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Graf, A.

Publications and source records attributed to Graf, A..

12 recordsLinked to original sources
bioRxiv2026

The MAPK phosphatase VHP-1 buffers pharynx-to-body proportions against tissue-specific growth imbalance in C. elegans

Maintaining appropriate organ size ratios in the face of growth fluctuations is critical for the development of a reproducible body plan. Yet the mechanisms involved remain poorly understood. Here, we investigated how pharynx-to-body proportions are maintained in Caenorhabditis elegans, combining tissue-specific perturbations, genetic screening, and longitudinal live imaging. A genome-wide RNAi screen revealed that knock-down of the dual-specificity MAPK phosphatase VHP-1 turns animals hypersensitive to inter-tissue growth imbalance caused by pharyngeal or epidermal depletion of the mTORC1 activator RAGA-1 or the ribosomal protein RPL-22. In contrast, vhp-1 mutants tolerated global raga-1 loss, indicating a specific requirement for vhp-1 under tissue growth imbalance. Knock-down of the p38 pathway suppressed the imbalance-specific defects of vhp-1 mutants. In contrast, JNK knock-down effectively rescued the pleiotropic phenotypes of vhp-1 mutants but only weakly reduced their sensitivity to RAGA-1 imbalance, indicating that these two stress-MAPK pathways make distinct contributions to the response to growth imbalance. Finally, whole-animal VHP-1 levels increased upon epidermal RAGA-1 depletion, and epidermal VHP-1 depletion did not reproduce the sensitivity caused by global vhp-1 loss, consistent with a contribution from VHP-1 outside the growth-perturbed epidermis in buffering against local growth imbalance.

developmental biology↗
bioRxiv2026

CIP2A tetramerization is required for mitotic DNA repair

DNA lesions that persist in mitosis threaten genome stability. These lesions recruit TOPBP1-CIP2A, a complex crucial to tether and process damaged DNA on mitotic chromosomes. Importantly, CIP2A is synthetic lethal in BRCA1/2 mutant cancer cells. However, mechanistic insight into the function of CIP2A is lacking. Here we provide first structural insights into full-length CIP2A and reveal how CIP2A dimers, 46 nm in length, assemble via conserved C-terminal motifs into dumbbell-like tetramers. DNA repair signatures demonstrate that CIP2A tetramerization is crucial for mitotic DNA repair by polymerase theta (POLQ). Tetramerization of CIP2A is needed to recruit POLQ to mitotic DNA lesions and to prevent micronucleation upon replication-born DNA lesions. Tetramerization-defective CIP2A mutants specifically decrease survival of BRCA1/2 mutant cancer cells in a dominant negative manner. Combined, we demonstrate that CIP2A tetramers are crucial components of a mitotic DNA-tethering and repair scaffold, which processes replication-mediated DNA lesions that persist in mitosis.

cell biology↗
bioRxiv2026

Dbf4-dependent kinase promotes meiotic DNA end resection through cyclin-dependent kinase 12 and DNA-2 in Caenorhabditis elegans

Meiotic crossovers depend on the accurate processing of programmed DNA double-strand breaks by homologous recombination, which involves generating by resection the single-stranded DNA ends required for the subsequent strand invasion step. How regulatory kinases control DNA end resection in animal meiosis remains poorly understood. Here, we identify the Caenorhabditis elegans Dbf4-dependent kinase (DDK) complex, composed of CDC-7 and DBF-4, and identify CDK-12 as a candidate substrate. DDK promotes efficient meiotic DNA end resection, while crossover formation remains largely preserved in its absence. Loss of DDK function leads to SPO-11-dependent chromosome bridges at diakinesis that arise from recombination intermediates formed early in recombination. In addition to chromosome bridges, we observed elevated heterologous recombination that occurs independently of the canonical or alternative end-joining pathways. Interestingly, CDC-7 and DBF-4 localize dynamically during prophase I and load onto DNA in a hierarchical manner that depends on the BRC-1-BRD-1 complex. Epistasis analysis placed CDC-7 and CDK-12 within the DNA-2-dependent branch of long-range DNA resection. Together, our findings reveal an unidentified DDK/CDK-12/DNA-2 signaling axis that promotes efficient meiotic DNA resection to avoid the generation of aberrant recombination outcomes.

