Biology subjects

Berger, H.

Publications and source records attributed to Berger, H..

16 recordsLinked to original sources

bioRxiv2025

Aberrant brain-type neuronal programs in large-cell pancreatic neuroendocrine carcinoma

High-grade pancreatic neuroendocrine carcinoma (panNEC) is a rare, aggressive cancer with limited tissue availability. Single-nucleus transcriptomics of five large-cell panNECs revealed two clinically relevant neuroendocrine cell states: one highly proliferative with an aberrant brain-specific neuronal differentiation program and another stress-responsive state enriched for heat stress, hypoxia, and glycolysis. Our findings suggest potentially new therapeutic vulnerabilities, highlighting the need to evaluate the efficacy of combination therapies.

cancer biology↗

bioRxiv2025

Inactivation of CDK4/6, CDK2, and ERK in G1-phase triggers differentiation commitment

Terminal cell differentiation, a process vital for tissue development and regeneration where progenitor cells acquire specialized functions and permanently exit the cell cycle, is still poorly understood at the molecular level. Using live-cell imaging and adipogenesis as a model, we demonstrate that the initial stage involves a variable number of cell divisions driven by redundant CDK4/6 or CDK2 activation.. Subsequently, a delayed decrease in cyclin D1 and an increase in p27 levels leads to the attenuation of CDK4/6 and CDK2 activity. This results in G1 lengthening and the induction of PPARG, the master regulator of adipogenesis. PPARG then induces p21, and later p18, culminating in the irreversible inactivation of CDK4/6 and CDK2, and thus, permanent cell cycle exit. However, contrary to expectation, CDK inactivation alone is not sufficient to trigger commitment to differentiation and functional specialization; ERK inactivation is also required. Our study establishes that the coordinated activation and subsequent delayed inactivation of CDK4/6, CDK2, and ERK are crucial determinants for irreversible cell cycle exit and differentiation commitment in terminal cell differentiation.

cell biology↗

bioRxiv2024

Copper acquisition is essential for plant colonization and virulence in a root-infecting vascular wilt fungus

Phytopathogenic fungi provoke devastating agricultural losses and are difficult to control. How fungal pathogens adapt to the plant environment to cause disease and complete their life cycle on the host remains poorly understood. Here we show that efficient acquisition of copper, mediated by the transcriptional regulator Mac1, is crucial for plant colonization and virulence in Fusarium oxysporum, a soilborne ascomycete that causes vascular wilt on more than 150 different crops. RNA-seq and ChIP-seq establish a direct role of Mac1 in activation of copper deficiency response genes, many of which are induced during plant infection. Loss of Mac1 impairs growth of F. oxysporum under copper-limiting condition as well as vascular colonization and virulence on tomato plants. Importantly, Mac1-independent overexpression of a copper reductase and a copper transporter restores growth under copper limitation and virulence in the mac1 null mutant background. These findings establish a key role for copper acquisition in fungal pathogenicity on plants and reveal new ways to protect crops from phytopathogens.

genetics↗

bioRxiv2023

Helicobacter pylori cancer associated CagA protein drives intestinal metaplastic transition in human gastric organoids

Gastric intestinal metaplasia (GIM) constitutes a pre-neoplastic stage in the development of stomach cancer. While strong evidence points to a role of infection with CagA positive Helicobacter pylori in the development of GIM, currently available experimental models have not provided mechanistic clues on this association. Here, we ectopically expressed the H. pylori CagA protein in human gastric organoids derived from normal, primary epithelial cells. Native CagA protein was produced and rapidly processed to yield a tyrosine-phosphorylated C-terminal fragment of [~]35 kDa. It led to an activation of the STAT3 pathway and aberrant elevation of CDX2 expression, a marker of intestinal type of cells, as well as other intestinal markers. Thus, CagA drives re-programming of gastric cells towards an intestinal-like phenotype, towards GIM. In summary, we describe a cooperative mechanism of CagA-induced STAT3 signaling and intestinal-like trans-differentiation, promoting a pre-neoplastic state. Our model provides mechanistic evidence for a direct role of CagA in driving premalignancy in gastric pathogenesis.

