Biology subjects

Grün, D.

Publications and source records attributed to Grün, D..

12 recordsLinked to original sources

bioRxiv2026

BARe-seq enables high-throughput dissection of cis-regulatory control of transcriptional bursting

Transcriptional bursts determine RNA output through two kinetic parameters: burst size and burst frequency. How cis-regulatory DNA encodes these kinetic parameters remains unclear, in part because existing approaches do not combine scalable sequence perturbation with allele-resolved burst inference. Here, we developed Bulk Allele Resolution Sequencing (BARe-seq), an allele-resolved massively parallel reporter assay that enables inference of transcriptional burst parameters from bulk sequencing. Applying BARe-seq to libraries of 1000 promoters and 1000 enhancers in Drosophila S2 cells revealed distinct kinetic properties of promoters and enhancers. Promoter-dependent mean expression was driven by both burst size and burst frequency: TATA-box promoters showed larger bursts, whereas DPE promoters showed higher burst frequency. In contrast, enhancer strength was primarily driven by burst frequency, although specific transcription factor motifs were also associated with burst size. Thus, BARe-seq dissects cis-regulatory control of transcriptional bursting and extends allele-resolved measurements to pooled reporter assays in bulk sequencing experiments.

molecular biology↗

bioRxiv2025

An immunobiliary single-cell atlas resolves crosstalk of type 2 cDCs and γδT cells in cholangitis

Background and aimsThe immunobiliary niche serves as a reservoir of tissue-resident immune cells, yet the role of unconventional T cells during cholangitis remains poorly understood. Here, we connect cell state dynamics of type 2 conventional dendritic cells (cDC2) in cholangitis with site-specific{gamma}{delta} T17 responses in liver and draining lymph nodes (LN). MethodsThe 0.1% Diethoxycarbonyl-1,4-Dihydrocollidine (DDC) diet was used to generate an immunobiliary DC- and{gamma}{delta} T-enriched mouse liver and LN single-cell RNA-sequencing (scRNA-seq) atlas covering temporal disease dynamics, resolution and cDC2B depletion. cDC2 trajectories were inferred using VarID2 and{gamma}{delta} T cell transcription factor (TF) regulon activity was predicted by SCENIC. The human biliary niche was resolved by integrating human liver scRNA-seq data with available spatial transcriptomics data. Functional studies were conducted using Tcrd knockout, Il17a/f knockout and Tcrd reporter mice. ResultsA disease trajectory of Mgl2+ cDC2B was identified connecting DC maturation, homing and the expression of Il17-inducing genes. Disease progression was associated with numeric exhaustion of mature cDC2B and recruitment of DC precursors.{gamma}{delta} T cells were the main Il17 producers and were subtyped into Il17ahigh Scart1+ V{gamma}6+ and Il17low Scart2+ V{gamma}4+ populations exhibiting cDC2-directed communication and divergent TF regulon activity. Spatial proximity and conserved molecular interactions of cDC2 and{gamma}{delta} T cells were confirmed in human cholangitis. Il17-deficiency resulted in reduced liver fibrosis in mice, while cDC2B depletion attenuated{gamma}{delta} T17 cell states. ConclusionsIn cholangitis, a profibrogenic function of{gamma}{delta} T cells is contingent on the induction by peribiliary cDC2B, thereby highlighting relevant disease determinants within the immunobiliary and liver-draining LN niche. Impact and ImplicationsThe immunobiliary niche contains rare immune cells such as conventional dendritic cells and unconventional T cells, however, the function of these cell types in liver inflammation remains poorly understood. Mirroring human biliary diseases such as primary sclerosing cholangitis, we induced experimental cholangitis in a mouse model to generate a site-specific single-cell sequencing atlas resource resolving underexplored cell populations. Our data capture a profibrotic hepatic disease state trajectory of Mgl2+ cDC2B inducing a{gamma}{delta} T cell-specific Il17-response, which is attenuated upon cDC2B depletion and in DC precursors, which are characterized by reduced genomic accessibility of{gamma}{delta} T cell-interacting genes. These results highlight the importance of portal niche residing underexplored immune cell populations and the necessity to further resolve immunobiliary niche responses and crosstalk in inflammatory settings such as in cholangitis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/664083v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1d21193org.highwire.dtl.DTLVardef@7ff3dcorg.highwire.dtl.DTLVardef@630c78org.highwire.dtl.DTLVardef@c79f9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Spatio-temporal dynamics of the fibrotic niche in cardiac repair

