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Publications in Biomedical Research by NOMIS researchers

Persistence of spike protein at the skull-meninges-brain axis may contribute to the neurological sequelae of COVID-19

NOMIS Researcher(s)

Published in

December 11, 2024

SARS-CoV-2 infection is associated with long-lasting neurological symptoms, although the underlying mechanisms remain unclear. Using optical clearing and imaging, we observed the accumulation of SARS-CoV-2 spike protein in the skull-meninges-brain axis of human COVID-19 patients, persisting long after viral clearance. Further, biomarkers of neurodegeneration were elevated in the cerebrospinal fluid from long COVID patients, and proteomic analysis of human skull, meninges, and brain samples revealed dysregulated inflammatory pathways and neurodegeneration-associated changes. Similar distribution patterns of the spike protein were observed in SARS-CoV-2-infected mice. Injection of spike protein alone was sufficient to induce neuroinflammation, proteome changes in the skull-meninges-brain axis, anxiety-like behavior, and exacerbated outcomes in mouse models of stroke and traumatic brain injury. Vaccination reduced but did not eliminate spike protein accumulation after infection in mice. Our findings suggest persistent spike protein at the brain borders may contribute to lasting neurological sequelae of COVID-19.

Research field(s)
Molecular Biology, Virology, Immunology

DOI
10.1016/j.chom.2024.11.007

Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes

NOMIS Researcher(s)

Published in

December 4, 2024

Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75–99%) and silencing (76–92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrPC, screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and ‘post-pooling’ individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.

Research field(s)
Bioinformatics, Biotechnology, Biochemistry & Molecular Biology, Genetics & Heredity

DOI
10.1038/s41551-024-01278-4

Genome editing with the HDR-enhancing DNA-PKcs inhibitor AZD7648 causes large-scale genomic alterations

NOMIS Researcher(s)

Published in

November 27, 2024

The DNA-PKcs inhibitor AZD7648 enhances CRISPR–Cas9-directed homology-directed repair efficiencies, with potential for clinical utility, but its possible on-target consequences are unknown. We found that genome editing with AZD7648 causes frequent kilobase-scale and megabase-scale deletions, chromosome arm loss and translocations. These large-scale chromosomal alterations evade detection through typical genome editing assays, prompting caution in deploying AZD7648 and reinforcing the need to investigate multiple types of potential editing outcomes.

Research field(s)
Biochemistry & Molecular Biology, Genetics & Heredity

DOI
10.1038/s41587-024-02488-6

Archaeal type six secretion system mediates contact-dependent antagonism

NOMIS Researcher(s)

Published in

November 15, 2024

Microbial communities are shaped by cell-cell interactions. Although archaea are often found in associations with other microorganisms, the mechanisms structuring these communities are poorly understood. Here, we report on the structure and function of haloarchaeal contractile injection systems (CISs). Using a combination of functional assays and time-lapse imaging, we show that Halogeometricum borinquense exhibits antagonism toward Haloferax volcanii by inducing cell lysis and inhibiting proliferation. This antagonism is contact-dependent and requires a functional CIS, which is encoded by a gene cluster that is associated with toxin-immunity pairs. Cryo–focused ion beam milling and imaging by cryo–electron tomography revealed that these CISs are bound to the cytoplasmic membrane, resembling the bacterial type six secretion systems (T6SSs). We show that related T6SS gene clusters are conserved and expressed in other haloarchaeal strains, which exhibit antagonistic behavior. Our data provide a mechanistic framework for understanding how archaea may shape microbial communities and affect the food webs they inhabit.

Research field(s)
Microbiology

DOI
10.1126/sciadv.adp7088

Mechanism of bacterial predation via ixotrophy

NOMIS Researcher(s)

Published in

October 18, 2024

Ixotrophy is a contact-dependent predatory strategy of filamentous bacteria in aquatic environments for which the molecular mechanism remains unknown. We show that predator-prey contact can be established by gliding motility or extracellular assemblages we call “grappling hooks.” Cryo–electron microscopy identified the grappling hooks as heptamers of a type IX secretion system substrate. After close predator-prey contact is established, cryo–electron tomography and functional assays showed that puncturing by a type VI secretion system mediated killing. Single-cell analyses with stable isotope–labeled prey revealed that prey components are taken up by the attacker. Depending on nutrient availability, insertion sequence elements toggle the activity of ixotrophy. A marine metagenomic time series shows coupled dynamics of ixotrophic bacteria and prey. We found that the mechanism of ixotrophy involves multiple cellular machineries, is conserved, and may shape microbial populations in the environment.

