chromosomes are similar between cell types. Concordantly, changes in autosomal DNA methylation are mainly driven by the presence of a Y chromosome. These findings highlight the cell-type specificity of sex-linked and autosomal gene regulation in SCA conditions. HighlightsO_LISex chromosome aneuploidy induces cell-type specific changes in gene expression C_LIO_LIDampening of the inactive X chromosome in XXY alleviate X overexpression C_LIO_LIHigh Y-linked gene expression in XXY neuronal precursor cells but not cardiomyocytes C_LIO_LISex chromosome aneuploidy disrupts sex biases in autosomal gene expression C_LI

Interpretable representations of gene expression are used to define cellular identities and the molecular programs active within cells, two related, but distinct phenomena. In the case of microglia, a cell type with high transcriptomic, functional, and morphological heterogeneity, the predominant representation of transcriptomic data presumes the adoption of distinct molecular identities, despite a lack of easily separable transcriptional states. Here, we explore alternative transcriptomic representations by comparing two single-cell analysis methods: differential expression analysis for identities and co-expression network analysis for molecular programs. For microglia, co-expression network analysis identifies highly significant functional ontologies not resolved by differential expression analysis. The identified co-expression modules are preserved across transcriptomic datasets and suggest reducible functional programs that activate and modulate depending on context. We conclude that co-expression analysis constitutes a best practice for single cell analysis of an individual cell type and describing microglia function as concurrent molecular programs offers a more parsimonious model of microglia function.

The interleukin-17 (IL-17) family of cytokines comprises structurally distinct ligands and receptors which mediate immune responses at mucosal surfaces. The growing understanding of its regulatory functions beyond immunity, together with extensive genetic variation in protein-coding genes, raises the possibility that IL-17 cytokines participate in an even wider network of biologic processes. Despite successes of experimental approaches to chart IL-17 functions, inherent signaling complexities and crosstalk with multiple physiologic pathways obscure a full appreciation of the biological potential of IL-17. Here, we integrated comparative genomics, evolutionary rate covariation (ERC), and signatures of natural selection to resolve phylogenetic relationships between IL-17 ligands and receptors and discovered evidence for hidden signaling interactions. ERC analysis revealed putative ligand-receptor interactions for IL-17D and IL-17RC and suggested uncharacterized potential signaling mediator for the receptor IL-17REL, such as IL-17B. Signals of covariation extended beyond the IL-17 family to other genes encoding neurodevelopmental effectors and growth factors, emphasizing recurrent co-evolutionary patterns that delineate the immune and neuromodulatory roles of IL-17. These connections are underlined by signatures of positive selection in the disordered N-terminal domain of IL-17E and its cognate receptor, IL-17RB, key modulators of both type 2 immune response and neuronal function, suggesting functional consequences of this understudied domain. Together, our findings suggest that IL-17 biology is repeatedly impacted by lineage-specific selective pressures that dictate both immune and non-immune functions. By anchoring the expanding IL-17 field in an evolutionary framework, we propose a model for understanding the diversification and functional expansion of this and other cytokine families.

Spinal muscular atrophy (SMA), traditionally defined as a neuromuscular disorder characterized by degeneration of lower motor neurons, is increasingly recognized as a multi-organ disease. SMA is caused by deficiency of the survival motor neuron (SMN) protein below a critical threshold required for cellular homeostasis. While motor neurons are particularly vulnerable, the ubiquitous expression and fundamental functions of SMN result in widespread perturbations across multiple tissues. Here, we generated a label-free quantitative proteomics atlas of spinal cord, heart, and gastrocnemius muscle from wild-type, heterozygous, and SMA mice at the symptomatic stage, including cohorts treated, at postnatal day 1 (P1), with a systemic suboptimal dose of SMN antisense oligonucleotides (SMN-ASOs), resulting in partial SMN restoration. SMN deficiency induced pronounced, tissue-specific proteome remodeling, with peripheral tissues exhibiting broader molecular alterations than spinal cord. Cross-tissue analyses revealed limited overlap, although heart and muscle showed partial convergence in metabolic and mitochondrial-associated pathways. SMN-ASO treatment partially repositioned these proteomes toward control states; however, restoration was incomplete and strongly tissue-dependent, with persistent dysregulation of mitochondrial and metabolic pathways. These findings demonstrate that SMN deficiency drives systemic yet heterogeneous proteome remodeling and that partial SMN restoration does not fully reverse established molecular alterations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/715402v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@114d73borg.highwire.dtl.DTLVardef@13e8c13org.highwire.dtl.DTLVardef@15e4ba0org.highwire.dtl.DTLVardef@1b70fb8_HPS_FORMAT_FIGEXP M_FIG C_FIG

