Biology subjects

Kremer, A.

Publications and source records attributed to Kremer, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Perturbation of the replication stress-ATR axis accelerates human erythroid differentiation but blocks terminal maturation

Replication stress (RS) can act as an input to differentiation: in erythroid and myeloid cells it is sufficient to accelerate commitment. Yet genotoxic chemotherapy, which imposes replication stress on the bone marrow can cause anemia; a frequent dose-limiting toxicity whose deficit of mature red cells is often attributed to progenitor depletion rather than to any effect on differentiation itself. Here, we ask how RS-induced accelerated commitment gives rise to impaired red cell output, using in vitro erythroid differentiation of cord-blood CD34+ progenitors perturbed with clinically relevant chemotherapy agents. We find that treated erythroblasts differentiate faster than controls but fail to mature, measured by lack of enucleated cells. Inhibition of ATR, but not ATM or DNA-PK, reproduces this phenotype. Mechanistically, single-cell profiling of drug-treated cells shows convergence on a transcriptional program, marked by loss of MYB and MYC activity. Treated cultures also accumulate aberrant late erythroblasts depleted for globin and heme programs and enriched for p53 and proteotoxic stress signatures. Together, these results propose that defective maturation is an alternative mechanism for chemotherapy-induced anemia, in addition to depletion of progenitors.

cell biology↗

bioRxiv2026

Climate adaptation across space and time: lessons from oak populations

Tree populations can respond to climate change through migration, phenotypic plasticity, or genetic evolution. Despite long generation times of forest tree species, recent studies suggest that their evolutionary responses may occur rapidly. Using oaks as a model system, we synthesize evidence from 88 common garden studies and from historical, retrospective and longitudinal approaches to explore how populations have adapted to climatic variability across different biomes, and assess the consistency and pace of evolutionary responses across spatial and temporal climatic gradients. We found that approximately 61% of the studies exhibited significant differences among populations but climatic drivers and adaptive strategies differed among biomes. Temperature-related clines predominated in temperate regions, with populations from warmer origins showing longer growing seasons and higher growth potential. In seasonally dry biomes, aridity favored increased drought tolerance in Mediterranean populations but drought avoidance in tropical populations. Allochronic studies revealed genetic changes over decades to millenia in response to climate changes, with warming associated with increased growth and reduced specific leaf area in temperate oaks. Thus, spatial differentiation and temporal evolution were generally congruent in direction for most traits except for leaf unfolding, while short-term evolutionary rates exceeded long-term estimates by two to three orders of magnitude. In summary, provenance trials can provide useful information on the direction of climate-driven evolution for some traits, but may underestimate its contemporary pace. More studies are needed to evaluate whether standing genetic variation of forest tree species is sufficient to track current climate change.

evolutionary biology↗

bioRxiv2025

Cryopreservation of siRNA-treated cells is feasible

Cryopreservation is a routine step in the manufacturing process of adoptive cell therapies, providing critical logistic flexibility. RNAi-based therapies are increasingly being explored as enhancers or modulators of adoptive cell therapies. However, the impact of cryopreservation on cells treated with RNAi-based therapies has not been investigated before. In this study, we addressed this knowledge gap by examining silencing efficacy in siRNA-treated cells that undergo cryopreservation. Our findings demonstrate that silencing in cryopreserved cells is comparable to that in cells maintained continuously in culture. Moreover, we found that the duration of siRNA exposure plays a significant role in cells that later undergo cryopreservation, with extended exposure improving silencing efficiency. However, this effect diminishes at higher siRNA concentrations. Additionally, we showed that siRNA treatment is feasible at low temperatures (2-8{degrees}C), and siRNA-treated cells can be cryopreserved for extended periods (at least one month) without loss of efficacy. Furthermore, we demonstrated the feasibility of cryopreserving siRNA-treated primary cells, including those resembling leukapheresis material. Our work establishes the feasibility of integrating siRNA treatments into current manufacturing processes for adoptive cell therapies.

bioengineering↗

bioRxiv2025

Corelative light and electron microscopy (CLEM) image registration by blood vessel axes alignment

Correlative light and electron microscopy (CLEM) is a multi-modal microscopy approach that contains complementary information from light (LM) and electron microscopy (EM). CLEM image alignment can be a challenging task for both the biological expert as well as for existing automated methods. This work introduces a novel method that focuses on aligning the medial axes of blood vessels in both the LM and EM modalities. Evaluation of 3D CLEM image registration problems shows that this approach obtains an accurate alignment in an intuitive and robust way. This method holds potential as a valuable tool for global and local alignment of a subset of challenging CLEM image alignment problems, both as guidance in a manual setting or in a more automated workflow.

