Biology subjects

Buchholz, F.

Publications and source records attributed to Buchholz, F..

13 recordsLinked to original sources

bioRxiv2026

Loss of UBP1 drives oxaliplatin resistance through a targetable dependency on translation initiation

Oxaliplatin is a common component of various chemotherapeutic regimens for the treatment of gastrointestinal cancers. However, the majority of patients exhibit resistance to oxaliplatin-based therapy. Here, we integrated knockout and transcription-activation CRISPR screens in patient-derived gastric cancer organoids (GC PDOs) to comprehensively profile major genetic and transcriptomic changes observed over the course of resistance acquisition. Our screens identified UBP1, a transcription factor frequently lost in GC, as a critical determinant of oxaliplatin resistance development. Leveraging a large GC organoid biobank from a co-clinical trial and primary tumor omics data, we reveal that downregulation of specifically MYC-driven ribosome biogenesis drives oxaliplatin resistance, highlighting the drugs role as a ribosome biogenesis stressor. Mechanistically, UBP1 loss reduced expression of its direct target, MAX, a MYC cofactor, leading to downregulation of ribosome biogenesis and protection against nucleolar stress. Crucially, we discover that such downregulation is inevitably followed by a compensatory reliance on translation initiation, making it a therapeutic vulnerability in oxaliplatin-resistant tumors. Consequently, the resistance could be overcome by a synergistic action of the translation initiation repressor 4EGI, and the effect was also maintained in PDO that acquired resistance in vivo under clinically relevant conditions. Our data uncover a common marker of oxaliplatin resistance and identify a novel therapeutic strategy to reverse resistance to one of the most frequently used anticancer drugs.

cancer biology↗

bioRxiv2025

Robust antibiotic sensitization of pathogenic Pseudomonas aeruginosa via negative hysteresis in the cell envelope

Antibiotic combination in time and space is a key strategy to combat antimicrobial resistance. The success of such treatment designs requires their robust efficacy across treatment conditions and a pathogens genomic diversity. This study found that an initial treatment with a {beta}-lactam antibiotic causes robust cellular sensitization towards an aminoglycoside antibiotic across the high-risk human pathogen Pseudomonas aeruginosa, including resistant strains. This phenomenon of cellular sensitization, termed negative hysteresis, is modulated by the Cpx envelope stress response system and linked to membrane stress during growth. The increase in efficacy is achieved through a {beta}-lactam induced elevated cellular uptake of the subsequently administered aminoglycoside. Negative hysteresis and the Cpx system are linked in several cases to the expression of synergistic drug interactions, thus enhancing efficacy of antibiotic combinations. Overall, our study identifies the phenomenon of negative hysteresis as a robustly inducible phenotype and thus a unique focus for optimizing antimicrobial therapy.

evolutionary biology↗

bioRxiv2025

Resistance variation and bacterial interactions shape the adaptation of a genetically diverse bacterial population to antimicrobial treatment

Bacterial infections are often polymicrobial and subject to the evolution of antimicrobial resistance (AMR). Existing knowledge on AMR in such polymicrobial infections usually relies on observational patient data, for which cause-effect relationships are difficult to infer, or on studying interactions between different bacterial species, ignoring the commonly encountered variation within species. Here, we therefore asked how mixed populations with strains from the same species evolve under antibiotic treatment. We used a genetically diverse population of the high-risk human pathogen Pseudomonas aeruginosa, and first identified strain variation in both AMR and pairwise bacteria-bacteria interactions, the latter ranging from beneficial, neutral, to competitive. Using experimental evolution, we subsequently demonstrate that the response to selection by different antibiotic treatments is significantly influenced by AMR strain variation, bacterial interactions, and also spatial population structure. Moreover, de novo AMR evolution was additionally impacted by variation in resistance rates towards the two considered antibiotics. A second evolution experiment emphasized the central role of strain variation and bacterial interactions in determining the evolutionary outcome. We conclude that ecological dynamics in genetically diverse pathogen populations are key for our general understanding of infection characteristics and AMR evolution, and, therefore, deserve particular attention during treatment of polymicrobial infections.

evolutionary biology↗

bioRxiv2025

NCBP2-AS2 is a mitochondrial microprotein, regulates energy metabolism and neurogenesis, and is downregulated in Alzheimer's disease