The human neonatal immune system is developmentally specialized to balance the unique requirements of perinatal transition. Disruption of this finely tuned balance, as in preterm birth, may have profound consequences for immunity and overall health. However, the impact of prematurity on immune composition and functional responsiveness across gestational ages (GA) remains incompletely understood. Single-cell profiling has advanced our understanding of neonatal immunity, yet most studies were limited to unimodal readouts, narrow GA windows, or baseline function. Here, we present a comprehensive human neonatal CITE-seq atlas (82 samples from 25 neonates and 10 adults as controls) at the first days of life covering a wide GA range and integrating baseline and stimulated conditions. Most notably, we identify a GA-dependent immune transition point centered around 32 weeks of GA, which discriminates extremely and very preterm neonates (GA <32wks) from those of higher GA ([&ge;]32wks). In particular, early-life immunity in extremely and very preterm infants showed CD15+ granulocytic myeloid derived suppressor cell-like predominance, whereas more mature neonates exhibited interferon-primed transcriptional profiles. This resulted in divergent myeloid-to-lymphocyte signaling networks and qualitatively distinct NK- and T-cell bystander responses upon activation. Together, these findings show that intrauterine development imprints GA-specific immune programs. By defining a developmental transition around a GA of 32 weeks that regulates baseline and induced responses of neonatal immune cells, our atlas provides a framework for understanding the vulnerability of preterm infants and thus may pave the way for developing GA-adapted immunomodulatory strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/715643v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1db4534org.highwire.dtl.DTLVardef@9c9665org.highwire.dtl.DTLVardef@55f063org.highwire.dtl.DTLVardef@190a52_HPS_FORMAT_FIGEXP M_FIG C_FIG

Large-scale single-cell atlases such as the Human Cell Atlas have transformed our understanding of human biology. Yet, the lack of a robust framework that standardises quality control, expands cellular annotation, and adds normalisation and analytical layers, limits multi-study analyses and the usefulness of this resource. Here we present cellNexus, a comprehensive tool and resource that converts the Human Cell Atlas collection into analysis-ready data by linking quality control layers, metadata enrichment, expression normalisation, analysis and data aggregation. These enhancements enable robust statistical modelling across studies, exemplified by a multi-tissue map of immune cell communication during ageing, which reveals macrophage-muscle axes as among the most depleted regenerative interactions with age. All harmonised layers, including pseudobulk and cell-cell communication summaries, are accessible via a public web interface and with R and Python APIs. By providing continuous integration with CELLxGENE releases, cellNexus transforms large cell atlas corpora into an accessible, reproducible, interoperable foundation for large-scale biological discovery and the next generation of single-cell foundation models.

Twice as many women develop Alzheimers disease (AD) compared to men. Several key aspects, such as genetic risk factors, hormonal vulnerability, social responsibilities, and differences in longevity, contribute to the strong female bias in AD. To assess whether sex differences can be detected during the onset of AD, we examined the amyloid-{beta} (A{beta}) plaque burden--one of the hallmarks of AD--and microglial states in young 5XFAD mouse models of amyloid pathology. We hypothesized that an increase in microglial cell number and phagocytic activity will directly correlate with an elevated A{beta} burden and shape the appearance of compact dense-core plaques in the cortex from 2 to 6 months of age. As expected, no change in microglial density and phenotype was found in A{beta} plaque-free hypothalamus of 5XFAD male and female mice when compared to age-matched wildtype controls. By quantifying the number and coverage of diffuse and dense-core plaques in the cortex, we discovered a pronounced increase in A{beta} plaques and microglial clustering in 4-month-old female 5XFAD compared to male mice. By 6 months, no sex difference in plaque load and microglial density was observed. Our spatiotemporal characterization of microglial Clec7a/Dectin-1 and CD68 expression revealed sex differences in the upregulation of these phagocytic markers in plaque-proximal microglia. In 2-months-old males, greater phagolysosomal activity around diffuse plaques may benefit A{beta} clearance. However, in females, the lower initial microglial reactivity and subsequent rise in Dectin-1-driven phagocytic activity may have led to the increase in dense-core plaques at 4 months. Our results suggest that during early amyloidosis, sex differences in CD68-associated lysosomal activity and microglia-driven plaque compaction may cause disproportionate AD risk and severity that is compounded by other exacerbating factors during aging. Taken together, sex-specific targeting of microglial proliferation and phagocytic activity may be a promising intervention in presymptomatic patients with known AD risks.