bioinformatics↗

bioRxiv2025

miRNA Mimic Optimization: Chemical Structure - Activity - Targetome Relationship to Engineer Selective Anti-Tumor Immunity in T cells

MicroRNA (miRNA) mimic therapies act through a broad and complex targetome in disease contexts. However, both the composition of these targetomes and the impact of chemical modifications on them remain poorly understood. In this study, we investigate eight fully chemically modified miRNA scaffolds across three miRNA sequences in a model immune disorder, graft-versus-host-disease (GvHD), which is an off-tumor effect of allogeneic T cell therapy. We demonstrate that conventional silencing assays fail to predict the functional performance of miRNA mimics in GVHD and graft-versus-leukemia (GvL), the on-tumor effect of allogeneic T cells. Moreover, we find that chemical scaffolds influence the duration of silencing mediated by miRNA mimics. We identify a miR-374b version as a lead miRNA candidate that not only inhibits GVHD but also enhances GVL--an unprecedented and desired improvement in on-versus-off-tumor selectivity of clinical relevance. Further analysis reveals distinct responder and non-responder groups to miR-374b therapy. While responders exhibit significant transcriptomic shifts, non-responders show virtually no changes, despite intact miRNA pathway function, underscoring a strong correlation between transcriptome reprogramming and therapeutic efficacy. RNA sequencing reveals that miR-374b acts via metabolic reprogramming of T cells and induces partially distinct transcriptional programs in GVHD and GVL. Collectively, our findings demonstrate that fully chemically modified miRNA mimic therapies are feasible to rewrite the T-cell transcriptome, profoundly improving on-versus-off-tumor specificity in T-cell-mediated cancer therapies.

bioengineering↗

bioRxiv2024

Structure - silencing duration relationships in RNAi medicines in rapidly dividing cells

RNA interference (RNAi)-based medicines offer precise targeting of virtually any transcript, making them an appealing new drug class for addressing unmet needs in immune-oncological applications. While RNAi therapies show exceptional duration of effect in non-dividing cells, their efficacy in rapidly dividing cells, crucial for immune-oncology, remains largely unexplored. Unlike in non-dividing cells, full chemical modification in rapidly dividing cells has not consistently extended silencing duration, according to limited data available. In this study, we investigated key factors affecting the duration of effect for three main types of RNAi-based therapeutics (siRNA, miRNA mimics, and miRNA inhibitors) in rapidly dividing cancer and immune cells. Saturation of intracellular depots by multiple loading doses, a common strategy to prolong silencing duration in non-dividing hepatocytes, had minimal impact on siRNA duration of effect in rapidly dividing cells. However, modifying the antisense strand with a 5-(E)-vinylphosphonate (5-VP) to protect siRNAs from exonucleases and enhance AGO2 binding significantly extended siRNA silencing duration to over 30 days both in vitro and in vivo. For miRNA mimics, extensive stabilization of the antisense strand with phosphorothioates was not effective and led to reduced potency and silencing duration. Interestingly, a shorter duplex region commonly seen in therapeutic siRNAs partially rescued duration of silencing in miRNA mimics with extended phosphorothioate modifications. On the other hand, miRNA inhibitors demonstrated robust reversal of miRNA activity for an impressive 25 days in cancer cell lines. Our findings enable the rational design of the chemical architecture and administration regimens of RNAi-based therapies in oncology and immunology.

pharmacology and toxicology↗

bioRxiv2024

Newly discovered base barrier cells provide compartmentalization of choroid plexus, brain and CSF

