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Holland, A.

Publications and source records attributed to Holland, A..

16 recordsLinked to original sources

Self-supervised learning for a gene program-centric view of cell states

Single-cell omics has extended the biological interrogation of cell state from examining the expression of individual genes to unbiased profiling of tens of thousands of genes at once. However, extracting biological insights from such high-dimensional data remains challenging. To enable downstream analyses, many computational approaches compress cell state into a single latent representation. This can obscure the structure of underlying gene programs (GP), defined as coordinated sets of biologically related genes, such as signalling pathway response modules or transcription factor targets. Here, we present Tripso, a self-supervised transformer deep learning model which learns multiple GP-specific embeddings from predefined GPs, while also enabling the discovery of novel, data-driven GPs. Tripso facilitates principled comparisons across development, disease, and experimental systems. Firstly, in a dataset of human hematopoietic cells spanning prenatal development through adulthood and aging and including newly generated data, Tripso resolved age-specific GP patterns, including elevated JAK-STAT activity in pediatric hematopoietic cells and postnatal shifts in IKZF1 GP activity during B cell differentiation. Secondly, leveraging Tripso GP embeddings and comparing in vivo to in vitro data, we hypothesized and experimentally validated that inhibition of the SEC61 translocon improved maintenance of hematopoietic stem cells in culture. Finally, Tripsos capacity for data-driven GP discovery revealed a previously uncharacterized tissue-resident memory T cell GP with increased activity in atopic dermatitis. Its spatial co-localization with sebaceous gland-associated immune niches was demonstrated in spatial transcriptomic and proteomic data. Thus, by moving beyond single embeddings of cellular states, Tripso enables interpretable and actionable discoveries, demonstrating how GP-centric modelling can generate hypotheses with substantial biomedical relevance. By anchoring cellular representations in meaningful GPs, Tripso establishes a principled and biologically grounded framework towards the development of interpretable virtual cell models.

bioinformatics↗

Predicting how perturbations reshape cellular trajectories with PerturbGen

A major challenge in biology is predicting how cells transition between states over time and how perturbations disrupt these transitions. Understanding such dynamics is critical for identifying interventions that reverse pathological programs or reprogram cells toward desired states. Although recent computational approaches can predict single-cell perturbation responses in silico, they cannot predict responses across dynamic cell trajectories, for example how early perturbations reconfigure later cell states. To address this gap, we introduce PerturbGen, a generative foundation model trained on over 100 million single-cell transcriptomes that predicts perturbation responses along cellular trajectories. PerturbGen predicts how genetic perturbation at source state shapes downstream states, alters gene programs and trajectories across time, for example in differentiation or disease progression. We apply PerturbGen to three newly generated multi-condition human single-cell datasets spanning immune responses, hematopoiesis and skin development. In an in vivo immune challenge, PerturbGen predicts that knocking out an IL1B signal in myeloid cells attenuates later cytokine-interferon programs, with downstream changes consistent with a reversal of IL-1{beta} stimulation signature. In hematopoiesis, anchoring perturbation-induced programs to human genetics enables simulation of monogenic blood disorders, recapitulates established disease-associated biology whilst systematically revealing lineage-specific programs, including in lineages where this was not previously possible. In skin organoids, PerturbGen predicted that Wnt activation enhances stromal differentiation recapitulating the trajectory observed in human prenatal skin, findings that were functionally validated by experimentally activating Wnt signaling. Together, PerturbGen extends modeling of gene perturbations from static to dynamic cellular systems. We envision PerturbGen enabling the creation of in silico, trajectory-aware perturbation atlases and virtual cells across diverse biological scenarios, supporting optimization of disease models and prioritization of candidate molecular interventions for therapeutic discovery.

bioinformatics↗

EXOSC3 S1-domain variants implicated in PCH1b alter RNA exosome cap subunit abundance and thermal stability disrupting rRNA processing and targeting of AU-rich mRNA