Microproteins, short functional peptides encoded by small genes, are emerging as critical regulators of cellular processes, yet their roles in mitochondrial function and neurodegeneration remain underexplored. In this study, we identify NCBP2-AS2 as an evolutionarily conserved mitochondrial microprotein with significant roles in energy metabolism and neurogenesis. Using a combination of cellular and molecular approaches, including CRISPR/Cas9 knockout models, stoichiometric co- immunoprecipitation, and advanced imaging techniques, we demonstrate that NCBP2-AS2 localizes to the inner mitochondrial space and interacts with translocase of the inner membrane (TIM) chaperones. These interactions suggest a role in ATPase subunit transport, supported by the observed reductions in ATPase subunit levels and impaired glucose metabolism in NCBP2-AS2-deficient cells. In zebrafish, NCBP2-AS2 knockout led to increased astroglial proliferation, microglial abundance, and enhanced neurogenesis, particularly under amyloid pathology. Notably, we show that NCBP2-AS2 expression is consistently downregulated in human Alzheimers disease brains and zebrafish amyloidosis models, suggesting a conserved role in neurodegenerative pathology. These findings reveal a novel link between mitochondrial protein transport, energy metabolism, and neural regeneration, positioning NCBP2-AS2 as a potential therapeutic target for mitigating mitochondrial dysfunction and promoting neurogenesis in neurodegenerative diseases such as Alzheimers disease.

cell biology↗

bioRxiv2024

Variation in the response to antibiotics and life-history across the major Pseudomonas aeruginosa clone type (mPact) panel

Pseudomonas aeruginosa is a ubiquitous, opportunistic human pathogen. Since it often expresses multidrug resistance, it is ranked by the World Health Organization among the top 3 high priority pathogens, for which new treatment options are urgently required. An evaluation of new treatments is usually performed experimentally with one of the canonical laboratory strains (e.g., PAO1 or PA14). However, these two strains are unlikely representative of the strains infecting patients, because they have adapted to laboratory conditions and do not capture the enormous genomic diversity of the species. Here, we characterized the major P. aeruginosa clone type (mPact) panel. This panel consists of 20 strains, which reflect the genomic diversity of the species, cover all major clone types, and have both patient and environmental origins. We found significant strain variation in distinct responses towards antibiotics and general growth characteristics. Only few of the measured traits are related, and if so, only for specific antibiotics. Moreover, high levels of resistance were only identified for clinical mPact isolates and could be linked to known AMR (antimicrobial resistance) genes in the sequenced genomes. One strain also produced highly unstable AMR, indicating an evolutionary cost to resistance expression. By linking isolation source, growth, and virulence to life history traits, we further identified specific adaptive strategies for individual mPact strains towards either host processes or degradation pathways. Overall, the mPact panel provides a reasonably sized set of distinct strains, enabling in-depth analysis of new treatment designs or evolutionary dynamics in consideration of the species genomic diversity. ImportanceNew treatment strategies are urgently needed for high risk pathogens such as the opportunistic and often multidrug resistant pathogen Pseudomonas aeruginosa. Here, we characterize the major P. aeruginosa clone type (mPact) panel. It consists of 20 strains with different origins that cover the major clone types of the species as well as its genomic diversity. This mPact panel shows significant variation in (i) resistance against distinct antibiotics, including several last resort antibiotics, (ii) related traits associated with the response to antibiotics, and (iii) general growth characteristics. We further developed a novel approach that integrates information on resistance, growth, virulence, and life-history characteristics, allowing us to demonstrate the presence of distinct adaptive strategies of the strains that focus either on host interaction or resource processing. In conclusion, the mPact panel provides a manageable number of representative strains for this important pathogen for further in-depth analyses of treatment options and evolutionary dynamics.

microbiology↗

bioRxiv2023

TOBF1 modulates mouse embryonic stem cell fate through co-transcriptional regulation of alternative splicing

Embryonic stem (ES) cells retain the ability to undergo lineage-specific differentiation that can eventually give rise to different cell types that constitute an organism. Although stem cell specific biological networks of transcription factors and epigenetic modifiers are well established, how the ES cell specific transcriptional and alternative splicing (AS) machinery regulate their expression has not been sufficiently explored. In this study, we show that the lncRNA associated protein TOBF1 regulates the co-transcriptional alternative splicing of transcripts necessary for maintaining stem cell identity in mouse ES cells. Overlaying information derived from TOBF1 chromatin occupancy, the distribution of its pluripotency-associated OCT-SOX binding motifs, and transcripts undergoing differential expression and alternative splicing upon its disruption unmasked local nuclear territories where these distinct events converge, ultimately leading to the maintenance of mouse ES cell identity.

