Biology subjects

Zaugg, J. B.

Publications and source records attributed to Zaugg, J. B..

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bioRxiv2024

Integration of genetic and epigenetic data pinpoints autoimmune specific remodelling of enhancer landscape in CD4+ T cells

CD4+ T cells play a crucial role in adaptive immune responses and have been implicated in the pathogenesis of autoimmune diseases (ADs). Despite numerous studies, the molecular mechanisms underlying T cell dysregulation in ADs remain incompletely understood. Here, we used transcriptomic and epigenomic data from CD4+ T cells of healthy donors and patients with systemic lupus erythematosus (SLE), psoriasis, juvenile idiopathic arthritis (JIA), and Graves disease to investigate the role of enhancers in AD pathogenesis. By generating enhancer-based gene regulatory networks (eGRNs), we identified disease-specific dysregulated pathways and potential downstream target genes of enhancers harbouring AD-associated single-nucleotide polymorphisms, which we also validated using CRISPRi in primary CD4+ T cells. Our results suggest that alterations in the regulatory landscapes of CD4+ T cells, including enhancers, contribute to the development of ADs and provide a basis for developing new therapeutic approaches.

genomics↗

bioRxiv2023

Scalable ultra-high-throughput single-cell chromatin and RNA sequencing reveals gene regulatory dynamics linking macrophage polarization to autoimmune disease

Enhancers and transcription factors (TFs) are crucial in regulating cellular processes, including disease-associated cell states. Current multiomic technologies to study these elements in gene regulatory mechanisms lack multiplexing capability and scalability. Here, we present SUM-seq, a cost-effective, scalable Single-cell Ultra-high-throughput Multiomic sequencing method for co-assaying chromatin accessibility and gene expression in single nuclei. SUM-seq enables profiling hundreds of samples at the million cell scale and outperforms current high-throughput single-cell methods. We applied SUM-seq to dissect the gene regulatory mechanisms governing macrophage polarization and explored their link to traits from genome-wide association studies (GWAS). Our analyses confirmed known TFs orchestrating M1 and M2 macrophage programs, unveiled key regulators, and demonstrated extensive enhancer rewiring. Integration with GWAS data further pinpointed the impact of specific TFs on a set of immune traits. Notably, inferred enhancers regulated by the STAT1/STAT2/IRF9 (ISGF3) complex were enriched for genetic variants associated with Crohns disease, ulcerative colitis and multiple sclerosis, and their target genes included known drug targets. This highlights the potential of SUM-seq for dissecting molecular disease mechanisms. SUM-seq offers a cost-effective, scalable solution for ultra-high-throughput single-cell multiomic sequencing, excelling in unraveling complex gene regulatory networks in cell differentiation, responses to perturbations, and disease studies.

genomics↗

bioRxiv2023

A single-cell multi-omic and spatial atlas of nodal B-cell lymphomas reveals B-cell maturation drives intratumor heterogeneity

Intratumor heterogeneity underpins cancer pathogenesis and evolution, although it is typically considered independent from the differentiation processes that drive physiological cell-type diversity. As cancer types and subtypes arise from different cell types, we investigated whether cellular differentiation influences intratumor heterogeneity. Nodal B-cell non-Hodgkin lymphomas are a diverse set of cancers originating from different stages of B-cell maturation. Through single-cell transcriptome and surface epitope profiling (CITE-Seq) of diffuse large B-cell, mantle cell, follicular, and marginal zone lymphomas in addition to reactive lymph nodes from 51 patients, we found multiple B-cell maturation states within tumors. Intratumor maturation states emerged from the same clone, revealing divergent differentiation from a shared cell of origin. Maturation state composition varied across subtypes and tumors, which encompassed mixed cell-of-origin diagnostic subtypes. Through highly multiplexed immunohistochemistry (CODEX) of samples from 19 of these patients, we found that intratumor maturation states inhabited distinct spatial niches, displaying cellular interactions and regulatory networks typical of their maturation states while harboring different genetic variants. By deconvoluting intratumor maturation states from a microarray dataset of 507 patients, we identified risk groups within diagnoses with striking differences in survival, including IgM memory-enriched germinal center B-cell (M = 1.9 vs >10 years, p = 0.00039) and activated B-cell (M = 2.4 vs 9.6 years, p = 0.016) diffuse large B-cell lymphoma, and dark zone-enriched follicular lymphoma (M = 8.6 vs 13 years; p = 0.0019). Our findings reveal cellular differentiation remains plastic in B-cell lymphomas, driving tumor variation, evolution, and response. Key PointsO_LICellular differentiation remains plastic in B-cell lymphomas, driving tumor variation, evolution, and response. C_LIO_LIIntratumor maturation states occupy unique immune niches, harbor distinct genetic variants, and are tied to different survival outcomes. C_LI