Image-based spatial transcriptomics depends on cell segmentation to assign transcripts to individual cells, but how segmentation algorithms perform across tissues with distinct cellular architectures is poorly understood. This study presents the broadest independent benchmark to date of cell segmentation algorithms for spatial transcriptomics, comparing five approaches across ten mouse tissues using a 5,006-gene Xenium panel. To quantify segmentation errors, Co-expression Rejection in Segmentation Purity (CRISP) was developed, an open-source tool available in R and Python that measures cell purity through tissue-specific mutually exclusive marker co-expression without requiring ground truth annotations. This benchmark revealed that segmentation algorithms face a fundamental tradeoff between maximizing transcript capture and maintaining cell purity, and that the severity of this tradeoff is tissue-dependent. Proseg achieved the highest average performance across tissues, though the magnitude of its advantage varies with tissue architecture. Overall, CRISP provides per-tissue performance profiles as a practical resource for algorithm selection.

Changes in regulatory sequences controlling the timing and activity of gene products underlie much of natural phenotypic variation. Yet, what these changes are and how they impact gene expression remain largely unknown. To address this question, we investigated how transcriptional activity and homeostatic responsiveness of orthologous promoters of the metabolic gene TDH3 evolved among Saccharomyces yeast. We found that promoter expression level increased specifically in the S. cerevisiae lineage and that a substantial part of this increase was caused by genetic variants located between the well-characterized, conserved binding sites for two direct transcriptional regulators. These nucleotide changes altered the promoters expression levels while leaving the expression dynamics conserved. Further, the effects of these nucleotide changes were only seen in the presence of a third transcription factor, TYE7p, which is recruited by the other transcription factors through protein-protein interactions. These results suggest that the cis-regulatory changes act through their influence on the collective assembly/activation of the transcription factors, and that changes acting through such a mechanism can allow distinct parts of gene expression, such as expression level and dynamics, to be tuned separately.

Systemic inflammation is implicated in various diseases, yet its upstream determinants remain poorly examined. We conducted a large scale two-sample Mendelian randomisation (MR) study to systematically evaluate the potential causal effects of 3,213 molecular (metabolomic, proteomic), physiological and disease traits on circulating interleukin-6 (IL-6) and C-reactive protein (CRP) levels. Genetic instruments were derived from genome wide association studies and analysed using inverse variance weighted (IVW), weighted median, and MR-Egger methods with multiple testing correction. Bidirectional MR was performed to assess reverse causation. After Bonferroni correction, evidence of potential causal effects was observed for 72 traits on CRP and 9 traits on IL-6. CRP was predominantly influenced by metabolomic traits, especially lipid and fatty acid measures. Genetically proxied adiposity (body mass index and obesity), triglyceride rich lipoproteins, glycoprotein acetyls (GlycA), and apolipoprotein E increased CRP levels, whereas HDL-related cholesterols, polyunsaturated fatty acids, and glutamine decreased CRP. Most associations were consistent across MR methods, supporting the robustness of these results. As expected, IL-6 had a large effect on CRP. IL-6 was influenced by primarily adiposity and HDL-related lipid measures, with generally smaller effect sizes and limited support across sensitivity analyses. Bidirectional analyses indicated little evidence that CRP directly drives metabolic traits when restricting to cis-acting instruments, whereas genetically proxied IL-6 signalling showed consistent downstream effects on HDL particle concentration and composition. Adiposity is a shared upstream determinant of both inflammatory biomarkers, with stronger and broader effects on CRP. These findings suggest that CRP acts as an integrated downstream readout of systemic inflammatory burden, whereas IL-6 reflects a more tightly regulated and context-dependent process. Our work clarifies traits that may causally influence systemic inflammation and highlights biological pathways linking inflammation to cardiometabolic and inflammatory diseases. By mapping upstream determinants of IL-6 and CRP, we also provide a resource to prioritise key drivers for mechanistic study and therapeutic targeting.

Germ cells are the only cells of an organism that pass onto the next generation and, hence perpetuate the species. To ensure this, germ cells need dedicated transcriptional repertoire, that ensure specification, proliferation, differentiation and fate maintenance. We previously characterized LSL-1, a conserved zinc-finger transcription factor that acts as a major direct transcriptional activator of genes involved in germ cell development, fate specification, meiosis and genome stability. Here, we show that LSL-1 interacts with the transcription factor HIM-17, the chromatin proteins BRA-2 and XND-1. These proteins are functionally related to LSL-1 and they colocalize at germline gene promoters, forming most likely a transcription-promoting complex. Furthermore, LSL-1 lies in close proximity to members of the COMPASS and the MOF complexes, corroborating the observation that HIM-17 and LSL-1 are required to maintain normal level of H3K4 methylation in the gonad. Finally, we show that LSL-1 interacting partners are necessary to maintain germ cell fate. Altogether, we propose that LSL-1 interacts with transcription regulators and chromatin modifiers to ensure the establishment of the transcriptional repertoire appropriate for germline function as well as for cell fate maintenance.