Missense variants in EXOSC3, an RNA exosome subunit, have been identified in patients with PCH1b. We investigated three missense variants in the S1 domain of EXOSC3, including one variant of uncertain significance (VUS) and two pathogenic variants (hence S1 variants). EXOSC3 S1 variant cell lines were generated using CRISPR-Cas9 resulting in widespread proteome changes including decreases in some RNA exosome subunits paired with increases in the catalytic subunit DIS3. Thermal stability, analyzed by PISA, revealed extensive destabilization of RNA exosome cap subunits and the cap-associated exonuclease EXOSC10. Functionally, S1 variants altered rRNA processing with corresponding protein compensation observed in rRNA processing proteins outside the RNA exosome. Exogenous overexpression of EXOSC3 rescues many molecular defects caused by S1 variants suggesting that protein destabilization and turnover strongly contribute to molecular defects. Overall, our findings define the mechanisms through which cells respond to EXOSC3 S1 variant disruption of RNA processing homeostasis.

systems biology↗

Mechanoimmunological Control of Metastatic Site Selection

Cancer cells alter their mechanical properties in response to the rigidity of their environment. Here, we explored the implications of this environmental mechanosensing for anti-tumor immunosurveillance using single cell biophysical profiling and metastasis models. Cancer cells stiffened in more rigid environments, a biophysical change that sensitized them to cytotoxic lymphocytes. In immunodeficient mice, this behavior manifested in the outgrowth of stiffer metastatic cells in the rigid bone than in the soft lung, while in immunocompetent hosts, it led to preferential elimination of stiffer cancer cells and suppression of bone metastasis. Environmentally-induced cell stiffening and immune sensitization both required Osteopontin, a secreted glycoprotein that is upregulated during bone colonization. Analysis of patient metastases spanning mechanically distinct tissues revealed associations between environmental rigidity, immune infiltration, and cancer cell stiffness consistent with mechanically driven immunosurveillance. These results demonstrate how environmental mechanosensing modulates anti-tumor immunity and suggest a mechanoimmunological basis for metastatic site selection.

cancer biology↗

Large serine integrases utilise scavenged phage proteins as directionality cofactors

Recombination directionality factors (RDFs) for large serine integrases (LSIs) are cofactor proteins that control the directionality of recombination to favor excision over insertion. Although RDFs are predicted to bind their cognate LSIs in similar ways, there is no overall common structural theme across LSI RDFs, leading to the suggestion that some of them may be moonlighting proteins with other primary functions. To test this hypothesis, we searched for characterized proteins with structures similar to the predicted structures of known RDFs. Our search shows that the RDFs for two LSIs, TG1 integrase and Bxb1 integrase, show high similarities to a single stranded DNA binding (SSB) protein and an editing exonuclease, respectively. We present experimental data to show that TG1 RDF is a functional SSB protein. We used mutational analysis to validate the integrase-RDF interface predicted by AlphaFold2 multimer for TG1 integrase and its RDF, and establish that control of recombination directionality is mediated via protein-protein interaction at the junction of recombinases second DNA binding domain and the base of the coiled coil domain.

molecular biology↗

Identification of cognate recombination directionality factors for large serine recombinases by virtual pulldown

Integrases from the "large serine" family are simple, highly directional site-specific DNA recombinases that have great promise as synthetic biology and genome editing tools. Integrative recombination (mimicking phage or mobile element insertion) requires only integrase and two short ([~]40 - 50) DNA sites. The reverse reaction, excisive recombination, does not occur until it is triggered by the presence of a second protein termed a Recombination Directionality Factor (RDF), which binds specifically to its cognate integrase. Identification of RDFs has been hampered due to their lack of sequence conservation and lack of synteny with the phage integrase gene. Here we use Alphafold2-multimer to identify putative RDFs for more than half of a test set of 98 large serine recombinases, and experimental methods to verify predicted RDFs for 4 of 5 integrases chosen as test cases. We find no universally conserved structural motifs among known and predicted RDFs, yet they are all predicted to bind a similar location on their cognate integrase, suggesting convergent evolution of function. Our methodology greatly expands the available genetic toolkit of cognate integrase - RDF pairs.

synthetic biology↗

Variable orthogonality of RDF-large serine integrase interactions within the phiC31 family