cancer biology↗

bioRxiv2023

HIPSD&R-seq enables scalable genomic copy number and transcriptome profiling

Single-cell DNA-sequencing (scDNA-seq) enables decoding somatic cancer variation. Existing methods are hampered by low throughput or cannot be combined with transcriptome sequencing in the same cell. We propose HIPSD&R-seq (HIgh-throughPut Single-cell Dna and Rna-seq), a scalable yet simple assay to profile low-coverage DNA and RNA in thousands of cells in parallel. Our approach builds on an accessible modification of the 10X Genomics platform for scATAC and multiome profiling. In applications to human cell models and primary tissue, we demonstrate the feasibility to detect rare clones and we combine the assay with combinatorial indexing to profile over 16,000 cells.

cancer biology↗

bioRxiv2023

Histone H3.3 lysine 9 and 27 control repressive chromatin states at cryptic cis-regulatory elements and bivalent promoters in mouse embryonic stem cells

Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutated histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleashed cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupted deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal level of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs.

genomics↗

bioRxiv2022

Convolutional networks for supervised mining of molecular patterns within cellular context

Cryo-electron tomograms capture a wealth of structural information on the molecular constituents of cells and tissues. We present DeePiCt (Deep Picker in Context), an open-source deep-learning framework for supervised structure segmentation and macromolecular complex localization in cellular cryo-electron tomography. To train and benchmark DeePiCt on experimental data, we comprehensively annotated 20 tomograms of Schizosaccharomyces pombe for ribosomes, fatty acid synthases, membranes, nuclear pore complexes, organelles and cytosol. By comparing our method to state-of-the-art approaches on this dataset, we show its unique ability to identify low-abundance and low-density complexes. We use DeePiCt to study compositionally-distinct subpopulations of cellular ribosomes, with emphasis on their contextual association with mitochondria and the endoplasmic reticulum. Finally, by applying pre-trained networks to a HeLa cell dataset, we demonstrate that DeePiCt achieves high-quality predictions in unseen datasets from different biological species in a matter of minutes. The comprehensively annotated experimental data and pre-trained networks are provided for immediate exploitation by the community.

bioinformatics↗

bioRxiv2021

GRaNIE and GRaNPA: Inference and evaluation of enhancer-mediated gene regulatory networks applied to study macrophages

Among the biggest challenges in the post-GWAS (genome-wide association studies) era is the interpretation of disease-associated genetic variants in non-coding genomic regions. Enhancers have emerged as key players in mediating the effect of genetic variants on complex traits and diseases. Their activity is regulated by a combination of transcription factors (TFs), epigenetic changes and genetic variants. Several approaches exist to link enhancers to their target genes, and others that infer TF-gene connections. However, we currently lack a framework that systematically integrates enhancers into TF-gene regulatory networks. Furthermore, we lack an unbiased way of assessing whether inferred regulatory interactions are biologically meaningful. Here we present two methods, implemented as user-friendly R packages: GRaNIE (Gene Regulatory Network Inference including Enhancers) for building enhancer-based gene regulatory networks (eGRNs) and GRaNPA (Gene Regulatory Network Performance Analysis) for evaluating GRNs. GRaNIE jointly infers TF-enhancer, enhancer-gene and TF-gene interactions by integrating open chromatin data such as ATAC-Seq or H3K27ac with RNA-seq across a set of samples (e.g. individuals), and optionally also Hi-C data. GRaNPA is a general framework for evaluating the biological relevance of TF-gene GRNs by assessing their performance for predicting cell-type specific differential expression. We demonstrate the power of our tool-suite by investigating gene regulatory mechanisms in macrophages that underlie their response to infection and cancer, their involvement in common genetic diseases including autoimmune diseases, and identify the TF PURA as putative regulator of pro-inflammatory macrophage polarisation. Availability- GRaNIE: https://bioconductor.org/packages/release/bioc/html/GRaNIE.html - GRaNPA: https://git.embl.de/grp-zaugg/GRaNPA Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