cell biology↗
bioRxiv2026

Advances in Thin-Film Graphene Neurotechnology for Chronic Nerve Stimulation and Recording

Neurotechnology is being explored for restoring sensorimotor functions after paralysis or amputation, which requires peripheral nerve interfaces that are selective, bidirectional, and chronically stable. Reduced graphene oxide (rGO)-based microelectrodes offer low impedance and a high charge-injection limit; however, long-term in vivo performance has been limited by the durability of encapsulation. Here, we introduce a 10 {micro}m-thick transverse intrafascicular multichannel electrode (TIME) with a hybrid polyimide-Al2O3 encapsulation engineered to improve fabrication yield, electrode-to-electrode uniformity, and device stability. In vitro, devices maintained near-ideal capacitive behaviour after accelerated ageing (3 months in PBS at 57 {degrees}C) and sustained 109 biphasic stimulation pulses without detectable electrochemical degradation. In vivo, the arrays recorded compound nerve action potentials after one month and enabled selective activation of distinct peripheral nerve fibres with comparatively low current thresholds during four months of follow-up, remaining below the device maximum injectable current. Together, these results demonstrate that combining graphene microelectrodes with a thin hybrid encapsulation improves chronic reliability of intraneural thin-film interfaces and helps to close the gap between laboratory prototypes and clinically relevant neuroprosthetic systems.

bioengineering↗
bioRxiv2025

Respiratory viruses activate autophagy via the IFN-STAT1/STAT5B-SOCS1 axis

Autophagy is an ancient catabolic process that has emerged as part of the innate immunity. Upon infection, autophagy is activated but key factors responsible remained unclear. Here, we show that interferon (IFN) released during viral infections subsequently activates autophagy via STAT1/5B-mediated upregulation of SOCS1. Our data shows that scavenging of IFNs diminishes autophagy induced by several respiratory viruses. All types of IFN (I, II and III) mediated robust autophagic flux activation in both cell lines and primary human lung fibroblasts in a JAK1-3 dependent manner. Depletion or pharmacological inhibition of individual STAT transcription factors demonstrated that both STAT1 and STAT5B are required for IFN-induced autophagy. Upon IFN stimulation STAT1 and STAT5B associate and translocate to the nucleus. Transcriptome analyses revealed that inhibition of STAT5 only reduces expression of a subset of IFN-stimulated genes, whereas most known anti-viral IFN-stimulated genes remain induced to high levels. Among the STAT5 dependent genes was Suppressor of Cytokine Signaling 1 (SOCS1). Overexpression of SOCS1 stimulated autophagy, whereas its depletion impaired IFN-induced autophagy. Successful viruses like measles virus (MeV) or respiratory syncytial viruses (RSV) evolved strategies to exploit autophagy to promote their own replication. Uncoupling IFN-mediated ISG defenses from autophagy induction using by STAT5 inhibition reduced virus-induced autophagy, and inhibited efficient replication of autophagy-dependent MeV and RSV. Taken together, our data show that IFN promotes autophagy via STAT1/STAT5B-SOCS1 in viral infections and reveal that targeting of this axis allows inhibition of autophagy-dependent viruses without compromising innate immune defenses. Author summaryAutophagy, like the Interferon (IFN) system, is considered part of the innate immune defenses that are activated upon viral infection. However, some viruses--like measles (MeV) and respiratory syncytial virus (RSV)--have learned to hijack this process to promote their replication. Our study shows that autophagy activation upon viral infections is promoted by the IFN system. Consequently, blocking IFNs reduced virus-induced autophagy. Mechanistic analyses demonstrated that various types of IFN induce expression of SOCS1, via the transcription factors STAT1 and STAT5B in cell lines and primary human lung cells. Pharmacological inhibition or depletion of STAT1, STAT5B or SOCS1 impaired IFN-induced autophagy. Importantly, disrupting STAT5 signaling does not impact IFN-mediated induction of the large majority of ISGs. Thus, selective disruption of IFN-induced autophagy by inhibiting STAT5 restricted replication of MeV and RSV. Our findings reveal an underappreciated mechanistic link between the IFN system and autophagy and suggest that targeting of the STAT5-SOCS1 axis may inhibit autophagy-dependent viruses without compromising IFN-mediated defenses.

microbiology↗
bioRxiv2025

Replication-competent SIVcpz CRISPR screen identifies barriers to successful cross-species transmission