Research field(s)
Microbiology

DOI
10.1126/science.adp0614

Synaptic neoteny of human cortical neurons requires species-specific balancing of SRGAP2-SYNGAP1 cross-inhibition

NOMIS Researcher(s)

Published in

October 14, 2024

Human-specific (HS) genes have been implicated in brain evolution, but their impact on human neuron development and diseases remains unclear. Here, we study SRGAP2B/C, two HS gene duplications of the ancestral synaptic gene SRGAP2A, in human cortical pyramidal neurons (CPNs) xenotransplanted in the mouse cortex. Downregulation of SRGAP2B/C in human CPNs led to strongly accelerated synaptic development, indicating their requirement for the neoteny that distinguishes human synaptogenesis. SRGAP2B/C genes promoted neoteny by reducing the synaptic levels of SRGAP2A,thereby increasing the postsynaptic accumulation of the SYNGAP1 protein, encoded by a major intellectual disability/autism spectrum disorder (ID/ASD) gene. Combinatorial loss-of-function experiments in vivo revealed that the tempo of synaptogenesis is set by the reciprocal antagonism between SRGAP2A and SYNGAP1, which in human CPNs is tipped toward neoteny by SRGAP2B/C. Thus, HS genes can modify the phenotypic expression of genetic mutations leading to ID/ASD through the regulation of human synaptic neoteny.

Research field(s)
Genetics & Heredity

DOI
10.1016/j.neuron.2024.08.021

If you please, draw me a neuron — linking evolutionary tinkering with human neuron evolution

NOMIS Researcher(s)

Published in

October 1, 2024

Animal speciation often involves novel behavioral features that rely on nervous system evolution. Human-specific brain features have been proposed to underlie specialized cognitive functions and to be linked, at least in part, to the evolution of synapses, neurons, and circuits of the cerebral cortex. Here, we review recent results showing that, while the human cortex is composed of a repertoire of cells that appears to be largely similar to the one found in other mammals, human cortical neurons do display specialized features at many levels, from gene expression to intrinsic physiological properties. The molecular mechanisms underlying human species-specific neuronal features remain largely unknown but implicate hominid-specific gene duplicates that encode novel molecular modifiers of neuronal function. The identification of human-specific genetic modifiers of neuronal function brings novel insights on brain evolution and function and, could also provide new insights on human species-specific vulnerabilities to brain disorders.

Research field(s)
Genetics & Heredity, Evolutionary Biology

DOI
10.1016/j.gde.2024.102260

Docking a flexible basket onto the core of the nuclear pore complex

NOMIS Researcher(s)

Published in

August 13, 2024

The nuclear basket attaches to the nucleoplasmic side of the nuclear pore complex (NPC), coupling transcription to mRNA quality control and export. The basket expands the functional repertoire of a subset of NPCs in Saccharomyces cerevisiae by drawing a unique RNA/protein interactome. Yet, how the basket docks onto the NPC core remains unknown. By integrating AlphaFold-based interaction screens, electron microscopy and membrane-templated reconstitution, we uncovered a membrane-anchored tripartite junction between basket and NPC core. The basket subunit Nup60 harbours three adjacent short linear motifs, which connect Mlp1, a parallel homodimer consisting of coiled-coil segments interrupted by flexible hinges, and the Nup85 subunit of the Y-complex. We reconstituted the Y-complex•Nup60•Mlp1 assembly on a synthetic membrane and validated the protein interfaces in vivo. Here we explain how a short linear motif-based protein junction can substantially reshape NPC structure and function, advancing our understanding of compositional and conformational NPC heterogeneity.