genomics↗

bioRxiv2021

Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition

Recent progress in the generation of bona-fide Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding in detail this developmental process. Here, we sought to elucidate the function of the hematopoietic regulators Tal1, Lmo2 and Lyl1 in the Endothelial to Hematopoietic Transition (EHT), the process through which HSPCs are generated from endothelial precursors during embryogenesis. We used a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development, and combined gain-of-function experiments in sorted vascular smooth muscle cells (VSM) with multi-omics to obtain mechanistic insights into the mode of action of Tal1, Lmo2 and Lyl1. We found that these factors promote the silencing of the VSM transcriptional program and the activation of the hematopoietic one. Through this approach and the use of a Tet-on system to control the expression of Tal1 during hematopoietic specification from mESCs, we discovered that its expression in endothelial cells is crucial for the EHT to occur.

developmental biology↗

bioRxiv2021

Enhancer-priming in ageing human bone marrow mesenchymal stromal cells contributes to immune traits

Bone marrow mesenchymal stromal cells (BMSCs) can differentiate into adipocytes and osteoblasts, and are important regulators of the haematopoietic system. Ageing associates with an increased ratio of bone marrow adipocytes to osteoblasts and immune dysregulation. Here, we carried out an integrative multiomics analysis of ATAC-Seq, RNA-Seq and proteomics data from primary human BMSCs in a healthy cohort age between 20 - 60. We identified age-sensitive elements uniquely affecting each molecular level where transcription is mostly spared, and characterised the underlying biological pathways, revealing the interplay of age-related gene expression mechanism changes spanning multiple gene regulatory layers. Through data integration with enhancer-mediated gene regulatory network analysis, we discovered that enhancers and transcription factors influence cell differentiation potential in the ageing BMSCs. By combining our results with genome-wide association study data, we found that age-specific changes could contribute to common traits related to BMSC-derived tissues such as bone and adipose tissue, and to immune-related traits on a systemic level such as asthma. We demonstrate here that a multiomics approach is crucial for unravelling complex information, providing new insights on how ageing contributes to bone marrow- and immune-related disorders.

systems biology↗

bioRxiv2021

Pathological LSD1 mutations cause HDAC-mediated aberrant gene repression during early cell differentiation

Lysine-specific demethylase 1 (LSD1/KDM1A) demethylates both histone and non-histone substrates, recruits repressive chromatin complexes, and is increased in cancers. De novo LSD1 mutations impairing protein function lead to a rare developmental disorder, but the molecular details of the pathology remains unclear. Using patient-derived fibroblasts, reprogrammed pluripotent stem cells, and differentiated cells, we found over 4000 differentially expressed genes and 68 transcription factors (TFs) whose motif accessibilities changed upon LSD1 mutation. An enhancer-mediated gene regulatory network approach identified transcriptional repressors with impaired activity in fibroblast and stem cells, leading to erroneous activation of their target genes. We also revealed overall decreases in TF target gene expression during early lineage differentiation of LSD1 mutant stem cells, likely caused by increased activity of repressive histone deacetylases (HDACs), co-factors of LSD1. Indeed, an HDACs inhibitor restored changes in gene expression including downregulation phenotype. Our findings characterize the molecular pathogenesis of LSD1 mutations and suggest potential therapeutic strategies for the developmental disorder and cancers caused by LSD1 dysregulations.

genomics↗

bioRxiv2021

Mapping drug-microenvironment-genetic interplay in CLL reveals trisomy 12 as a modulator of microenvironmental signals