The choroid plexus (ChP) is a highly understudied structure of the central nervous system (CNS). The structure hangs in the brain ventricles, is composed of an epithelial cell layer, which produces the cerebrospinal fluid (CSF) and forms the blood-CSF barrier. It encapsulates a stromal mix of fenestrated capillaries, fibroblasts and a broad range of immune cells. Here, we report that the ChP base region harbors unique fibroblasts that cluster together, are connected by tight junctions and seal the ChP stroma from brain and CSF, thereby forming ChP base barrier cells (ChP BBCs). ChP BBCs are derived from meningeal mesenchymal precursors, arrive early during embryonic development, are maintained throughout life and are conserved across species. Moreover, we provide transcriptional profiles and key markers to label ChP BBCs and observe a striking transcriptional similarity with meningeal arachnoid barrier cells (ABCs). Finally, we provide evidence that this fibroblast cluster functions as a barrier to control communication between CSF and the ChP stroma and between the latter and the brain parenchyma. Moreover, loss of barrier function was observed during an inflammatory insult. Altogether, we have identified a novel barrier that provides functional compartmentalization of ChP, brain and CSF. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/601696v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@181e787org.highwire.dtl.DTLVardef@1875f33org.highwire.dtl.DTLVardef@7b2bcdorg.highwire.dtl.DTLVardef@78baa6_HPS_FORMAT_FIGEXP M_FIG Newly discovered base barrier cells provide compartmentalization of choroid plexus, brain and CSF The choroid plexus (ChP) hangs in the brain ventricles and is composed of an epithelial cell layer which produces the cerebrospinal fluid (CSF) and forms the blood-CSF barrier. The ChP epithelial cells are continuous with the ependymal cells lining the ventricle wall. At this base region, we identified and characterized a novel subtype of fibroblasts coined the ChP base barrier cells (BBCs). ChP BBCs express tight junctions (TJs), cluster together and seal the ChP stroma from CSF and brain parenchyma. The subarachnoid space (SAS) CSF penetrates deep into choroid plexus invaginations where it is halted by ChP BBCs. Abbreviations: E9-16.5 (embryonic day 9-16.5); P1-4 (postnatal day 1-4). C_FIG

neuroscience↗

bioRxiv2023

Systematic optimization of siRNA productive uptake into resting and activated T cells ex vivo

RNA-based medicines are ideally suited for precise modulation of T cell phenotypes in anti-cancer immunity, in autoimmune diseases and for ex vivo modulation of T-cell-based therapies. Therefore, understanding productive siRNA uptake to T cells is of particular importance. Most studies used unmodified siRNAs or commercially available siRNA with undisclosed chemical modifications patterns to show functionality in T cells. Despite being an active field of research, robust siRNA delivery to T cells still represents a formidable challenge. Therefore, a systematic approach is needed to further optimize and understand productive siRNA uptake pathways to T cells. Here we compared conjugate-mediated and nanoparticle-mediated delivery of siRNAs to T cells in the context of fully chemically modified RNA constructs. We showed that lipid-conjugate-mediated delivery outperforms lipid-nanoparticle-mediated and extracellular-vesicle-mediated delivery in activated T cells ex vivo. Yet, ex vivo manipulation of T cells without the need of activation is of great therapeutic interest for CAR-T, engineered TCR-T and allogeneic donor lymphocyte applications. We are first to report productive siRNA uptake into resting T cells using lipid-conjugate mediated delivery. Interestingly, we observed strong dependence of silencing activity on lipid-conjugate-identity in resting T cells but not in activated T cells. This phenomenon is consistent with our early uptake kinetics data. Lipid-conjugates also enabled delivery of siRNA to all mononuclear immune cell types, including both lymphoid and myeloid lineages. These findings are expected to be broadly applicable for ex vivo modulation of immune cell therapies.

molecular biology↗

bioRxiv2023

Identification of inter- and intra-tumoral molecular phenotypes steering temozolomide resistance in patient-derived glioblastoma cells.

BackgroundRadiation therapy and chemotherapy using Temozolomide are the standard adjuvant treatments for patients with glioblastoma. Despite maximal treatment prognosis is still poor largely due to the emergence of Temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. This study aims to investigate the diverse molecular mechanisms involved in temozolomide resistance. MethodsTo induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell sequencing on four of the cell cultures to track the evolution of temozolomide resistance. ResultsThe induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. ConclusionsThe adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumors ability to survive. Depending on the tumors adaptability potential, subpopulations with acquired resistance mechanisms may arise. Further research is necessary to deepen our understanding of these mechanisms and develop strategies to overcome them.

cancer biology↗

bioRxiv2023

Extensive variation of leaf specialized metabolite production in sessile oak (Quercus petraea) populations is to a large extent genetically determined but not locally adaptive.

