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Jung, M.

Publications and source records attributed to Jung, M..

71 records · Page 4Linked to original sources

Cell cycle gene regulation dynamics revealed by RNA velocity and deep-learning

The cell cycle is a fundamental process of life, however, a quantitative understanding of gene regulation dynamics in the context of the cell cycle is still far from complete. Single-cell RNA-sequencing (scRNA-seq) technology gives access to its dynamics without externally perturbing the cell. Here, we build a high-resolution map of the cell cycle transcriptome based on scRNA-seq and deep-learning. By generating scRNA-seq libraries with high depth, in mouse embryonic stem cells and human fibroblasts, we are able to observe cycling patterns in the unspliced-spliced RNA space for single genes. Since existing methods in scRNA-seq are not efficient to measure cycling gene dynamics, we propose a deep learning approach to fit these cycling patterns sorting single cells across the cell cycle. We characterize the cell cycle in asynchronous pluripotent and differentiated cells identifying major waves of transcription during the G1 phase and systematically study the G1-G0 transition where the cells exit the cycle. Our work presents to the scientific community a broader understanding of RNA velocity and cell cycle maps, that we applied to pluripotency and differentiation. Our approach will facilitate the study of the cell cycle in multiple cellular models and different biological contexts, such as cancer and development.

systems biology↗

Transcriptional silencing of ALDH2 in acute myeloid leukemia confers a dependency on Fanconi anemia proteins

Hundreds of genes become aberrantly silenced in acute myeloid leukemia (AML), with most of these epigenetic changes being of unknown functional consequence. Here, we demonstrate how gene silencing can lead to an acquired dependency on the DNA repair machinery in AML. We make this observation by profiling the essentiality of the ubiquitin conjugation and ligation machinery in cancer cell lines using domain-focused CRISPR screening, which revealed Fanconi anemia (FA) proteins UBE2T (an E2) and FANCL (an E3) as unique dependencies in AML. We demonstrate that these dependencies are due to a synthetic lethal interaction between FA proteins and Aldehyde Dehydrogenase 2 (ALDH2), which function in parallel pathways to counteract the genotoxic effects of endogenous aldehydes. We provide evidence that DNA hypermethylation and transcriptional silencing of ALDH2 occur in a recurrent manner in human AML patient samples, which is sufficient to confer FA pathway dependency in this disease. Taken together, our study suggests that targeting of the ubiquitination reaction catalyzed by FA proteins can eliminate ALDH2-deficient AML.

cancer biology↗

Two Piwis with Ago-like functions silence somatic genes at the chromatin level

Most sRNA biogenesis mechanisms involve either RNAseIII cleavage or ping-pong amplification by different Piwi proteins harboring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from endogenous remote loci. Our data does not indicate any signatures from ping-pong amplification but Dicer cleavage of long dsRNA. We show that Ptiwi13 and 14 have different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and Ptiwi14 loaded siRNAs show a 5'-U signature. Both Ptiwis show in addition a general preference for Uridine-rich sRNAs along the entire sRNA length. Our data indicates both Ptiwis and 2-O-methylation to contribute to strand selection of Dicer cleaved siRNAs. This unexpected function of two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. As both Ptiwis show differential subcellular localisation, Ptiwi13 in the cytoplasm and Ptiwi14 in the vegetative macronucleus, we conclude that cytosolic and nuclear silencing factors are necessary for efficient chromatin silencing.

genetics↗

Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility

PEX14 functions as peroxisomal docking protein for the import receptor PEX5. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human {beta}-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the {beta}-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.

biochemistry↗

Fatty acid oxidation makes the difference: Lineage-selective disturbance by DEHP via ROS in myeloid hematopoiesis

Endocrine disrupting compounds (EDC) are ubiquitous in the human environment, displaying a highly relevant research topic. The impact of EDC on the differentiation of primitive cells, e.g. in hematopoiesis, is of particular interest. We found profound inhibitory effects of di-2-ethylhexyl phthalate (DEHP) on erythropoiesis and dendropoiesis, mediated via reactive oxygen species (ROS) generation. Neutrophil differentiation, however, was not affected by DEHP. ROS leads to a shift from glycolysis to the pentose phosphate pathway and diminishes ATP generation from glycolysis, ultimately resulting in apoptosis in both cell types. In neutrophils, ATP generation is held constant by active fatty acid oxidation (FAO), rendering these cells highly resistant against ROS. This relationship also holds true in HUVEC and HepG2 cells, also in combination with other organic peroxides. We, therefore, uncover a key mechanism for ROS quenching which further explains the distinct ROS quenching ability of different tissues.

pharmacology and toxicology↗

Genome-Enabled Discovery of Anthraquinone Biosynthesis in Senna tora

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we sequenced and annotated the genome of S. tora at the chromosome level with contig N50 and super-scaffold N50 of 4.03 Mb and 41.7 Mb. Comparison among related plant species showed that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora. Combining genomics, transcriptomics, metabolomics, and biochemistry, we identified a CHS-L responsible for biosynthesis of anthraquinones, the first example in plants. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants. One Sentence SummaryThe chromosome-scale reference genome of a medicinal plant Senna tora, transcriptomics, metabolomics, and biochemical analysis provide new insights into anthraquinone biosynthesis in plants.