BackgroundTau aggregation is the defining feature of tauopathies, however, the mechanisms by which distinct tau strains drive disease-specific responses remain unclear. Existing models largely rely on recombinant tau seeding or tau overexpression, which fail to capture the biochemical diversity of pathological tau. The aim of this study was to develop a robust and reproducible human cell-based model of disease-specific tau pathology and to use this model to determine how tau from unique diseases impact tau accumulation and lysosomal dysfunction. MethodsPatient-derived tau aggregates were enriched from post-mortem brain tissue obtained from sporadic Alzheimers disease (AD), Picks disease (PiD), progressive supranuclear palsy (PSP), and control cases using phosphotungstic acid precipitation. Patient-derived tau preparations were biochemically characterised by immunoblotting and mass spectrometry and normalised for tau content prior to seeding. Patient-derived tau aggregates were seeded into multiple human immortalised cell lines (SH-SY5Y, M03.13, U-87 MG, and U-118 MG cells) and iPSC-derived astrocytes. Tau seeding efficiency, aggregate morphology, and integrity of the autophagy-lysosomal pathway was assessed using quantitative imaging approaches. ResultsPatient-derived tau seeds retained disease-specific phosphorylation patterns and isoform composition and led to reproducible, dose-dependent insoluble tau accumulation in all cell lines tested. Despite equivalent tau input and similar background protein composition, PiD-derived tau had the most aggressive pathological signature, showing the highest number of tau aggregates per cell and inducing system wide disruptions in the autophagy lysosomal system including increased SQSTM1 puncta and lysosomal damage markers. Seeding with AD-derived tau led to a high number of tau aggregates per cell and more specifically depleted the lysosomal protease CTSD and uniquely co-seeded A{beta} pathology. Seeding with PSP-derived tau resulted in only a moderate number of tau aggregates per cell and uniquely caused increased lysosomal biogenesis. ConclusionsTogether, these results demonstrate that intrinsic properties of human tau strains drive disease-specific cellular responses and establish a scalable, physiologically relevant platform for dissecting tau-cell interactions and screening therapeutics across tauopathies.

Ewing sarcoma (EwS) is an aggressive, human-exclusive tumor typically driven by the EWS::FLI1 fusion protein. To assess whether the neomorphic functions of EWS::FLI1 are fundamentally dependent on evolutionarily recent cofactors such as ETS transcription factors (ETS-TFs), Plycomb group (PcG) proteins, CBP/p300, or specific subunits of the BAF complex, we expressed EWS::FLI1 in the model organism Saccharomyces cerevisiae. This minimal system was chosen because several key EWS::FLI 's cofactors possess greatly reduced sequence homology (e.g., BAF) or are lacking altogether (e.g., ETS-TFs, PcG, or CBP/p300). We used co-IP/MS to map the yeast interactome, Chip-Seq to identify gDNA binding sequences, RNA-Seq for global gene expression, and engineered reporters to test conversion of (GGAA) tandem repeats (GGAASat) into neoenhancers. We found that the yeast EWS::FLI1 interactome was more limited and qualitatively distinct from its human counterpart, sharing core machinery (e.g. RNA Polymerase II, FACT) but lacking the BAF/SWI-SNF and spliceosome complexes, and showing strong enrichment for the SAGA chromatin remodeling complex. We also found that EWS::FLI1 binds to hundreds of sites in the yeast genome with a clear preference for putative ETS-TF consensus sequences and (CA) dinucleotide repeats. Yet, EWS::FLI1 expressing cells presented only minimal transcriptional dysregulation, a stark contrast to the extensive changes observed in humans and Drosophila cells. Finally, we found that EWS::FLI1 successfully converted silent GGAASat sequences into active enhancers in yeast. This remarkable result occurs despite the absence of homologs for key human activators, such as CBP/p300, strongly suggesting that EWS::FLI1 can mobilize functionally related, non-homologous pathways to establish neoenhancers at GGAASat sites. Altogether, our results indicate that EWS::FLI1's core ability to drive GGAASat-dependent gene expression is a conserved, ancient property, while GGAASat-independent extensive transcriptome reprogramming is dependent on co-factors and pathways specific to animal cells.