Biology subjects

Schmitz, M.

Publications and source records attributed to Schmitz, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Stemness overcomes IFNγ-induced antitumour exhaustion in human macrophages

Macrophages hold great promise for antitumour therapies (1-4), particularly owing to their ability to infiltrate solid tumours (5). Yet therapeutic success has been limited (6,7), because macrophages exhibit rapid exhaustion of antitumorigenic activity in the tumour (5,8,9). Here, we induced a stem-like self-renewal programme in human induced pluripotent stem cell (iPSC)-derived macrophages by deleting the self-renewal restricting transcription factors MAFB and MAF (MAF-DKO). Human MAF-DKO macrophages showed activated stem cell signatures, increased clonogenicity and proliferative potential. Increased self-renewal capacity was accompanied by enhanced mitochondrial fitness and invigorated energy metabolism. Importantly, these stemness features prevented exhaustion, cellular aging-associated parameters and loss of energy efficiency that are typically associated with proinflammatory macrophage activation. In addition, activated MAF-DKO macrophages showed a broad loss of IFN{gamma}-triggered immunosuppressive pathways (10,11), induced a super-activated state with robust cytotoxic activity and resisted exhaustion upon tumour cell exposure. Consequently, activated MAF-DKO macrophages showed intratumoral persistence with a stable antitumoral phenotype in vivo, conferred robust reduction of tumour burden and extended survival of tumour-bearing mice. Together, this demonstrates that the induction of stemness in human macrophages can overcome critical roadblocks of antitumour therapy by conferring resistance to activation-triggered exhaustion of antitumour activity in the TME.

immunology↗

bioRxiv2026

Germline removal reprograms somatic genome maintenance towards faster, energy-efficient, high-fidelity DNA defence and repair

Germ cells maintain their genomes with greater fidelity than somatic cells, yet how germline status systemically shapes somatic genome protection in vertebrates remains poorly understood. Combining germline ablation with multi-organ transcriptomics in zebrafish (Danio rerio), we dissected germline control of somatic DNA repair and genotoxic stress responses. Using CRISPR/Cas9 mediated knockout of the germline determinant gene dnd-1, we compared germline free (GLF) males and germline carrying (GLC) siblings across five somatic organs at baseline and following sub-lethal {gamma}-irradiation, with sampling at 3 h and 24 h post-irradiation. Germline ablation profoundly reorganised baseline somatic transcription, favouring shorter, exon rich genes with functions in chromatin integrity, cell cycle control, and high-fidelity DNA repair, and attenuating DREAM complex repression of canonical repair targets. After irradiation, GLF fish mounted a faster response that rapidly engaged homologous recombination, checkpoint control, and proteostasis, whereas GLC fish remained biased toward biosynthetic and translational programmes. Hedgehog signalling acted as a transient regulatory node coordinating the strain-specific DNA damage response, and a repeat-rich region of chromosome 4 emerged as a hotspot of coordinated gene transposable element (TE) activity. GLF soma showed reduced early TE expression after irradiation, with LTR retrotransposons showing the strongest concordance with nearby differentially expressed genes. Our findings identify germline status as a systems level switch that tunes somatic genome maintenance and TE dynamics across multiple organs and provide a molecular framework for understanding how the germline regulates somatic ageing in vertebrates.

genetics↗

bioRxiv2026

Binary node clustering via contrastive learning for haplotype phasing in de novo genome assembly

Accurate haplotype phasing is essential for high-quality genome assembly, yet de novo phasing of complex genomes without parental data remains challenging. We formulate haplotype phasing as a node clustering problem with overlapping clusters on augmented unitig graphs, where nodes represent contiguous, non-branching DNA sequence fragments and two edge types can encode sequence overlap or Hi-C proximity information. We introduce a contrastive learning framework with a custom objective function and train a graph-transformer-based model, termed grapHiC, to phase paternal, maternal, and homozygous unitig nodes. grapHiC; is the first machine-learning-based method to perform reference-free haplotype phasing and the first approach to directly phase raw unitig graphs without prior simplification. We show that grapHiCaccurately clusters nodes on human-genome-scale graphs and that its predictions can effectively guide phased de novo genome assembly, producing human assemblies with contiguity and phasing quality comparable to the state of the art when integrated with the DipGNNome assembler.

bioinformatics↗

bioRxiv2026

Structural basis of RNA-guided DNA integration by type I CRISPR-associated transposases

CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex1,2. Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools3-6. The type I-F Pseudoalteromonas CAST (PseCAST) displays the highest activity in mammalian cells to date7 and has been the subject of extensive directed evolution8, but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation9. Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the PseCAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa PseCAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.

molecular biology↗

bioRxiv2025

Cross-species consensus atlas of the primate basal ganglia

The basal ganglia (BG) are conserved brain regions essential for motor control, learning, emotion, and cognition, and are implicated in neurological and psychiatric disease. Yet a unified cross-species taxonomy of BG cell types is lacking, limiting translation of BG circuit mechanisms, interpretation of human genetic risk, and development of cell type-targeted tools. We present a multiomic consensus atlas of 1.8 million nuclei from human, macaque, and marmoset spanning eight BG structures. Integrating cross-species gene expression, open chromatin, and spatial profiling enables definition of conserved and divergent cell types. Alignment to existing mouse and human atlases identifies 61 homologous cell types conserved over 80 million years. We identify a STRd D2 StrioMat Hybrid medium spiny neuron (MSN) type with molecular, electrophysiological, and morphological features that clarify hybrid MSN identities. Comparative cis-regulatory analysis reveals conserved sequence grammars that encode cell identity and inform viral targeting strategies, providing a foundational resource for BG evolution, function, and disease.

neuroscience↗

bioRxiv2025

DipGNNome: Diploid de novo genome assembly with geometric deep learning and beam-search

De novo genome assembly remains a central challenge in computational biology, particularly for diploid genomes where maternal and paternal haplotypes must be accurately resolved. Existing assemblers achieve impressive results through carefully designed heuristics, yet modern deep learning methods remain largely unexplored in the diploid setting. We present DipGNNome, the first deep learning-based framework for diploid de novo genome assembly. Our approach formulates assembly as an edge classification and traversal problem on haplotype-aware assembly graphs, training graph neural networks (GNNs) to guide contig construction. To enable this, we establish a novel pipeline for generating diploid graphs with ground-truth edge labels, providing the first systematic way to produce training data for machine learning models in this domain. This framework creates a foundation for applying and extending graph-based deep learning to diploid assembly. DipGNNome creates assemblies comparable to state-of-the-art and demonstrates the feasibility of deep learning for diploid assembly and introduces a paradigm that bridges algorithmic genomics with graph representation learning. Our code, dataset and trained model is openly available at https://github.com/lbcb-sci/DipGNNome.

genomics↗

bioRxiv2025

Proteomic and Kinetic Characterization of Prion Seeding in Distinct Human CJD Strains Unveils Early Diagnostic Biomarkers

To enhance understanding of early diagnosis, differential disease progression rates, and the molecular profiles of human prion strains, we analyzed prion seeding activity over time in Creutzfeldt-Jakob disease (CJD)-infected mice using the real-time quaking-induced conversion (RT-QuIC) assay. Our previous work highlighted pre- clinical alterations in endocytic machinery and cytoskeleton-associated responses in CJD-affected brain regions. In this study, infectious prion strains derived from human CJD-MM1 and VV2 brain tissues were inoculated into tg340, tg361 (expressing approximately four times the human PrP-M129 and PrP-V129, respectively), and tg650 (expressing approximately six times the human PrP-M129) mice. A total of 188 brain samples (cortex) were analyzed from confirmed CJD-infected mice and control mice at both pre-clinical and clinical stages of the disease. Notably, we observed region- specific and PrP strain-specific differences in seeding activity at the pre-clinical stage of disease in CJD-MM1 and VV2 infected mice. The lag phase between the positive response ranged from 7.5 to 24.5 hours across all regions and disease stages. In the cortex, CJD-MM1-infected tg340 mice exhibited a prolonged lag phase ([~]24.5 hours), while CJD-VV2-infected tg341 mice showed minimal seeding response and relative fluorescence unit (RFU) signal rates. In contrast, the cerebellum of VV2 clinical stage mice exhibited a shorter lag phase, and VV2 preclinical stage mice showed significantly lower RFU signal rates. Proteomic profiling via SWATH-MS identified 500 and 682 differentially expressed proteins in the MM1 and VV2 models, respectively. Key proteins such as Gnl1, Stxbp1, Pllp, Gps1, Nefh, Ahsa1, Rala, Cacybp, Pdk1, Unc13a, Rab21, Rraga, Ppp1r9a, Eif4b, Atp2b2, Vps51, H2afx in CJD-MM1 and Gm10358, Tnc, Calb2, Ppm1h, Dnaja1, Gm45808, Hpcal1, Prkca, Dock3, Syn2, Agap2, Tmem126a, Fdps, Ndufa4 in CJD- VV2 showed significant alterations at the early pre-clinical stages, correlating with detectable prion replication. These molecular shifts highlight potential early-stage diagnostic biomarkers. Functional analyses revealed that both MM1 and VV2 subtypes engage early compensatory responses; however, MM1 primarily involves metabolic reprogramming and enhanced vesicle clearance, while VV2 is characterized by pronounced disturbances in calcium signaling and structural integrity at early stage of the disease. These findings emphasize the utility of RT-QuIC and proteomics in characterizing prion seeding and progression, providing valuable insights into the molecular mechanisms underlying prion diseases and potential early diagnostic markers.