cell biology↗

bioRxiv2023

γδ T cell-mediated cytotoxicity against patient-derived healthy and cancer cervical organoids

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to {beta} T cells and natural killer (NK) cells, {gamma}{delta} T cells represent a cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of {gamma}{delta} T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded {gamma}{delta} T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to {gamma}{delta} T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential {gamma}{delta} T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for {gamma}{delta} T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of {gamma}{delta} T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.

immunology↗

bioRxiv2022

Polaramycin B, and not physical interaction, is the signal that rewires fungal metabolism in the Streptomyces - Aspergillus interaction.

Co-culturing the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid (ORS) and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact is a prerequisite for the metabolic reaction that involves a fungal amino acid starvation response and activating chromatin modifications at the biosynthetic gene cluster (BGC). Here we show that not physical contact, but a guanidine containing macrolide, named polaramycin B, triggers the response. The substance is produced constitutively by the bacterium and provokes the production of ORS above a certain concentration. In addition, several other secondary metabolites were induced by polaramycin B. Our genome-wide transcriptome analysis showed that polaramycin B treatment causes down-regulation of fungal genes necessary for membrane stability, general metabolism and growth. A compensatory genetic response can be observed in the fungus that included up-regulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a novel chemical communication, in which the antifungal bacterial metabolite polaramycin B leads to the production of antibacterial defence chemicals and to the up-regulation of genes necessary to compensate for the cellular damage caused by polaramycin B.

microbiology↗

bioRxiv2022

DNA methylation in human gastric epithelial cells allows cell type-related plasticity and defines regional identity

BackgroundEpigenetic modifications in mammalian DNA are commonly manifested by DNA methylation. In the stomach, altered DNA methylation patterns have been observed following chronic Helicobacter pylori infections and in gastric cancer. In the context of epigenetic regulation, the regional nature of the stomach has been rarely considered in detail. ResultsHere, we describe the DNA methylation landscape across the phenotypically different regions of the healthy human stomach (i.e., antrum, corpus, fundus) together with the corresponding transcriptomes. We show that stable regional DNA methylation differences translate to a limited extent into regulation of the transcriptomic phenotype, indicating a largely permissive epigenetic regulation. We identify a small number of transcription factors with novel region-specific activity and likely epigenetic impact in the stomach, including GATA4, IRX5, IRX2, PDX1, and CDX2. Detailed analysis of the Wnt pathway reveals differential regulation along the craniocaudal axis, which involves non-canonical Wnt signaling in determining cell fate in the proximal stomach. ConclusionsBy extending our analysis to pre-neoplastic lesions and gastric cancers, we conclude that epigenetic dysregulation already characterizes intestinal metaplasia as a founding basis for functional changes in gastric cancer. Finally, our study provides a well-defined resource of regional stomach transcription and epigenetics as a starting point for further studies.

molecular biology↗

bioRxiv2021

Resurrecting essential amino acid biosynthesis in a mammalian cell

Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids1. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly2,3, raising the possibility that these pathways were lost from organisms with access to abundant EAAs in the environment4,5. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in a mammalian cell. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese Hamster Ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation, and thus represents a successful example of metazoan EAA biosynthesis restoration. This prototrophic CHO line grows in valine-free medium, and metabolomics using labeled precursors verified de novo biosynthesis of valine. RNA-seq profiling of the valine prototrophic CHO line showed that the synthetic pathway minimally disrupted the cellular transcriptome. Furthermore, valine prototrophic cells exhibited transcriptional signatures associated with rescue from nutritional starvation. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, thus paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.

synthetic biology↗

bioRxiv2021

Chlamydia coinfection inhibits HPV-induced safeguards of the cellular and genomic integrity in patient-derived ectocervical organoids