developmental biology↗

bioRxiv2022

Phylogroup-specific variation shapes pangenome dynamics in Pseudomonas aeruginosa

BackgroundPseudomonas aeruginosa is an opportunistic pathogen consisting of three phylogroups (hereafter named A, B, and C) of unevenly distributed size. Here, we assessed phylogroup-specific evolutionary dynamics in a collection of P. aeruginosa genomes. MethodsIn this genomic analysis, using phylogenomic and comparative genomic analyses, we generated 18 hybrid assemblies from a phylogenetically diverse collection of clinical and environmental P. aeruginosa isolates, and contextualised this information with 1991 publicly available genomes of the same species. We explored to what extent antimicrobial resistance (AMR) genes, defence systems, and virulence genes vary in their distribution across regions of genome plasticity (RGPs) and "masked" (RGP-free) genomes, and to what extent this variation differs among the phylogroups. FindingsWe found that members of phylogroup B possess larger genomes, contribute a comparatively larger number of pangenome families, and show lower abundance of CRISPR-Cas systems. Furthermore, AMR and defence systems are pervasive in RGPs and integrative and conjugative/mobilizable elements (ICEs/IMEs) from phylogroups A and B, and the abundance of these cargo genes is often significantly correlated. Moreover, inter- and intra-phylogroup interactions occur at the accessory genome level, suggesting frequent recombination events. Finally, we provide here a panel of diverse P. aeruginosa strains to be used as reference for functional analyses. InterpretationAltogether, our results highlight distinct pangenome characteristics of the P. aeruginosa phylogroups, which are possibly influenced by variation in the abundance of CRISPR-Cas systems and that are shaped by the differential distribution of other defence systems and AMR genes. FundingGerman Science Foundation, Max-Planck Society, Leibniz ScienceCampus Evolutionary Medicine of the Lung, BMBF program Medical Infection Genomics, Kiel Life Science Postdoc Award. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSTo date, pangenome studies exploring the epidemiology and evolution dynamics of bacterial pathogens have been limited due to the use of gene frequencies across whole species dataset without accounting for biased sampling or the population structure of the genomes in the dataset. We searched PubMed without language restrictions for articles published before September 1, 2021, that investigated the phylogroup-specific evolutionary dynamics across bacterial species. In this literature search we used the search terms "pangenome" and "phylogroup" or "uneven", which returned 14 results. Of these, only one study used a population structure-aware approach to explore pangenome dynamics in a bacterial species consisting of multiple phylogroups with unevenly distributed members. Added value of this studyTo our knowledge, this study is the first to assess phylogroup-specific evolutionary dynamics in a collection of genomes belonging to the nosocomial pathogen P. aeruginosa. Using a refined approach that challenges traditional pangenome analyses, we found specific signatures for each of the three phylogroups, and we demonstrate that members of phylogroup B contribute a comparatively larger number of pangenome families, have larger genomes, and have a lower prevalence of CRISPR-Cas systems. Additionally, we observed that antibiotic resistance and defence systems are pervasive in regions of genome plasticity and integrative and conjugative/mobilizable elements from phylogroups A and B, and that antibiotic resistance and defence systems are often significantly correlated in these mobile genetic elements. Implications of all the available evidenceThese results indicate that biases inherent to traditional pangenome approaches can obscure the real distribution of important cargo genes in a bacterial species with a complex population structure. Furthermore, our findings pave the way to new pangenome approaches that are currently under-explored in comparative genomics and, crucially, shed a new light on the role that integrative and conjugative/mobilizable elements may play in protecting the host against foreign DNA.

microbiology↗

bioRxiv2022

Prediction of designer-recombinases for DNA editing with generative deep learning

Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designer-recombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming. Here we present RecGen (Recombinase Generator), an algorithm for the intelligent generation of designer-recombinases. We gathered the sequence information of over two million Cre-like recombinase sequences evolved for 89 different target sites with which we trained Conditional Variational Autoencoders for recombinase generation. Experimental validation demonstrated that the algorithm can predict recombinase sequences with activity on novel target-sites, indicating that RecGen is useful to accelerate the development of future designer-recombinases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/486669v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c08326org.highwire.dtl.DTLVardef@e26984org.highwire.dtl.DTLVardef@7d7542org.highwire.dtl.DTLVardef@9d6642_HPS_FORMAT_FIGEXP M_FIG C_FIG Teaser Figure: Recombinase prediction generates active recombinases for a desired target site, while existing libraries with a similar target site need to go through directed evolution to achieve activity on the new site. While evolution takes weeks, prediction and synthesis of recombinases can be done in days.

bioinformatics↗

bioRxiv2020

Correction of a Factor VIII genomic inversion with designer-recombinases

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct a 140 kb genomic inversion of the F8 gene, which is frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we developed a fused heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, resulted in prominent correction of the inversion and restored Factor VIII mRNA expression. Our data suggests that designer-recombinases may represent efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

molecular biology↗

bioRxiv2020

USP28 and SPINT2 mediate cell cycle arrest after whole genome doubling

Tetraploidy is frequent in cancer and whole genome doubling shapes the evolution of cancer genomes, thereby driving the transformation, metastasis and drug resistance. Yet, human cells usually arrest when they become tetraploid due to p53 activation that leads to CDKN1A expression, cell cycle arrest, senescence or apoptosis. To uncover the barriers that block proliferation of tetraploids, we performed an RNAi mediated genome-wide screen in a human cancer cell line. We identified 140 genes whose depletion improved survival of tetraploids and characterized in depth two of them: SPINT2 and USP28. We show that SPINT2 is a general regulator of CDKN1A, regulating its transcription via histone acetylation. By mass spectrometry and immunoprecipitation, we show that USP28 interacts with NuMA1 and affects centrosome clustering. Moreover, tetraploid cells accumulate DNA damage and loss of USP28 reduces checkpoint activation. Our results indicate three aspects that contribute to survival of tetraploid cells: i) increased mitogenic signaling and reduced expression of cell cycle inhibitors, ii) the ability to establish functional bipolar spindle, and iii) reduced DNA damage signaling.

cell biology↗

bioRxiv2019

Large-scale dissection suggests that ultraconserved elements are dispensable for mouse embryonic stem cell survival and fitness

Withdrawal statementThe authors have withdrawn their manuscript from bioRxiv regretfully. During additional experiments for the revision we noticed inconsistencies in some results, which may affect the main conclusion of the manuscript. In particular, we noticed that using different primer pairs, some clones that were classified as homozygous knockouts, are actually not knockouts. These were unexpected findings, but might reflect the recently described larger rearrangements observed using CRISPR technology1,2,3,4. Follow up experiments are ongoing, but we would like to withdraw the manuscript at this point in time to avoid misunderstandings. Therefore, the manuscript should currently not be cited. Any questions regarding the manuscript should be addressed to the corresponding author.

genetics↗

bioRxiv2018

Comparative RNAi Screens in Isogenic Human Stem Cells Reveal SMARCA4 as a Differential Regulator

Large-scale RNAi screens are a powerful approach to identify functions of genes in a cell-type specific manner. For model organisms, genetically identical (isogenic) cells from different cell-types are readily available, making comparative studies meaningful. For humans, however, screening isogenic cells is not straightforward. Here, we show that RNAi screens are possible in genetically identical human stem cells, employing induced pluripotent stem cell as intermediates. The screens revealed SMARCA4 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 4) as a stemness regulator, while balancing differentiation distinctively for each cell type. SMARCA4 knockdown in hematopoietic stem progenitor cells (HSPC) caused impaired self-renewal in-vitro and in-vivo with skewed myeloid differentiation; whereas in neural stem cells (NSC), it impaired selfrenewal while biasing differentiation towards neural lineage, through combinatorial SWI/SNF subunit assembly. Our findings pose a powerful approach for deciphering human stem cell biology and attribute distinct roles to SMARCA4 in stem cell maintenance.

molecular biology↗

bioRxiv2017

MS Western, a method of multiplexed absolute protein quantification is a practical alternative to western blotting

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the sub-femtomole level in whole cell or tissue lysates without metabolic or chemical labelling and without using specific antibodies. MS Western relies upon GeLC-MS/MS and quantifies proteins by in-gel co-digestion with an isotopically labelled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

biochemistry↗