Specialized or secondary metabolites play a key role in plant resistance against abiotic stresses and defences against bioaggressors. For example, in sessile oaks Quercus petraea, phenolics contribute to reduce herbivore damage and improve drought resistance. Here, we explored the natural variation of specialized metabolites in nine European provenances of sessile oaks and aimed to detect its underlying genetic bases. We sampled mature leaves from high and low branches on 225 sessile oak trees located in a common garden and used untargeted metabolomics to characterise the variation of 217 specialized metabolites. In addition, we used whole genome low-depth sequencing to genotype individuals for 1.4M genetic markers. We found that leaf specialized metabolites displayed extensive within-provenance variation, but very little differentiation between provenances. In addition, a genome-wide association study allowed detecting significant associations for 42% of these metabolites. Hence, our results suggest that genetic variation for most leaf specialized metabolites is unlikely to be locally adaptive, however lack of differentiation among populations suggests selection acts locally to maintain diversity at loci associated with leaf specialized metabolites variation.

evolutionary biology↗

bioRxiv2021

Locally adapted oak populations along an elevation gradient display different molecular strategies to regulate bud phenology.

Research conductedWith the ongoing global warming, there are serious concerns about the persistence of locally adapted populations. Indeed, with the raising of temperature, the phenological cycle of tree species may be strongly affected since higher winter temperatures may have a negative impact on endodormancy release if chilling requirements are not fulfilled during winter and late frost in spring may expose trees if buds flush too early. Thus, Environmental gradients (showing continuous variations of environmental conditions) constitute a design of choice to analyze the effect of winter dormancy in locally adapted population. MethodsIn the present study, we used an elevation gradient in the Pyrenees to explore the gene expression network involved in dormancy regulation in natural populations of sessile oak locally adapted to temperature. Terminal buds were harvested during dormancy induction and release at different elevations. Then, gene expression was quantified using RNAseq and we used a likelihood ratio test to identify genes displaying significant dormancy, elevation or dormancy-by-elevation interaction effects. Key resultsOur results highlight molecular processes in locally adapted populations along this elevation cline, and made it possible to identify key dormancy-by-elevation responsive genes revealing that locally adapted populations have evolved distinct molecular strategies to adapt their bud phenology in response to environmental variation (i.e. temperature).

plant biology↗

bioRxiv2019

Adaptive Introgression Promotes Fast Adaptation In Oaks Marginal Populations

Range shifts and species range limits are two fundamental, related processes in population and evolutionary genetics that have received much attention since a large impact of climate change in species distributions was predicted. In general, there is a broad consensus on the effects of abiotic interactions on range limits, but comprehensive evidence supporting/rejecting the impact of biotic interactions is lacking. Hybridization has long been recognized as a biotic interaction favoring marginal populations establishment and range expansion through transgressive segregation or adaptive introgression, but recently new roles have been claimed for hybridization, such as the trigger of adaptive radiations, or indirect effects on population sizes that would allow persistence until new mutations arises or the environment changes. In this work, we selected two Mediterranean oak species with ecological discrimination based on soil pH, and intensively sampled three interspecific pairs of marginal populations from taxon-extreme environments under heterogeneous climate conditions. We genotyped 110 EST-SSR markers evenly distributed across their genomes and applied a variety of population and landscape genetics models to validate candidate genes for local adaptation. Then, several introgression screens on shared candidates showed that the three inter-specific population pairs contain evidences of adaptive introgression and that events occur in both directions. Other significant findings from our work are: (i) Aproximate Bayesian Computation coupled to coalescent simulations supports small hybridization rates since recent secondary contact in two population pairs affected by Quaternary climatic oscillations but continuous old interspecific gene flow in the pair less affected by climate, (ii) introgression at loci involved in local adaptations leads to strong geographic structure of marginal oak populations when sampling is large enough, and (iii) sampling efforts can be targeted to reveal different components of populations structure. Finally, we review evidences that support our conclusions and discuss some evolutionary implications of adaptive introgression on range expansion.

evolutionary biology↗

bioRxiv2019

A ‘Human-in-the-Loop’ Approach for Semi-automated Image Restoration in Electron Microscopy

The recent advent of 3D in Electron Microscopy (EM) has allowed for detection of detailed sub-cellular nanometer resolution structures. While being a scientific breakthrough, this has also caused an explosion in dataset size, necessitating the development of automated workflows. Automated workflows typically benefit reproducibility and throughput compared to manual analysis. The risk of automation is that it ignores the expertise of the microscopy user that comes with manual analysis. To mitigate this risk, this paper presents a hybrid paradigm. We propose a human-in-the-loop (HITL) approach that combines expert microscopy knowledge with the power of large-scale parallel computing to improve EM image quality through advanced image restoration algorithms. An interactive graphical user interface, publicly available as an ImageJ plugin, was developed to allow biologists to use our framework in an intuitive and user-friendly fashion. We show that this plugin improves visualization of EM ultrastructure and subsequent (semi-)automated segmentation and image analysis.