Research field(s)
Biochemistry & Molecular Biology, Biology

DOI
10.1038/s41556-024-01484-x

Removal of TREX1 activity enhances CRISPR–Cas9-mediated homologous recombination

NOMIS Researcher(s)

Published in

August 12, 2024

CRISPR–Cas9-mediated homology-directed repair (HDR) can introduce desired mutations at targeted genomic sites, but achieving high efficiencies is a major hurdle in many cell types, including cells deficient in DNA repair activity. In this study, we used genome-wide screening in Fanconi anemia patient lymphoblastic cell lines to uncover suppressors of CRISPR–Cas9-mediated HDR. We found that a single exonuclease, TREX1, reduces HDR efficiency when the repair template is a single-stranded or linearized double-stranded DNA. TREX1 expression serves as a biomarker for CRISPR–Cas9-mediated HDR in that the high TREX1 expression present in many different cell types (such as U2OS, Jurkat, MDA-MB-231 and primary T cells as well as hematopoietic stem and progenitor cells) predicts poor HDR. Here we demonstrate rescue of HDR efficiency (ranging from two-fold to eight-fold improvement) either by TREX1 knockout or by the use of single-stranded DNA templates chemically protected from TREX1 activity. Our data explain why some cell types are easier to edit than others and indicate routes for increasing CRISPR–Cas9-mediated HDR in TREX1-expressing contexts.

Research field(s)
Biochemistry & Molecular Biology, Genetics & Heredity

DOI
10.1038/s41587-024-02356-3

CCAR1 promotes DNA repair via alternative splicing

NOMIS Researcher(s)

Published in

July 25, 2024

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

Research field(s)
Genetics & Heredity, Biomedical Research

DOI
10.1016/j.molcel.2024.06.011

Deep 3D histology powered by tissue clearing, omics and AI

NOMIS Researcher(s)

Published in

July 12, 2024

To comprehensively understand tissue and organism physiology and pathophysiology, it is essential to create complete three-dimensional (3D) cellular maps. These maps require structural data, such as the 3D configuration and positioning of tissues and cells, and molecular data on the constitution of each cell, spanning from the DNA sequence to protein expression. While single-cell transcriptomics is illuminating the cellular and molecular diversity across species and tissues, the 3D spatial context of these molecular data is often overlooked. Here, I discuss emerging 3D tissue histology techniques that add the missing third spatial dimension to biomedical research. Through innovations in tissue-clearing chemistry, labeling and volumetric imaging that enhance 3D reconstructions and their synergy with molecular techniques, these technologies will provide detailed blueprints of entire organs or organisms at the cellular level. Machine learning, especially deep learning, will be essential for extracting meaningful insights from the vast data. Further development of integrated structural, molecular and computational methods will unlock the full potential of next-generation 3D histology.

Research field(s)
Biochemistry & Molecular Biology, Chemistry

DOI
10.1038/s41592-024-02327-1

Single cell transcriptomes and multiscale networks from persons with and without Alzheimer’s disease

NOMIS Researcher(s)

Published in

July 10, 2024

The emergence of single nucleus RNA sequencing (snRNA-seq) offers to revolutionize the study of Alzheimer’s disease (AD). Integration with complementary multiomics data such as genetics, proteomics and clinical data provides powerful opportunities to link cell subpopulations and molecular networks with a broader disease-relevant context. We report snRNA-seq profiles from superior frontal gyrus samples from 101 well characterized subjects from the Banner Brain and Body Donation Program in combination with whole genome sequences. We report findings that link common AD risk variants with CR1 expression in oligodendrocytes as well as alterations in hematological parameters. We observed an AD-associated CD83(+) microglial subtype with unique molecular networks and which is associated with immunoglobulin IgG4 production in the transverse colon. Our major observations were replicated in two additional, independent snRNA-seq data sets. These findings illustrate the power of multi-tissue molecular profiling to contextualize snRNA-seq brain transcriptomics and reveal disease biology.

Research field(s)
Genetics & Heredity, Biology

DOI
10.1038/s41467-024-49790-0

FilamentID reveals the composition and function of metabolic enzyme polymers during gametogenesis

NOMIS Researcher(s)

Published in

June 20, 2024

Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a “filament identification” (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.

Research field(s)
Microbiology, Biology

DOI
10.1016/j.cell.2024.04.026

APOE3 Christchurch Heterozygosity and Autosomal Dominant Alzheimer’s Disease

NOMIS Researcher(s)

Published in

June 19, 2024

Variants in APOE and PSEN1 (encoding apolipoprotein E and presenilin 1, respectively) alter the risk of Alzheimer’s disease. We previously reported a delay of cognitive impairment in a person with autosomal dominant Alzheimer’s disease caused by the PSEN1E280A variant who also had two copies of the apolipoprotein E3 Christchurch variant (APOE3Ch). Heterozygosity for the APOE3Ch variant may influence the age at which the onset of cognitive impairment occurs. We assessed this hypothesis in a population in which the PSEN1E280A variant is prevalent.