Large serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse, excisive reaction, generating new recombination sites, attR and attL. In addition to promoting attR x attL reaction, the RDF inhibits attP x attB recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases ({phi}C31, {phi}BT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both attP x attB and attR x attL reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases - RDF pairs: {phi}C31 integrase was the least selective, with all three RDFs activating it for attR x attL recombination. Similarly, {phi}C31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate {phi}C31 integrase. {phi}BT1 and TG1 RDFs were noticeably more effective than {phi}C31 RDF at inhibiting attP x attB recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase - RDF pair. The binding surface on RDF is much more conserved than the binding surface on integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase - RDF pairs. Additionally, the ability of a particular integrases preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches.

molecular biology↗

Within-host adaptation of a foliar pathogen, Xanthomonas, on pepper in presence of quantitative resistance and ozone stress

O_LIThe evolving threat of new pathogen variants in the face of global environmental changes poses a risk to the plant health and can impact the efficacy of resistance-based disease management. C_LIO_LIHere, we studied short-term eco-evolutionary response of the pathogen, Xanthomonas perforans, on quantitative resistant and susceptible pepper during a single growing season in open-top chambers under the influence of elevated Ozone (O3). C_LIO_LIWe observed increased disease severity, accompanied by higher variation on resistant cultivar under elevated O3, with no apparent change on the susceptible cultivar. This altered resistance response under elevated O3 is linked to altered eco-evolutionary dynamics of pathogen. While a single pathogen genotype remained prevalent on susceptible cultivar, resistant cultivar supported heterogenous pathogen population, with the evidence of short- term evolutionary modifications seeded by de novo parallel mutations. Altered O3 levels led to strain turnover on resistant cultivar with higher within-host polymorphism containing higher proportion of random de novo mutations lacking parallelism. C_LIO_LIPopulation heterogeneity is a mechanism of pathogen adaptation in response to the stressors. While parallel mutations in response to quantitative resistance may provide clues to predicting long-term pathogen evolution, high proportion of transient mutations suggest less predictable pathogen evolution under climatic alterations. C_LI

evolutionary biology↗

Fish-microbe systems in the hostile but highly biodiverse Amazonian blackwaters

Amazonian blackwaters are extremely biodiverse systems containing some of the most naturally acidic, dissolved organic carbon-rich and ion-poor waters on Earth. Physiological adaptations of fish facing these ionoregulatory challenges are unresolved but could involve microbially-mediated processes. Here, we characterize the physiological response of 964 fish-microbe systems from four blackwater Teleost species along a natural hydrochemical gradient, using dual RNA-Seq and 16S rRNA of gill samples. We find that responses to blackwaters are host-species-specific, but occasionally include the overexpression of Toll-receptors and integrins associated to interkingdom communication. Blackwater gill microbiomes are characterized by a transcriptionally-active betaproteobacterial cluster potentially interfering with epithelial permeability. We explore further blackwater fish-microbe interactions by analyzing transcriptomes of 320 axenic zebrafish larvae exposed to sterile, non-sterile and inverted (non-native bacterioplankton) blackwater. We find that axenic zebrafish do not survive well when exposed to sterile/inverted blackwater, suggesting an essential role of endogenous symbionts in blackwater fish physiology.

ecology↗

Genomic and environmental factors shape gill microbiome activity in an Amazonian teleost holobiont