bioinformatics↗

bioRxiv2019

Genomic landscape of the global oak phylogeny

O_LIThe tree of life is highly reticulate, with the history of population divergence buried amongst phylogenies deriving from introgression and lineage sorting. In this study, we test the hypothesis that there are regions of the oak (Quercus, Fagaceae) genome that are broadly informative about phylogeny and investigate global patterns of oak diversity.\nC_LIO_LIWe utilize fossil data and restriction-site associated DNA sequencing (RAD-seq) for 632 individuals representing ca. 250 oak species to infer a time-calibrated phylogeny of the worlds oaks. We use reversible-jump MCMC to reconstruct shifts in lineage diversification rates, accounting for among-clade sampling biases. We then map the > 20,000 RAD-seq loci back to a recently published oak genome and investigate genomic distribution of introgression and phylogenetic support across the phylogeny.\nC_LIO_LIOak lineages have diversified among geographic regions, followed by ecological divergence within regions, in the Americas and Eurasia. Roughly 60% of oak diversity traces back to four clades that experienced increases in net diversification due to climatic transitions or ecological opportunity.\nC_LIO_LIThe support we find for the phylogeny contrasts with high genomic heterogeneity in phylogenetic signal and introgression. Oaks are phylogenomic mosaics, and their diversity may in fact depend on the gene flow that shapes the oak genome.\nC_LI

evolutionary biology↗

bioRxiv2019

Adaptive introgression as a driver of local adaptation to climate in European white oaks

Latitudinal and elevational gradients provide valuable experimental settings for studies of the potential impact of global warming on forest tree species. The availability of long-term phenological surveys in common garden experiments for traits associated with climate, such as bud flushing, for sessile oaks (Quercus petraea), provides an ideal opportunity to investigate this impact.\n\nWe sequenced 18 sessile oak populations, and used available sequencing data for three other closely related European white oak species (Q. pyrenaica, Q. pubescens, Q. robur), to explore the evolutionary processes responsible for shaping the genetic variation across latitudinal and elevational gradients in extant sessile oaks. We used phenotypic surveys in common garden experiments and climatic data for the population of origin, to perform genome-wide scans for population differentiation, genotype-environment (GEA) and genotype-phenotype associations (GPA).\n\nThe inferred historical relationships between Q. petraea populations suggest that interspecific gene flow occurred between Q. robur and Q. petraea populations from cooler or wetter areas. A genome-wide scan of differentiation between Q. petraea populations identified SNPs displaying strong interspecific relative divergence between these two species. These SNPs followed genetic clines along climatic or phenotypic gradients, providing further support for the likely contribution of introgression to the adaptive divergence of Q. petraea populations.\n\nOverall, the results of this study indicate that adaptive introgression of Q. robur alleles has occurred. We discuss the results of this study in the framework of the post-glacial colonization scenario, in which introgression and diversifying selection have been proposed as essential drivers of Q. petraea microevolution.

evolutionary biology↗

bioRxiv2019

Inferring phenotypic plasticity and local adaptation to climate across tree species ranges using forest inventory data

AimTo test whether adaptive and plastic trait responses to climate across species distribution ranges can be untangled using field observations, under the rationale that, in natural forest tree populations, long-term climate shapes local adaptation while recent climate change drives phenotypic plasticity. LocationEurope. Time period1901-2014. TaxaSilver fir (Abies alba Mill.) and sessile oak (Quercus petraea (Matt.) Liebl.). MethodsWe estimated the variation of individual tree height as a function of long-term and short-term climates to tease apart local adaptation, plasticity and their interaction, using mixed-effect models calibrated with National Forest Inventory data (in-situ models). To validate our approach, we tested the ability of in-situ models to predict independently tree height observations in common gardens where local adaptation to climate of populations and their plasticity can be measured and separated. In-situ model predictions of tree height variation among provenances (populations of different geographical origin) and among planting sites were compared to observations in common gardens and to predictions from a similar model calibrated using common garden data (ex-situ model). ResultsIn Q. petraea, we found high correlations between in-situ and ex-situ model predictions of provenance and plasticity effects and their interaction on tree height (r > 0.80). We showed that the in-situ models significantly predicted tree height variation among provenances and sites for Abies alba and Quercus petraea. Spatial predictions of phenotypic plasticity across species distribution ranges indicate decreasing tree height in populations of warmer climates in response to recent anthropogenic climate warming. Main conclusionsOur modelling approach using National Forest Inventory observations provides a new perspective for understanding patterns of intraspecific trait variation across species ranges. Its application is particularly interesting for species for which common garden experiments do not exist or do not cover the entire climatic range of the species.