Cervical mucosa is continually confronted by coinfections with pathogenic microbes. In addition to human papillomavirus, coinfections with Chlamydia trachomatis have been associated with an increased risk of cervical cancer. However, the dynamics of coinfections, their impact on the epithelia, and their contribution to pathogenesis remain obscure. Using a novel human ectocervical squamous stratified epithelial organoids, we recapitulated the natural infections of the cervix by Chlamydia, HPV, and their coinfections. Towards this, we genetically manipulated the healthy organoids to mimic in vivo HPV persistence by introducing E6E7 oncogenes into the host genome. HPV persistent organoids show enhanced tissue regeneration, increased proliferation and differentiation of stem cells, and nuclear atypia resembling cervical intraepithelial neoplasia grade 1. We found that HPV interferes with normal Chlamydia development. Further, a unique transcriptional host response induced by Chlamydia and HPV leads to distinct reprogramming of host cell processes. Strikingly, in coinfections, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair (MMR). Distinct post-translational proteasomal-degradation and E2F-mediated transcriptional regulation delineate the inverse regulation of MMR during coinfections. Our study employing organoids demonstrates the jeopardy of multiple sequential infection processes and the unique cellular microenvironment they create, accelerating neoplastic progression.

cancer biology↗

bioRxiv2020

Activation of silent secondary metabolite gene clusters by nucleosome map-guided positioning of the synthetic transcription factor VPR-dCas9

Current methods for forced expression of selected target genes are based on promoter exchange or on overexpressing native or hybrid transcriptional activators in which gene-specific DNA binding domains are coupled to strong activation domains. While these approaches are very useful for promoters with known or synthetically introduced transcription factor binding sites, they are not suitable to turn on genes in biosynthetic gene clusters which often lack pathway-specific activators. To expand the discovery toolbox, we designed a Cas9-based RNA guided synthetic transcription activation system for Aspergillus nidulans based on enzymatically disabled dCas9 fused to three consecutive activation domains (VPR-dCas9). Targeting two biosynthetic gene clusters involved in the production of secondary metabolites, we demonstrate the utility of the system. Especially in silent regions facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to our targeted promoters we have created a genome-wide nucleosome map to identify the cognate nucleosome-free-regions (NFRs). Based on these maps, different single-guide RNAs (sgRNA) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to activate silent BGCs in A. nidulans, partially to very high expression levels and also open the opportunity to stepwise turn on individual genes within a BGC that allows to decipher the correlated biosynthetic pathway.

bioengineering↗

bioRxiv2019

Colibactin DNA damage signature indicates causative role in colorectal cancer

Colibactin, a potent genotoxin of Escherichia coli, causes DNA double strand breaks (DSBs) in human cells. We investigated if colibactin creates a particular DNA damage signature in infected cells by identifying DSBs in colon cells after infection with pks+ E.coli. Interestingly, genomic contexts of DSBs were enriched for AT-rich penta-/hexameric sequence motifs, exhibiting a particularly narrow minor groove width and extremely negative electrostatic potential. This corresponded with the binding characteristics of colibactin to double-stranded DNA, as elucidated by docking and molecular dynamics simulations. A survey of somatic mutations at the colibactin target sites of several thousand cancer genomes revealed significant enrichment of the identified motifs in colorectal cancers. Our work provides direct evidence for a role of colibactin in the etiology of human cancer.\n\nOne sentence summaryWe identify a mutational signature of colibactin, which is significantly enriched in human colorectal cancers.

microbiology↗

bioRxiv2019

Preservation of stemness in high-grade serous ovarian cancer organoids requires low Wnt environment

High-grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the parental tumor mutational profile and phenotype. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for generation of HGSOC organoids, while healthy FT organoids depend on BMP suppression by Noggin. Interestingly, FT organoids modified by stable shRNA knockdown (KD) of p53, PTEN, and Retinoblastoma (RB), also require a low Wnt environment for long-term growth, while FT organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotype of normal and KD organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation.