neuroscience↗

bioRxiv2025

HCN channels reveal conserved and divergent physiology in supragranular pyramidal neurons in primate species

The physiological properties of human and rodent neurons differ, yet the extent to which these differences reflect human specializations is often unclear. Compared with their rodent counterparts, human supragranular pyramidal neurons possess enriched HCN-channel-dependent intrinsic membrane properties and a related sensitivity to synaptic inputs containing delta/theta band frequencies. We tested whether other primate species possess enriched HCN-channel dependent membrane properties. We found ubiquitous HCN1 subunit gene expression in supragranular glutamatergic neurons across New World Monkeys, Old-World Monkeys, and great apes in single nucleus RNA-sequencing datasets. Using Patch-seq recordings from acute and cultured brain slices, we found robust HCN-dependent physiological properties in supragranular pyramidal neurons in a species of New-World monkey (Saimiri sciureus) and two species of Old-World Monkey (Macaca mulatta, Macaca nemestrina). In both human and macaque neocortex, HCN-related intrinsic properties increased in magnitude with increasing laminar depth, especially in one transcriptomic cell type. Within this type, HCN dependent properties were more pronounced in macaque than human neurons. These findings indicate that HCN-governed membrane properties and sensitivity to delta/theta band frequencies are roughly conserved in supragranular pyramidal neurons across at least 36 million years of primate evolution.

neuroscience↗

bioRxiv2025

EGFL7 promotes immune evasion in glioma through its interaction with integrin β2

Glioblastoma is the most aggressive form of malignant brain cancer, characterized by an immunosuppressive microenvironment and immune evasion. Despite the success of immune checkpoint inhibitors in other cancers, immunotherapies such as anti-PD1 have shown limited efficacy in glioblastoma, underscoring the need to identify tumor-intrinsic mechanisms that sustain this immunosuppressive microenvironment and to develop more effective therapeutic strategies targeting them. Previously, the secreted factor epidermal growth factor-like protein 7 (EGFL7) has been shown to promote brain tumor growth by affecting the glioblastoma microenvironment (GME). However, its impact on the immune system remained enigmatic. Here, we studied the role of EGFL7 in shaping the immune landscape in glioblastoma and identified the underlying molecular mechanisms it engages to drive glioma immune evasion. Single-cell transcriptomic profiling of immune cells derived of glioblastoma revealed that EGFL7 promotes an immunosuppressive GME, characterized by enhanced T cell exhaustion and polarization of macrophages towards a protumorigenic state. Proteomic profiling of EGFL7s interactome in glioma revealed its interaction with integrin {beta}2 (ITGB2), an immune cell surface receptor involved in cell adhesion and migration. Mechanistic studies uncovered the central role of this interaction for immune evasion, which promoted T cell exhaustion and the polarization of macrophages towards a pro-tumorigenic state. Genetic perturbation of the EGFL7-ITGB2 axis attenuated immunosuppression and prolonged the survival of glioblastoma-bearing mice. Remarkably, a combinatorial regimen of anti-EGFL7 and the checkpoint inhibitor anti-PD1 improved the efficacy of this drug, which by itself did not improve glioma patient survival so far. In conclusion, our study provides unequivocal evidence that EGFL7 mediates immune evasion in glioma and has great potential to serve as an add-on drug target to improve immunotherapies not functional in glioblastoma patients so far.