ecology↗

bioRxiv2018

In situ estimation of genetic variation of functional and ecological traits in Quercus petraea and Q. robur

BackgroundPredicting the evolutionary potential of natural tree populations requires the estimation of heritability and genetic correlations among traits on which selection acts, as differences in evolutionary success between species may rely on differences for these genetic parameters. In situ estimates are expected to be more accurate than measures done under controlled conditions which do not reflect the natural environmental variance.\n\nAimsThe aim of the current study was to estimate three genetic parameters (i.e. heritability, evolvability and genetic correlations) in a natural mixed oak stand composed of Quercus petraea and Quercus robur about 100 years old, for 58 traits of ecological and functional relevance (growth, reproduction, phenology, physiology, resilience, structure, morphology and defence).\n\nMethodsFirst we estimated genetic parameters directly in situ using realized genomic relatedness of adult trees and parentage relationships over two generations to estimate the traits additive variance. Secondly, we benefited from existing ex situ experiments (progeny tests and conservation collection) installed with the same populations, thus allowing comparisons of in situ heritability estimates with more traditional methods.\n\nResultsHeritability and evolvability estimates obtained with different methods varied substantially and showed large confidence intervals, however we found that in situ were less precise than ex situ esti-mates, and assessments over two generations (with deeper relatedness) improved estimates of heritability while large sampling sizes are needed for accurate estimations. At the biological level, heritability values varied moderately across different ecological and functional categories of traits, and genetic correlations among traits were conserved over the two species.\n\nConclusionWe identified limits for using realized genomic relatedness in natural stands to estimate the genetic variance, given the overall low variance of genetic relatedness and the rather low sampling sizes of currently used long term genetic plots in forestry. These limits can be overcome if larger sample sizes are considered, or if the approach is extended over the next generation.

evolutionary biology↗

bioRxiv2018

High-quality SNPs from genic regions highlight introgression patterns among European white oaks (Quercus petraea and Q. robur).

The Src homology-2 domain containing phosphatase SHP2 is a critical regulator of signal transduction, being implicated in cell growth and differentiation. Activating mutations cause developmental disorders and act as oncogenic drivers in hematologic cancers. SHP2 is activated by phosphopeptide binding to the N-SH2 domain, triggering the release of N-SH2 from the catalytic PTP domain. Based on early crystallographic data, it has been widely accepted that opening of the binding cleft of N-SH2 serves as the key "allosteric switch" driving SHP2 activation. To test the putative coupling between binding cleft opening and SHP2 activation as assumed by the "allosteric switch" model, we critically reviewed structural data of SHP2 and we used extensive molecular dynamics (MD) simulation and free energy calculations of isolated N-SH2 in solution, SHP2 in solution, and SHP2 in a crystal environment. Our results demonstrate that the binding cleft in N-SH2 is constitutively flexible and open in solution, and that a closed cleft found in certain structures is a consequence of crystal contacts. The degree of opening of the binding cleft has only a negligible effect on the free energy of SHP2 activation. Instead, SHP2 activation is greatly favored by the opening of the central {beta}- sheet of N-SH2. We conclude that opening of the N-SH2 binding cleft is not the key allosteric switch triggering SHP2 activation. Significance StatementSHP2 is a multi-domain protein, playing an important role in up-regulating cellular processes such as cell survival, proliferation, and programmed cell death. SHP2 mutations cause developmental disorders and were found in many cancer types, including neuroblastoma, breast cancer, and leukemia. In healthy cells, SHP2 mainly takes an autoinhibited, inactive form, and SHP2 is activated upon binding of phosphopeptides to the N-SH2 domain. For the past two decades, the widening of the binding cleft upon peptide binding has been considered as the key event driving SHP2 activation. Here, by analyzing crystallographic data and molecular simulations, we demonstrate that the binding cleft in N-SH2 is, instead, already open and accessible in solution, and its degree of opening does not influence SHP2 activation.

evolutionary biology↗