cancer biology↗

bioRxiv2018

Transition of Wnt signaling microenvironment delineates the squamo-columnar junction and emergence of squamous metaplasia of the cervix

The transition zones (TZ) between squamous and columnar epithelium constitute hotspots for the emergence of cancers. Carcinogenesis at these sites is often preceded by the development of metaplasia, where one epithelial type invades the neighboring one. It remains unclear how these niches are restrained at the boundary between the two epithelial types and what factors contribute to metaplasia. Here we show that the cervical squamo-columnar junction derives from two distinct stem cell lineages that meet at the TZ. In contrast to the prevailing notion, our analysis of cervical tissue showed that the TZ is devoid of any locally restricted, specialized stem cell population, which has been implicated as precursor of both cervical squamous cell carcinoma and adenocarcinoma. Instead, we reveal that these cancers originate from two separate stem cell lineages. We show that the switch in the underlying Wnt signaling milieu of the stroma is a key determinant of proliferation or quiescence of epithelial stem cell lineages at the TZ. Strikingly, while the columnar lineage of the endocervix is driven by Wnt signaling, the maintenance of squamous stratified epithelium of the ectocervix and emergence of squamous metaplasia requires inhibition of Wnt signaling via expression of Dickkopf2 (Dkk2) in the underlying stroma. Moreover, Notch signaling is required for squamous cell stratification. Thus, our results indicate that homeostasis at the TZ is not maintained by a transition from one epithelial type to another but rather results from alternative signals from the stromal compartment driving the differential proliferation of the respective cell lineages at the squamo-columnar junction.

cancer biology↗

bioRxiv2018

De novo assembly, delivery and expression of a 101 kb human gene in mouse cells

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assembled the 101 kb human HPRT1 gene in yeast, delivered it to mouse embryonic stem cells, and showed expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells. Significance StatementMammalian genomes consist of a tiny proportion of relatively well-characterized coding regions and vast swaths of poorly characterized "dark matter" containing critical but much less well-defined regulatory sequences. Given the dominant role of noncoding DNA in common human diseases and traits, the interconnectivity of regulatory elements, and the importance of genomic context, de novo design, assembly, and delivery can enable large-scale manipulation of these elements on a locus scale. Here we outline a pipeline for de novo assembly, delivery and expression of mammalian genes replete with native regulatory sequences. We expect this pipeline will be useful for dissecting the function of non-coding sequence variation in mammalian genomes.

genetics↗

bioRxiv2018

Synthetic chromosome fusion: effects on genome structure and function

As part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is [~]21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

synthetic biology↗

bioRxiv2018

Inter-species conservation of organisation and function between non-homologous regional centromeres

Despite the conserved essential function of centromeres, centromeric DNA itself is not conserved1-4. The histone-H3 variant, CENP-A, is the epigenetic mark that specifies centromere identity5-8. Paradoxically, CENP-A normally assembles on particular sequences at specific genomic locations. To gain insight into the specification of complex centromeres we took an evolutionary approach, fully assembling genomes and centromeres of related fission yeasts. Centromere domain organization, but not sequence, is conserved between Schizosaccharomyces pombe, S. octosporus and S. cryophilus with a central CENP-ACnp1 domain flanked by heterochromatic outer-repeat regions. Conserved syntenic clusters of tRNA genes and 5S rRNA genes occur across the centromeres of S. octosporus and S. cryophilus, suggesting conserved function. Remarkably, non-homologous centromere central-core sequences from S. octosporus are recognized in S. pombe, resulting in cross-species establishment of CENP-ACnp1 chromatin and functional kinetochores. Therefore, despite the lack of sequence conservation, Schizosaccharomyces centromere DNA possesses intrinsic conserved properties that promote assembly of CENP-A chromatin. Thus, centromere DNA can be recognized and function over unprecedented evolutionary timescales.

genomics↗