cancer biology↗

bioRxiv2025

Molecular and structural remodeling of stress granules in slowly and rapidly progressive Alzheimer's disease

Stress granules (SGs) are dynamic ribonucleoprotein condensates that modulate RNA metabolism during cellular stress. Although SG dysfunction has been increasingly linked to neurodegenerative diseases, their structural and molecular remodeling in Alzheimers disease (AD), particularly rapidly progressive AD (rpAD), remains poorly understood. Here, we present a comprehensive multi-omics characterization of SGs from postmortem frontal cortex tissues of control, slowly progressive AD (spAD), and rpAD subjects. SGs were immunoprecipitated using Anti-TIAR antibodies and analyzed via transmission electron microscopy (TEM), LCMS/MS-based proteomics, and RNA sequencing. Key protein findings were validated in human cortical brain homogenates and a 3xTg mouse model of A{beta} and tau pathology. TEM revealed disease-specific SG morphologies: small spherical granules in controls; moderate clustering in spAD; and large, amorphous aggregates in rpAD. Proteomic profiling identified 1,667 high-confidence SG-associated proteins, including RNA-binding proteins and disease-linked proteins such as MAPT, APP, and SNCA. SGs in rpAD were significantly enriched for pathways involved in MAPK signaling, proteostasis, and neuroinflammation, while showing reduced abundance of key cytoskeletal and translational regulators, such as TUBA1B and EEF1A2. Transcriptome analysis revealed widespread depletion of long, GC-rich, protein coding RNAs in rpAD SGs. Notably dynamic dysregulation of TUBA1B was also observed in the 3xTg mouse model and human cortical tissues, highlighting cytoskeletal vulnerability during disease progression. Together, these findings uncover profound structural and molecular remodeling of SGs in AD, with rpAD exhibiting a distinctive shift towards pathological SG composition and function. Our results highlight a link between SG alterations and aggressive AD subtypes, providing new mechanistic insights and suggesting new potential targets for therapeutic intervention.

neuroscience↗

bioRxiv2025

Beyond acute toxicity: evolutionary response by rapid polygenic adaptation to a complex environmental stressor in Chironomus riparius

Anthropogenic stressors, such as pollution, habitat degradation, and climate change, are altering selective pressures on natural populations, but the evolutionary consequences of chronic exposure to complex mixtures of contaminants remain poorly understood. Addressing this knowledge gap is critical to the emerging field of evolutionary ecotoxicology, which aims to understand how long-term exposure to environmental contaminants shapes adaptive evolution and genome-wide variation. In this study, we employed urban runoff sediment as complex and environmentally realistic model stressor to investigate how multigenerational exposure affects fitness and potentially drives genomic adaptation in the freshwater midge Chironomus riparius. We combined an evolutionary life-cycle test with the Evolve and Resequence (E&R) approach, exposing replicate populations over seven generations to three treatments: an uncontaminated control and two concentrations of urban runoff sediment (0.5% and 10%). Key fitness traits, including mortality, mean emergence time (EmT50), fertility, and population growth rate (PGR), were measured, while allele frequency changes (AFC) were tracked to identify genomic signatures of selection. The results revealed distinct and non-linear fitness responses across treatments, including transgenerational effects, recovery of performance, and evidence of life-history trade-offs. Candidate haplotypes were enriched for genes involved in membrane transport, metabolism, and gene regulation, suggesting selection on general stress-response pathways consistent with polygenic adaptation. Signals of selection were also detected in control populations, underscoring the evolutionary influence of laboratory conditions. Overall, our findings demonstrate how evolutionary ecotoxicology can reveal both the potential and the constraints of rapid adaptation to realistic environmental stressors and highlight the importance of integrating evolutionary perspectives into ecological risk assessment.

evolutionary biology↗

bioRxiv2025

Gpnmb Defines a Phagocytic State of Microglia Linked to Neuronal Loss in Prion Disease