plant biology↗

Areas of global importance for terrestrial biodiversity, carbon, and water

Summary paragraphTo meet the ambitious objectives of biodiversity and climate conventions, countries and the international community require clarity on how these objectives can be operationalized spatially, and multiple targets be pursued concurrently1. To support governments and political conventions, spatial guidance is needed to identify which areas should be managed for conservation to generate the greatest synergies between biodiversity and natures contribution to people (NCP). Here we present results from a joint optimization that maximizes improvements in species conservation status, carbon retention and water provisioning and rank terrestrial conservation priorities globally. We found that, selecting the top-ranked 30% (respectively 50%) of areas would conserve 62.4% (86.8%) of the estimated total carbon stock and 67.8% (90.7%) of all clean water provisioning, in addition to improving the conservation status for 69.7% (83.8%) of all species considered. If priority was given to biodiversity only, managing 30% of optimally located land area for conservation may be sufficient to improve the conservation status of 86.3% of plant and vertebrate species on Earth. Our results provide a global baseline on where land could be managed for conservation. We discuss how such a spatial prioritisation framework can support the implementation of the biodiversity and climate conventions.

ecology↗

Alternative splicing switches STIM1 targeting to specialized membrane contact sites and modifies SOCE

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator molecule of store-operated Ca2+ entry (SOCE) and a sorting regulator of certain ER proteins such as Stimulator of interferon genes (STING). Here, we characterize a conserved new variant, Stim1A, where splice-insertion translates into an additional C-terminal domain. We find prominent expression of exonA mRNA in testes, astrocytes, kidney and heart and confirm Stim1A protein in Western blot of testes. In situ, endogenous Stim1 with domain A, but not Stim1 without domain A localizes to unique adhesion junctions and to specialized membrane retrieval sites (tubulobulbar complexes) in testes. Functionally, using Ca2+ imaging and patch-clamp analysis, Stim1A shows a dominant-negative effect on SOCE and ICRAC, despite normal clustering and interaction with Orai1 investigated by combined TIRF and FRET analyses and as confirmed by an increased SOCE upon knock-down of endogenous Stim1A in astrocytes. Mutational analyses in conjunction with imaging and patch-clamp analyses of residues either in domain A or within the N-terminal region of Orai1 demonstrate a specific defect in stabilized channel gating. Our findings demonstrate that cell-type specific splicing of STIM1 adds both an intracellular targeting switch and adapts SOCE to meet the Ca2+ requirements of specific subcellular contact sites.

molecular biology↗

Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here we use three live-cell imaging approaches to assess dimerization of ORs at different expression levels. At high membrane densities, a split GFP assay reveals that {kappa}OR dimerizes, while OR and {delta}OR stay monomeric. In contrast, single-molecule imaging showed no {kappa}OR dimers at low receptor densities. To reconcile our seemingly contradictory results, we used a high-density single-molecule assay to assess membrane protein interactions at densities up to 100x higher than conventional single-molecule imaging. We observe that {kappa}OR is monomeric at low densities and forms dimers at densities that are considered physiological. In contrast, OR and {delta}OR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting {kappa}OR dimers but not higher-order aggregates suggests that ORs dimerize through a single, specific interface.

biochemistry↗

Finding the neural correlates of collaboration using a three-person fMRI hyperscanning paradigm

Humans have an extraordinary ability to interact and cooperate with others, which plays a pivotal role in societies at large. Despite its potential social and evolutionary significance, research on finding the neural correlates of collaboration has been limited partly due to restrictions on simultaneous neuroimaging of more than one participant (a.k.a. hyperscanning). A series of works now exists that used dyadic fMRI hyperscanning to examine the interaction between two participants. However, to our knowledge, no study to date has aimed at revealing the neural correlates of social interactions using a 3-person (or triadic) fMRI hyperscanning paradigm. Here, for the first time, we simultaneously measured the blood-oxygenation-level-dependent (BOLD) signal of triads (m=12 triads; n=36 participants), while they engaged in a joint drawing task based on the social game of Pictionary(R). General linear model (GLM) analysis revealed increased activation in the brain regions previously linked with the theory of mind (ToM) during the collaborative phase compared to the independent phase of the task. Furthermore, using intersubject brain synchronization (IBS) analysis, we revealed increased synchrony of the right temporo-parietal junction (R TPJ) during the collaborative phase. The increased synchrony in the R TPJ was observed to be positively associated with the overall team performance on the task. In sum, our novel paradigm revealed a vital role of the R TPJ among other ToM regions during a triadic collaborative drawing task.

neuroscience↗

Clinical severity in Fanconi anemia correlates with residual function of FANCB missense variants