Simian immunodeficiency viruses (SIVs) have crossed from apes to humans at least four times, but only one event gave rise to the AIDS pandemic. The host barriers that pandemic HIV-1 group M (major) strains overcame to spread efficiently in humans remain poorly understood. To identify such barriers, we performed CRISPR-Cas9 screens driven by the replication efficiency of SIVcpz, the chimpanzee precursor of HIV-1. Guide RNA libraries targeting more than 500 human genes encoding potential antiviral factors were inserted into the replication-competent SIVcpz MB897 molecular clone, which is phylogenetically closely related to HIV-1 group M strains. Propagation in Cas9-expressing human SupT1 T cells significantly enriched for sgRNAs targeting ADAR, AXIN1, CEACAM3, CD72, EHMT2, GRN, HMOX1, HMGA1, ICAM2, CD72, IFITM2, MEFV, PCED1B, SGOL2, SMARCA4, SUMO1 and TMEM173. These hits only partially overlapped with those identified in analogous HIV-1-based screens, indicating virus-specific restriction profiles. Functional analyses confirmed that IFITM2 (interferon-induced transmembrane protein 2), PCED1B (PC-esterase domain-containing protein 1B), MEFV (Mediterranean fever protein, pyrin/TRIM20), and AXIN1 (Axis inhibition protein 1), restrict replication of SIVcpz but not of HIV-1 group M strains in primary human CD4 T cells. These findings reveal previously unrecognized host factors that limit SIVcpz replication in human cells and highlight barriers that HIV-1 likely overcame during its adaptation for pandemic spread. One Sentence SummaryCRISPR screens with replication-competent SIVcpz identify human antiviral factors limiting efficient viral replication after zoonotic transmission.

immunology↗
bioRxiv2024

Back to the horns: a reconstruction of the ancestral horn state through distinct types of recombination events.

BackgroundBreeding of the genetically polled animals is the desirable approach in modern cattle husbandry. At least four different genetic variants associated with polledness in cattle have been identified, suggesting genetic heterogeneity. These dominant variants have been identified on chromosome 1 between the regions of approx. 2.42 to 2.73 Mb (reference: ARS-UCD1.2), also called the POLLED locus. Among these variants, Friesian (PF, 80 kbp duplication) and Celtic (PC, 212 bp complex InDel) are the most observed in the majority of breeds in the production systems globally, such as in Holstein-Friesian (HF) and Fleckvieh (FV). While a putative causal association of PC with polledness is proven, the presence of large duplication in PF makes it difficult to prove the causality. ResultsIn this study, we conduct whole-genome sequencing (WGS) analysis of two trios exhibiting unexpected inheritance patterns related to PC and PF variants. In both instances, horned offspring were produced from mating pairs where one parent was homozygous for the polled variant, and the other was homozygous for the ancestral horned variant. By analyzing the WGS data generated using long-read sequence technology, we show that de-novo generation of the ancestral horned variant in both the offspring was the result of distinct recombination events. Specifically, in case of the HF trio, it was the result of non-allelic homologous recombination in the gametes of the sire (PF/PF), while in case of the FV trio, it was the result of allelic homologous recombination in the gametes of the dam (PC/PF). The findings from the HF trio support the hypothesis that the 80-kbp duplication is the genetic variant responsible for the polled phenotype of Friesian origin. ConclusionHere we show that different genomic arrangements in POLLED locus can lead to the emergence of de-novo ancestral horn phenotypes. These kinds of arrangements can make a reliable gene test less reliable and the derivation of the phenotype difficult to predict. Therefore, it is important for the large POLLED-locus that any deviation from the expected result is critically analyzed. Possibly some of these cases can further narrow down the sequence motif that is essential for polledness in cattle.

genomics↗
bioRxiv2023

Traitor-virus-guided discovery of novel antiviral factors

Complex pathogen-host interactions govern the outcome of viral exposures but remain poorly understood because current methods to elucidate antiviral mechanisms are prone to artefacts and lack sensitivity. Here, we developed a virus-guided technology platform where the pathogen itself reveals its cellular opponents. To accomplish this, we engineered replication-competent HIV-1 expressing sgRNAs targeting potential antiviral genes in Cas9-expressing CD4+ T cells. Simultaneous analysis of HIV-1 constructs targeting >500 candidate genes revealed that sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B, RHOA and HMOX1 are strongly enriched over several rounds of replication. Overexpression and knock-out studies confirmed the antiretroviral activity of most factors but failed for some. Finally, we show that lack of the accessory nef gene increased enrichment of sgRNAs targeting SERINC5 and IFI16 demonstrating that this method allows identification of targets of accessory proteins. The versatile and effective HIV-guided CRISPR technology offers numerous possibilities for clarification of virus-host interactions and innate defense mechanisms.