Neurodegenerative conditions can induce the region-specific emergence of cell states relevant to their pathogenesis. To identify such phenomena, we generated a spatiotemporal transcriptomic atlas of mice infected with the RML prion strain. Thalamus and cerebellum experienced severe neuronal loss, developed intense microgliosis and, starting from 30 weeks post-inoculation, accumulated a novel microglial subpopulation characterized by strong expression of Glycoprotein non-metastatic melanoma protein B (Gpnmb). Elevated GPNMB levels were detected in the cerebrospinal fluid of sCJD patients, suggesting its possible usefulness as a biomarker of disease progression. The transcriptional profile of Gpnmb+ microglia reflected a state of enhanced phagocytic activity with upregulation of genes associated with lysosomal function, including vacuolar ATPase V0 domain subunit d2 (Atp6v0d2) and Galectin-3 (Lgals3). In microglia-like murine BV2 cells, Gpnmb upregulation was induced by soluble find-me signals released during apoptosis, but not by apoptotic bodies or prion accumulation. Likewise, human iPSC-derived microglia showed marked upregulation of GPNMB when co-cultured with apoptotic human neurons. Gpnmb ablation impaired the ability of BV2 cells to clear apoptotic cells, underscoring its role in maintaining microglial phagocytosis. Our findings define Gpnmb microglia as a distinct, apoptosis-driven phagocytic state, linking neuronal loss to microglial activation and positioning it as a key regulator of microglial responses to prion propagation.

neuroscience↗

bioRxiv2025

The MYCN/Aurora-A complex is a cyclin activating kinase for CDK12

Deregulated MYCN is a driver of aggressive pediatric and adult neuroendocrine tumors, but critical oncogenic processes downstream of MYCN remain poorly defined. In neuroblastoma, MYCN interacts with and activates the Aurora-A kinase. Here we show that Aurora-A is a CDK-activating kinase for CDK12 by phosphorylating T893 in the T-loop, thereby enhancing its kinase activity. Aurora-A-dependent activation of CDK12 controls phosphorylation of T4 of RNA polymerase and recruits transcription termination complexes, thereby preventing transcription-replication conflicts. Enhanced crosslinking and immunoprecipitation sequencing reveals that Aurora-A associates with splice sites on nascent RNA. RNA-bound Aurora-A is catalytically inactive. MYCN competes with RNA for binding to Aurora-A and displaces Aurora-A from RNA in cells, promoting its CDK12 kinase activity. Combining Aurora-A and CDK12 inhibition potently suppresses the growth of MYCN-amplified neuroblastoma cells and patient-derived xenografts. Our data demonstrate that an Aurora-A/CDK12-dependent transcription termination pathway is a critical and targetable dependency of MYCN-driven tumors.

cancer biology↗

bioRxiv2025

Detection of alpha synuclein seeding activity in tear fluid in patients with Parkinson's disease

Detection of alpha-synuclein seeding activity in tear fluid (TF) might provide a promising non-invasive biomarker for Parkinsons disease (PD) diagnosis. In this study, we applied the alpha-synuclein seeding amplification assay (aSynSAA) to detect misfolded alpha-synuclein (aSyn) aggregation in TF from PD patients. The discovery cohort included 11 PD patients and 13 controls, and the validation cohort consisted of 9 PD patients and 11 controls without synucleinopathies. The aSynSAA yielded positive results in over 55% of PD patients. These findings were confirmed in a second cohort, including patients with prion diseases as a negative control for synuclein pathology. Our results demonstrate for the first time the ability of aSynSAA to distinguish between PD and control groups in TF, with PD showing the highest seeding activity compared to prion disease and control groups. Further comparisons between cerebrospinal fluid (CSF) and TF samples from the same individuals revealed consistent seeding results across both biofluids. These findings highlight the potential of tear fluid as a novel, accessible medium for detecting Lewy body-specific misfolded synuclein aggregation in PD, which could aid in early diagnosis and disease progression monitoring.

neuroscience↗

bioRxiv2024

Inflammatory Mesenchymal Stromal Cells and IFN-responsive T cells are key mediators of human bone marrow niche remodeling in CHIP and MDS

Somatic mutations in hematopoietic stem/progenitor cells (HSPCs) can lead to clonal hematopoiesis of indeterminate potential (CHIP), potentially progressing to myelodysplastic syndromes (MDS). Here, we investigated how CHIP and MDS remodel the human bone marrow (BM) niche relative to healthy elderly donors, using single cell and anatomical analyses in a large BM cohort. We found distinct inflammatory remodeling of the BM in CHIP and MDS. Furthermore, the stromal compartment progressively lost its HSPC-supportive adipogenic CXCL12-abundant reticular cells while an inflammatory mesenchymal stroma cell (iMSCs) population emerged in CHIP, which expanded in MDS. iMSCs exhibited distinct functional signatures in CHIP and MDS, retaining residual HSPC-support and angiogenic activity in MDS, corresponding with an increase in microvasculature in the MDS niche. Additionally, an IFN-responsive T cell population was linked to fueling inflammation in the stroma. Overall, these findings open new avenues for early intervention in hematological malignancies.