The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited to small scale studies and systematic analyses are lacking. We chose Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in adults, as a model disease to study this complex interplay systematically. We performed a combinatorial assay using 12 drugs individually co-applied with each of 17 microenvironmental stimuli in 192 primary CLL samples, generating a comprehensive map of drug-microenvironment interactions in CLL. This data was combined with whole-exome sequencing, DNA-methylation, RNA-sequencing and copy number variant annotation. Our assay identified four distinct CLL subgroups that differed in their responses to the panel of microenvironmental stimuli. These subgroups were characterized by distinct clinical outcomes independently of known prognostic markers. We investigated the effect of CLL- specific recurrent genetic alterations on microenvironmental responses and identified trisomy 12 as an amplifier of multiple microenvironmental stimuli. We further quantified the impact of microenvironmental stimuli on drug response, confirmed known interactions such as Interleukin (IL) 4 mediated resistance to B cell receptor (BCR) inhibitors, and identified new interactions such as Interferon-{gamma} induced resistance to BCR inhibitors. Finally, we identified interactions which were limited to genetic subgroups. Resistance to chemotherapeutics, such as Fludarabine, induced by Toll-Like Receptor (TLR) agonists could be observed in IGHV unmutated patient samples and IGHV mutated samples with trisomy 12. In-vivo relevance was investigated in CLL-infiltrated lymph nodes, which showed increased IL4 and TLR signalling activity compared to healthy samples (p<0.001). High IL4 activity in lymph nodes correlated with faster disease progression (p=0.038). We provide a publicly available resource (www.dietrichlab.de/CLL_Microenvironment/) which uncovers tumour cell extrinsic influences on drug response and disease progression in CLL, and how these interactions are modulated by cell intrinsic molecular features.

cancer biology↗

bioRxiv2021

Comparative chromatin accessibility upon BDNF-induced neuronal activity delineates neuronal regulatory elements

Neuronal activity induced by brain-derived neurotrophic factor (BDNF) triggers gene expression, which is crucial for neuronal survival, differentiation, synaptic plasticity, memory formation, and neurocognitive health. However, its role in chromatin regulation is unclear. Here, using temporal profiling of chromatin accessibility and transcription in mouse primary cortical neurons upon either BDNF stimulation or depolarization (KCl), we identify features that define BDNF-specific chromatin-to-gene expression programs. Enhancer activation is an early event in the regulatory control of BDNF-treated neurons, where the bZIP motif-binding Fos protein pioneered chromatin opening and cooperated with co-regulatory transcription factors (Homeobox, EGRs, and CTCF) to induce transcription. Deleting cis-regulatory sequences decreased BDNF-mediated Arc expression, a regulator of synaptic plasticity. BDNF-induced accessible regions are linked to preferential exon usage by neurodevelopmental disorder-related genes and heritability of neuronal complex traits, which were validated in human iPSC-derived neurons. Thus, we provide a comprehensive view of BDNF-mediated genome regulatory features using comparative genomic approaches to dissect mammalian neuronal activity.

genomics↗

bioRxiv2020

Operation of a TCA cycle subnetwork in the mammalian nucleus

Nucleic acid and histone modifications critically depend on central metabolism for substrates and co-factors. Although a few enzymes related to the formation of these required metabolites have been reported in the nucleus, the corresponding metabolic pathways are considered to function elsewhere in the cell. Here we show that a substantial part of the mitochondrial tricarboxylic acid (TCA) cycle, the biosynthetic hub of epigenetic modification factors, is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass-spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins, supporting their nuclear location. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus warranting a revision of the canonical view on metabolic compartmentalization and gene expression regulation.

systems biology↗

bioRxiv2020

Plasmodium falciparum malaria drives epigenetic reprogramming of human monocytes toward a regulatory phenotype