microbiology↗
bioRxiv2021

The Delta variant of SARS-CoV-2 maintains high sensitivity to interferons in human lung cells

Interferons are a major part of the anti-viral innate defense system. Successful pathogens, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), need to overcome these defenses to establish an infection. Early induction of interferons (IFNs) protects against severe coronavirus disease 2019 (COVID-19). In line with this, SARS-CoV-2 is inhibited by IFNs in vitro, and IFN-based therapies against COVID-19 are investigated in clinical trials. However, SARS-CoV-2 continues to adapt to the human population resulting in the emergence of variants characterized by increased transmission fitness and/or decreased sensitivity to preventive or therapeutic measures. It has been suggested that the efficient spread of these so-called "Variants of Concern" (VOCs) may also involve reduced sensitivity to IFNs. Here, we examined whether the four current VOCs (Alpha, Beta, Gamma and Delta) differ in replication efficiency or IFN sensitivity from an early isolate of SARS-CoV-2. All viruses replicated in a human lung cell line and in iPSC-derived alveolar type II cells (iAT2). The Delta variant showed accelerated replication kinetics and higher infectious virus production compared to the early 2020 isolate. Replication of all SARS-CoV-2 VOCs was reduced in the presence of exogenous type I, II and III IFNs. On average, the Alpha variant was the least susceptible to IFNs and the Alpha, Beta and Gamma variants show increased resistance against type III IFN. Although the Delta variant has outcompeted all other variants in humans it remained as sensitive to IFNs as an early 2020 SARS-CoV-2 isolate. This suggests that increased replication fitness rather than IFN resistance may be a reason for its dominance. Our results may help to understand changes in innate immune susceptibility of VOCs, and inform clinical trials exploring IFN-based COVID-19 therapies.

microbiology↗
bioRxiv2017

AtRsgA from Arabidopsis thaliana controls maturation of the small subunit of the chloroplast ribosome

SummaryPlastid ribosomes are very similar in structure and function to ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favourable at biological conditions, it requires activity of many assembly factors. Here, we have characterized a homolog of bacterial rsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA-sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were upregulated, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signalling pathways. To conclude, this study reveals a new chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function.\n\nSignificance statementAtRsgA is an assembly factor necessary for maturation of the small subunit of the chloroplast ribosome. Depletion of AtRsgA leads to dwarfed, chlorotic plants and smaller, but more numerous chloroplasts. Large-scale transcriptomic and proteomic analysis revealed that chloroplast-encoded and - targeted proteins were less abundant, while the corresponding transcripts were upregulated in the mutant. We analyse the transcriptional responses of several retrograde signalling pathways to suggest a mechanism underlying this compensatory response.

molecular biology↗
bioRxiv2017

Photoperiodic control of the Arabidopsis proteome reveals a translational coincidence mechanism

Plants respond to seasonal cues, such as the photoperiod, to adapt to current conditions and to prepare for environmental changes in the season to come. To assess photoperiodic responses at the protein level, we quantified the proteome of the model plant Arabidopsis thaliana by mass spectrometry across four photoperiods. This revealed coordinated changes of abundance in proteins of photosynthesis, primary and secondary metabolism, including pigment biosynthesis, consistent with higher metabolic activity in long photoperiods. Higher translation rates in the daytime than the night likely contribute to these changes via rhythmic changes in RNA abundance. Photoperiodic control of protein levels might be greatest only if high translation rates coincide with high transcript levels in some photoperiods. We term this proposed mechanism {acute}translational coincidence{acute}, mathematically model its components, and demonstrate its effect on the Arabidopsis proteome. Datasets from a green alga and a cyanobacterium suggest that translational coincidence contributes to seasonal control of the proteome in many phototrophic organisms. This may explain why many transcripts but not their cognate proteins exhibit diurnal rhythms.

plant biology↗