Research field(s)
Health Sciences, Genetics & Heredity, Clinical Medicine

DOI
10.1056/NEJMoa2308583

Geometry-driven migration efficiency of autonomous epithelial cell clusters

NOMIS Researcher(s)

Published in

June 19, 2024

The directed migration of epithelial cell collectives through coordinated movements plays a crucial role in various physiological processes and is increasingly understood at the level of large confluent monolayers. However, numerous processes rely on the migration of small groups of polarized epithelial clusters in complex environments, and their responses to external geometries remain poorly understood. To address this, we cultivate primary epithelial keratocyte tissues on adhesive microstripes to create autonomous epithelial clusters with well-defined geometries. We show that their migration efficiency is strongly influenced by the contact geometry and the orientation of cell–cell contacts with respect to the direction of migration. A combination of velocity and polarity alignment with contact regulation of locomotion in an active matter model captures quantitatively the experimental data. Furthermore, we predict that this combination of rules enables efficient navigation in complex geometries, which we confirm experimentally. Altogether, our findings provide a conceptual framework for extracting the interaction rules of active systems from their interaction with physical boundaries, as well as design principles for collective navigation in complex microenvironments.

Research field(s)
Biophysics

DOI
10.1038/s41567-024-02532-x

Evolution of diapause in the African turquoise killifish by remodeling the ancient gene regulatory landscape

NOMIS Researcher(s)

Published in

May 28, 2024

Suspended animation states allow organisms to survive extreme environments. The African turquoise killifish has evolved diapause as a form of suspended development to survive a complete drought. However, the mechanisms underlying the evolution of extreme survival states are unknown. To understand diapause evolution, we performed integrative multi-omics (gene expression, chromatin accessibility, and lipidomics) in the embryos of multiple killifish species. We find that diapause evolved by a recent remodeling of regulatory elements at very ancient gene duplicates (paralogs) present in all vertebrates. CRISPR-Cas9-based perturbations identify the transcription factors REST/NRSF and FOXOs as critical for the diapause gene expression program, including genes involved in lipid metabolism. Indeed, diapause shows a distinct lipid profile, with an increase in triglycerides with very-long-chain fatty acids. Our work suggests a mechanism for the evolution of complex adaptations and offers strategies to promote long-term survival by activating suspended animation programs in other species.

Research field(s)
Molecular Biology, Biochemistry & Molecular Biology, Developmental Biology, Genetics & Heredity

DOI
10.1016/j.cell.2024.04.048

Lifelong persistence of nuclear RNAs in the mouse brain

NOMIS Researcher(s)

Published in

April 4, 2024

Genomic DNA that resides in the nuclei of mammalian neurons can be as old as the organism itself. The life span of nuclear RNAs, which are critical for proper chromatin architecture and transcription regulation, has not been determined in adult tissues. In this work, we identified and characterized nuclear RNAs that do not turn over for at least 2 years in a subset of postnatally born cells in the mouse brain. These long-lived RNAs were stably retained in nuclei in a neural cell type–specific manner and were required for the maintenance of heterochromatin. Thus, the life span of neural cells may depend on both the molecular longevity of DNA for the storage of genetic information and also the extreme stability of RNA for the functional organization of chromatin.

Research field(s)
Genetics & Heredity, Biology

DOI
10.1126/science.adf3481

G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes

NOMIS Researcher(s)

Published in

March 21, 2024

Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.

Research field(s)
Biochemistry & Molecular Biology, Biophysics, Virology

DOI
10.1093/nar/gkad1200

A common cis-regulatory variant impacts normal-range and disease-associated human facial shape through regulation of PKDCC during chondrogenesis

NOMIS Researcher(s)

Published in

March 14, 2024

Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960, a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC – a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, we demonstrate that the rs6740960 SNP is sufficient to confer chondrocyte-specific differences in PKDCC expression. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in the maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features.

Research field(s)
Developmental Biology, Genetics & Heredity, Evolutionary Biology

DOI
10.7554/eLife.82564

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