Fish microbiomes provide functions critical for their hosts survival in contrasting environments. These communities are sensitive to a range of environmental-specific factors (i.e. physicochemical parameters, free-living bacterioplankton) and host-specific factors (i.e. host genetic background). The relative contribution of these genomic and environmental factors shaping Amazonian fish microbiomes is still unknown. Here, we investigated this topic by analyzing the gill microbiomes of 240 wild flag cichlids (Mesonauta festivus) from four different populations (genetic clusters) distributed in 12 sites of two contrasting water types (ion-poor/acidic black water and ion-rich/circumneutral white water). The transcriptionally active gill microbiomes were characterized by a 16S rRNA metabarcoding approach carried on RNA extractions. They were analyzed in light of comprehensive datasets from the hosts genetic background (Genotyping-By-Sequencing), the bacterioplanktonic pool of bacteria (16S rRNA) and a set of 34 environmental parameters. Results show that the transcriptional activity of gill microbiome samples was significantly different between the genetic clusters and between water types. However, they suggest that the contribution of the hosts genetic background was relatively weak in comparison to the environment-related factors in structuring the relative abundance of different gill microbiome transcripts. This result was also confirmed by a mixed-effects modeling analysis, which suggested that the dissimilarity between the transcriptional activity of bacterioplanktonic communities possessed the best explicative power regarding the dissimilarity between gill microbiomes transcripts, while pairwise fixation indexes (FST) from the hosts genetic data only had a weak explicative power. We discuss these results in terms of microbiome assembly processes and flag cichlid fish ecology. ImportanceHost-associated microbial communities respond to a range of factors specific to the host physiology, genetic backgrounds and life history. However, these communities also show different degrees of sensitivity to environment-dependant factors such as abiotic physico-chemical parameters and ecological interactions. The relative importance of host-versus environment-associated factors in shaping teleost microbiomes is still understudied and is paramount for their conservation and aquaculture. Here, we studied the relative importance of host- and environment-associated factors structuring teleost microbiomes using gill samples from a wild Amazonian teleost model (Mesonauta festivus) sampled in contrasting habitats along a 1500 km section of the Amazonian basin, thus ensuring high genetic diversity. Results showed that the contribution of the hosts genetic background was weak compared to environment-related bacterioplanktonic communities in shaping gill microbiomes, thereby suggesting that our understanding of teleost microbiome assembly could benefit from further studies focused on the ecological interplay between host-associated and free-living communities.

ecology↗

Leveraging CD39 To Identify Tumor-Reactive CD8 T cells In Human Lung Cancer

The repertoire of tumor-infiltrating lymphocytes (TILs) can be vast, and many of these TILs are not endowed with tumor reactivity. While a number of reports have shown that tumor-reactive TILs express CD39, few reports have demonstrated that conversely, CD39 can be leveraged to serve as a proxy of tumor-reactive CD8 T cells. Using single-cell CITE/RNA/TCRseq, we show that CD39+ CD8 T cells in human lung cancers demonstrate transcriptional and proteomic features of exhaustion, tumor reactivity, and clonal expansion. Moreover, TCR cloning revealed that CD39 enriched for tumor-reactive CD8 T cell clones. Flow cytometry of 440 lung cancer specimens revealed that CD39 level on CD8 T cells is only weakly correlated with tumoral features that currently guide lung cancer therapy, such as histology, driver mutation, PD-L1 and tumor mutation burden. PD-1 axis blockade, but not cytotoxic chemotherapy, increased intratumoral CD39+ CD8 T cells. CD39 correlated with PD-1 expression on CD8 T cells and high pre-treatment/early-on-treatment levels were associated with improved clinical outcomes, but not immune-related adverse events, from immune checkpoint blockade therapy. This comprehensive profiling of the clinical, pathological and molecular features highlights the utility of CD39 as a proxy for tumor-reactive CD8 T cells in human lung cancer.

immunology↗

The Amazon River microbiome, a story of humic carbon.

The Amazon River basin sustains dramatic hydrochemical gradients defined by three water types: white, clear and black waters. Black waters contain important loads of allochthonous humic dissolved organic carbon (DOC), mostly coming from bacteria-mediated lignin degradation, a process that remains understudied. Here, we identified the main bacterial taxa and functions associated with contrasting Amazonian water types, and shed light on their potential implication in the lignin degradation process. We performed an extensive field bacterioplankton sampling campaign from the three Amazonian water types, and combined our observations to a meta-analysis of 90 Amazonian basin shotgun metagenomes used to build a tailored functional inference database. We showed that the overall quality of DOC is a major driver of bacterioplankton structure, transcriptional activity and functional repertory. We also showed that among the taxa mostly associated to differences between water types, Polynucleobacter sinensis particularly stood out, as its abundance and transcriptional activity was strongly correlated to black water environments, and specially to humic DOC concentration. Screening the reference genome of this bacteria, we found genes coding for enzymes implicated in all the main lignin degradation steps, suggesting that this bacteria may play key roles in the carbon cycle processes within the Amazon basin.