molecular biology↗

bioRxiv2024

Exposure to complex mixtures of urban sediments containing Tyre and Road Wear Particles (TRWPs) increases the germ-line mutation rate in Chironomus riparius

Tyre and road wear particles (TRWPs) are a significant yet often underestimated source of environmental pollution, contributing to the accumulation of microplastics and a complex mixture of contaminants in both terrestrial and aquatic ecosystems. Despite their prevalence, the long-term evolutionary effects of TRWPs, beyond their immediate toxicity, remain largely unknown. In this study, we assessed mutagenicity in the non-biting midge Chironomus riparius, upon exposure to urban sediment collected from a runoff sedimentation basin. To assess the extent of mutagenic effects over multiple generations, we combined the urban sediment exposure model with short-term mutation accumulation lines (MALs) and subsequent whole genome sequencing (WGS). Our results reveal that the exposure to urban sediment significantly increases mutation rates compared to control groups by 50%, independent of concentration (0.5% and 10%). To infer potential causal processes, we conducted a comparative analysis with known mutational spectra from experiments with other studies. This comparison showed that the mutation profiles induced by urban sediment clearly clustered with those caused by Benzo[a]Pyrene (BaP), a known polycyclic aromatic hydrocarbon (PAH). A comprehensive chemical characterization of the sediment confirmed a considerable impact of road runoff and traffic-related contamination, including PAHs of primarily petrogenic origin. This suggests that PAH-like compounds present in urban sediments may play a significant role in the observed mutagenic effects. Our study shows that urban sediments influence mutation rates and alter mutational spectra in exposed organisms, potentially compromising genomic stability and shaping evolutionary trajectories. Additionally, we show that comparatively analysing mutational spectra may provide valuable insights into mutational processes. These genetic changes may have profound long-term effects on population dynamics and ecosystem health, underscoring the importance of understanding the evolutionary consequences of environmental pollution.

evolutionary biology↗

bioRxiv2024

Geometric deep learning framework for de novo genome assembly

The critical stage of every de novo genome assembler is identifying paths in assembly graphs that correspond to the reconstructed genomic sequences. The existing algorithmic methods struggle with this, primarily due to repetitive regions causing complex graph tangles, leading to fragmented assemblies. Here, we introduce GNNome, a framework for path identification based on geometric deep learning that enables training models on assembly graphs without relying on existing assembly strategies. By leveraging symmetries inherent to the problem, GNNome reconstructs assemblies with similar or superior contiguity compared to the state-of-the-art tools across several species, sequenced with PacBio HiFi or Oxford Nanopore. With every new genome assembled telomere-to-telomere, the amount of reliable training data at our disposal increases. Combining the straightforward generation of abundant simulated data for diverse genomic structures with the AI approach makes the proposed framework a plausible cornerstone for future work on reconstructing complex genomes with different ploidy and aneuploidy degrees. To facilitate such developments, we make the framework and the best-performing model publicly available, provided as a tool that can directly be used to assemble new haploid genomes.

bioinformatics↗

bioRxiv2024

Structural basis of Cdk7 activation by dual T-loop phosphorylation

Cyclin-dependent kinase 7 (Cdk7) occupies a central position in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of the general transcription factor TFIIH. Cdk7 forms an active complex upon association with Cyclin H and Mat1, and its catalytic activity is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the fully activated human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates a set of basic residues conserved in other CDKs, pS164 nucleates an arginine network involving all three subunits that is unique to the ternary Cdk7 complex. We identify differential dependencies of kinase activity and substrate recognition on individual phosphorylations within the Cdk7 T loop. The CAK function of Cdk7 is not affected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by phosphorylation at T170. Moreover, dual T-loop phosphorylation at both T170 and S164 stimulates multi-site phosphorylation of transcriptional substrates--the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and the SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7-regulatory phosphorylation is a two-step process in which phosphorylation of S164 precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing Cdk7 processivity, while the canonical pT170 enhances kinase activity towards critical substrates involved in transcription.

biochemistry↗