In malaria-naive children and adults, Plasmodium falciparum-infected red blood cells (Pf-iRBCs) trigger fever and other symptoms of systemic inflammation. However, in endemic areas where individuals experience repeated Pf infections over many years, the risk of Pf-iRBC-triggered inflammatory symptoms decreases with cumulative Pf exposure. The molecular mechanisms underlying these clinical observations remain unclear. Age-stratified analyses of monocytes collected from uninfected, asymptomatic Malian individuals before the malaria season revealed an inverse relationship between age and Pf-iRBC-inducible inflammatory cytokine (IL-1{beta}, IL-6 and TNF) production, whereas Malian infants and malaria-naive U.S. adults produced similarly high levels of inflammatory cytokines. Accordingly, monocytes of Malian adults produced more IL-10 and expressed higher levels of the regulatory molecules CD163, CD206, Arginase-1 and TGM2. These observations were recapitulated in an in vitro system of monocyte to macrophage differentiation wherein macrophages re-exposed to Pf-iRBCs exhibited attenuated inflammatory cytokine responses and a corresponding decrease in the epigenetic marker of active gene transcription, H3K4me3, at inflammatory cytokine gene loci. Together these data indicate that Pf induces epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype that attenuates inflammatory responses during subsequent Pf exposure. These findings also suggest that past malaria exposure could mitigate monocyte-associated immunopathology induced by other pathogens such as SARS-CoV-2. Author SummaryThe malaria parasite is mosquito-transmitted and causes fever and other inflammatory symptoms while circulating in the bloodstream. However, in regions of high malaria transmission the parasite is less likely to cause fever as children age and enter adulthood, even though adults commonly have malaria parasites in their blood. Monocytes are cells of the innate immune system that secrete molecules that cause fever and inflammation when encountering microorganisms like malaria. Although inflammation is critical to initiating normal immune responses, too much inflammation can harm infected individuals. In Mali, we conducted a study of a malaria-exposed population from infants to adults and found that participants monocytes produced less inflammation as age increases, whereas monocytes of Malian infants and U.S. adults, who had never been exposed to malaria, both produced high levels of inflammatory molecules. Accordingly, monocytes exposed to malaria in the laboratory became less inflammatory when re-exposed to malaria again later, and these monocytes turned down their inflammatory genes. This study helps us understand how people become immune to inflammatory symptoms of malaria and may also help explain why people in malaria-endemic areas appear to be less susceptible to the harmful effects of inflammation caused by other pathogens such as SARS-CoV-2.

immunology↗

bioRxiv2020

APOBEC2 binds Chromatin and Represses Transcription during Myoblast Differentiation

The activation induced cytidine deaminase/apolipoprotein B editing complex (AID/APOBEC) family comprises several nucleic acid editors with roles ranging from antibody diversification to mRNA editing. APOBEC2, an evolutionarily conserved member of this family, has neither an established substrate nor a mechanism of action, however genetic evidence suggests functional relevance in tissues such as muscle. Here, we demonstrate that in muscle, APOBEC2 does not have any of the attributed molecular functions of the AID/APOBEC family, such as RNA editing, DNA demethylation, or DNA mutation. Instead, we show that APOBEC2 occupies chromatin at promoter regions of certain genes, whose expression is repressed during muscle cell differentiation. We further demonstrate that APOBEC2 on one hand binds promoter region DNA directly and in a sequence specific fashion, while on the other it interacts with HDAC transcriptional corepressor complexes. Therefore, APOBEC2, by actively repressing the expression of non-myogenesis pathway genes, plays a key role in enforcing the proper establishment of muscle cell fate.

molecular biology↗

bioRxiv2020

Integrative Single-cell RNA-Seq and ATAC-Seq Analysis of Human Foetal Liver and Bone Marrow Haematopoiesis

Regulation of haematopoiesis during human development remains poorly defined. Here, we applied single-cell (sc)RNA-Seq and scATAC-Seq analysis to over 8,000 human immunophenotypic blood cells from foetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream from haematopoietic stem cell/multipotent progenitors (HSC/MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSC/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSC/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental haematopoiesis in the context of blood pathologies and regenerative medicine.

cell biology↗

bioRxiv2020

Predictive features of gene expression variation reveal a mechanistic link between expression variation and differential expression

For most biological processes, organisms must respond to extrinsic cues, while maintaining essential gene expression programs. Although studied extensively in single cells, it is still unclear how variation is controlled in multicellular organisms. Here, we used a machine-learning approach to identify genomic features that are predictive of genes with high versus low variation in their expression across individuals, using bulk data to remove stochastic cell-to-cell variation. Using embryonic gene expression across 75 Drosophila isogenic lines, we identify features predictive of expression variation, while controlling for expression level. Genes with low variation fall into two classes, indicating they employ different mechanisms to maintain a robust expression. In contrast, genes with high variation seem to lack both types of stabilizing mechanisms. Applying the framework to human tissues from GTEx revealed similar predictive features, indicating that promoter architecture is an ancient mechanism to control expression variation. Remarkably, expression variation features could also predict differential expression upon stress in both Drosophila and human. Differential gene expression signatures may therefore be partially explained by genetically encoded gene-specific features, unrelated to the studied treatment.

genomics↗