ecology↗

Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation

SUMMARYCells have evolved a metabolic control of DNA replication to respond to a wide range of nutritional conditions. Accumulating data suggest that this poorly understood control depends, at least in part, on Central Carbon Metabolism (CCM). In Bacillus subtilis, the glycolytic pyruvate kinase (PykA) is intricately linked to replication. This 585 amino-acid-long enzyme comprises a catalytic (Cat) domain that binds to phosphoenolpyruvate (PEP) and ADP to produce pyruvate and ATP, and a C-terminal domain of unknown function. Interestingly, the C-terminal domain termed PEPut interacts with Cat and is homologous a domain that, in other metabolic enzymes, are phosphorylated at a conserved TSH motif at the expense of PEP and ATP to drive sugar import and catalytic or regulatory activities. To gain insights into the role of PykA in replication, DNA synthesis was analyzed in various Cat and PEPut mutants grown in a medium where the metabolic activity of PykA is dispensable for growth. Measurements of replication parameters (ori/ter ratio, C period and fork speed) and of the pyruvate kinase activity showed that PykA mutants exhibit replication defects resulting from side chain modifications in the PykA protein rather than from a reduction of its metabolic activity. Interestingly, Cat and PEPut have distinct commitments in replication: while Cat impacts positively and negatively replication fork speed, PEPut stimulates initiation through a process depending on Cat-PEPut interaction and growth conditions. Residues binding to PEP and ADP in Cat, stabilizing the Cat-PEPut interaction and belonging to the TSH motif of PEPut were found important for the commitment of PykA in replication. In vitro, PykA affects the activities of replication enzymes (the polymerase DnaE, helicase DnaC and primase DnaG) essential for initiation and elongation and genetically linked to pykA. Our results thus connect replication initiation and elongation to CCM metabolites (PEP, ATP and ADP), critical Cat and PEPut residues and to multiple links between PykA and the replication enzymes DnaE, DnaC and DnaG. We propose that PykA is endowed with a moonlighting activity that senses the concentration of signaling metabolites and interacts with replication enzymes to convey information on the cellular metabolic state to the replication machinery and adjust replication initiation and elongation to metabolism. This defines a new type of replication regulator proposed to be part of the metabolic control that gates replication in the cell cycle.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

YAP/TAZ as a Novel Regulator of cell volume

How mammalian cells regulate their physical size is currently poorly understood, in part due to the difficulty of accurately quantifying cell volume in a high throughput manner. Here, using the fluorescence exclusion method, we demonstrate that the mechanosensitive transcriptional regulators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are novel regulators of single cell volume. We report that the role of YAP/TAZ in cell volume regulation must go beyond its influence on total cell cycle duration or the cell shape to explain the observed changes in volume. Moreover, for our experimental conditions, volume regulation by YAP/TAZ is independent of mTOR. Instead, we find YAP/TAZ directly impacts the cell division volume. Based on the idea that YAP/TAZ is a mechanosensor, we find that inhibiting the assembly of myosin and cell tension slows cell cycle progression from G1 to S. These results suggest that YAP/TAZ and the Hippo pathway may be modulating cell volume in combination with cytoskeletal tension during cell cycle progression.

bioengineering↗

Applying the auxin-inducible degradation (AID) system for rapid protein depletion in mammalian cells

The ability to deplete a protein of interest is critical for dissecting cellular processes. Traditional methods of protein depletion are often slow-acting, which can be problematic when characterizing a cellular process that occurs within a short period of time. Furthermore, these methods are usually not reversible. Recent advances to achieve protein depletion function by inducibly trafficking proteins of interest to an endogenous E3 ubiquitin ligase complex to promote ubiquitination and subsequent degradation by the proteasome. One of these systems, the auxin-inducible degron (AID) system, has been shown to permit rapid and inducible degradation of AID-tagged target proteins in mammalian cells. The AID system can control the abundance of a diverse set of cellular proteins, including those contained within protein complexes, and is active in all phases of the cell cycle. Here we discuss considerations for the successful implementation of the AID system and describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of an AID degron in human cells. This method can also be adapted to insert other tags, such as fluorescent proteins, at defined genomic locations.

cell biology↗