Fanconi anemia (FA) is the most common genetic cause of bone marrow failure, and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry (IFAR). Those with FANCB deletion or truncation demonstrate earlier than average onset of bone marrow failure, and more severe congenital abnormalities compared to a large series of FA individuals in the published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization was associated with two missence variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays showed earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is linked to the extent of residual FANCD2 monoubiquitination.\n\nKEY POINTSO_LIX-linked FANCB pathogenic variants predominantly cause acute, early onset bone marrow failure and severe congenital abnormalities\nC_LIO_LIBiochemical and cell-based assays with patient variants reveal functional properties of FANCB that associate with clinical severity\nC_LI

molecular biology↗

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a novel target of NDGA and provide mechanistic insight into its role in longevity.

cell biology↗

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Cas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity but at the cost of the on-target activity. DNA unwinding is the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two guanines added were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5 end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

biophysics↗

Landscape of tumor-infiltrating immune cells in human immunogenic cancers: B cells are inversely associated with CD8T cells, but positively correlated with Treg cells

The composition of tumor-infiltrating immune cells may be a strong predictor of cancer treatment responses and survival outcomes. While B cells have been suggested to suppress T cell cytotoxicity in preclinical studies, it has been less understood whether B cells will exert immune-regulatory roles in human cancers. We explored immune cell landscapes in six human immunogenic cancers, including bladder cancer, head and neck cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, and renal cell carcinoma by calculating gene expression patterns of immune cell-specific metagenes in a total of 2951 cancers. We demonstrated that tumor-infiltrating activated B cells was correlated with regulatory T cell (Treg) infiltration, but had an inverse association with activated CD8 T cell infiltration consistently across all six human cancers. Tumors infiltrated by activated B cells (ActB+ tumors) demonstrated an elevated expression of regulatory cytokines and immune checkpoints, compared to tumors without infiltration by activated B cells (ActB-tumors). Activated B infiltration was not significantly associated with survival outcomes.\n\nPrecisThis human cancer tissue analysis showed that tumor infiltration by activated B cells correlates with decreased infiltration by activated CD8 T cells in immunogenic solid tumors, implicating B cell inhibition may enhance T cell-mediated cytotoxicity.

cancer biology↗

Unified single-cell analysis of testis gene regulation and pathology in 5 mouse strains

By removing the confounding factor of cellular heterogeneity, single cell genomics can revolutionize the study of development and disease, but methods are needed to simplify comparison among individuals. To develop such a framework, we assayed the transcriptome in 62,600 single cells from the testes of wildtype mice, and mice with gonadal defects due to disruption of the genes Mlh3, Hormad1, Cul4a or Cnp. The resulting expression atlas of distinct cell clusters revealed novel markers and new insights into testis gene regulation. By jointly analysing mutant and wildtype cells using a model-based factor analysis method, SDA, we decomposed our data into 46 components that identify novel meiotic gene regulatory programmes, mutant-specific pathological processes, and technical effects. Moreover, we identify, de novo, DNA sequence motifs associated with each component, and show that SDA can be used to impute expression values from single cell data. Analysis of SDA components also led us to identify a rare population of macrophages within the seminiferous tubules of Mlh3-/- and Hormad1-/- testes, an area typically associated with immune privilege. We provide a web application to enable interactive exploration of testis gene expression and components at http://www.stats.ox.ac.uk/~wells/testisAtlas.html

genomics↗

Brain Connectivity Tracks Effects of Chemotherapy Separately from Behavioral Measures

Several studies in cancer research have suggested that cognitive dysfunction following chemotherapy, referred to in lay terms as \"chemobrain\", is a serious problem. At present, the changes in integrative brain function that underlie such dysfunction remains poorly understood. Recent developments in neuroimaging suggest that patterns of functional connectivity can provide a broadly applicable neuromarker of cognitive performance and other psychometric measures. The current study used multivariate analysis methods to identify patterns of disruption in resting state functional connectivity of the brain due to chemotherapy and the degree to which the disruptions can be linked to behavioral measures of distress and cognitive performance. Sixty two women (22 healthy control, 18 patients treated with adjuvant chemotherapy, and 22 treated without chemotherapy) were evaluated with neurocognitive measures followed by self-report questionnaires and open eyes resting-state fMRI scanning at three time points: diagnosis (M0, pre-adjuvant treatment), at least 1 month (M1), and 7 months (M7) after treatment. The results indicated deficits in cognitive health of breast cancer patients immediately after chemotherapy that improved over time. This psychological trajectory was paralleled by a disruption and later recovery of resting-state functional connectivity, mostly in the parietal and frontal brain regions. The functional connectivity alteration pattern seems to be a separable treatment symptom from the decreased cognitive health. More targeted support for patients should be developed to ameliorate these multi-faceted side effects of chemotherapy treatment on neural functioning and cognitive health.

neuroscience↗

Directed evolution of CRISPR-Cas9 to increase its specificity

The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we have used a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without sacrificing on-target activities in human cells.

biochemistry↗