The heart is one of the least regenerative organs in humans, and ischemic heart disease is the leading cause of death worldwide. Understanding the cellular and molecular processes that occur during cardiac wound healing is an essential prerequisite to reducing health burden and improve cardiac function after myocardial tissue damage. By integrating single-cell RNA-sequencing with imaging-based spatial transcriptomics, we reconstructed the spatio-temporal dynamics of the fibrotic niche after ventricular injury in adult mice. Our analysis reveals dynamic regulation of local cell communication niches over time. We identified interactions that regulate cardiac repair, including fibroblast proliferation silencing by Trem2high macrophages that prevents excessive fibrosis. Moreover, we discovered a rare population of dedifferentiating cardiomyocytes during early post-lesion stages, which was sustained by signals from myeloid and lymphoid cells. Culturing non-regenerative mouse cardiomyocytes or human heart tissue with these niche factors reactivated progenitor gene expression and cell cycle activity. In summary, this spatio-temporal cell type atlas provides valuable insights into the heterocellular interactions that control cardiac repair. HighlightsO_LIscRNA-seq and in situ sequencing reveal spatio-temporal dynamics of heart repair C_LIO_LILocal heterocellular communication niches coordinate overall wound response C_LIO_LIFibroblast cell cycle silencing by Trem2high macrophages suppresses excessive fibrosis C_LIO_LICardiomyocyte plasticity is promoted by myeloid and lymphoid cells C_LI

systems biology↗

bioRxiv2024

Scalable identification of lineage-specific gene regulatory networks from metacells with NetID

The identification of gene regulatory networks (GRN) governing distinct cell fates in multilineage cellular differentiation systems is of critical importance for understanding cell fate decision. Single-cell RNA-sequencing (scRNA-seq) provides a powerful tool for the quantification of gene-level co-variation across the cell state manifold. However, accurate GRN reconstruction is hampered by the sparsity of scRNA-seq data introducing substantial technical noise. Moreover, the high dimensionality of typical scRNA-seq datasets limits the scalability of available approaches. To overcome these challenges, and to facilitate the inference of lineage-specific GRNs with directed regulator-target relations, we introduce NetID. This approach optimizes coverage of the cell state manifold by homogenous metacells and avoids spurious gene-gene correlations observed with available imputation methods. Benchmarking demonstrates superior performance of NetID compared to imputation-based GRN inference. By incorporating cell fate probability information, NetID facilitates prediction of lineage-specific GRNs and recovers known network motifs centered around lineage-determining transcription factors governing bone marrow hematopoiesis, making it a powerful toolkit for deciphering the gene regulatory control of cellular differentiation from large-scale single-cell transcriptome data.

bioinformatics↗

bioRxiv2024

NiCo Identifies Extrinsic Drivers of Cell State Modulation by Niche Covariation Analysis

Cell states are modulated by intrinsic driving forces such as gene expression noise and extrinsic signals from the tissue microenvironment. The distinction between intrinsic and extrinsic cell state determinants is essential for understanding the regulation of cell fate in tissues during development, homeostasis and disease. The rapidly growing availability of single-cell resolution spatial transcriptomics makes it possible to meet this challenge. However, available computational methods to infer topological tissue domains, spatially variable gene expression, or ligand-receptor interactions are limited in capturing cell state changes driven by crosstalk between individual cell types within the same niche. We present NiCo, a computational framework for integrating single-cell resolution spatial transcriptomics with matched single-cell RNA-sequencing reference data to infer the influence of the spatial niche on the cell state. By applying NiCo to mouse embryogenesis, adult small intestine and liver data, we demonstrate the capacity to predict novel niche interactions that govern cell state variation underlying tissue development and homeostasis. In particular, NiCo predicts a feedback mechanism between Kupffer cells and neighboring stellate cells limiting stellate cell activation in the normal liver. NiCo provides a powerful tool to elucidate tissue architecture and to identify drivers of cellular states in local niches.

bioinformatics↗

bioRxiv2022

Slow integrin-dependent migration organizes networks of tissue-resident mast cells

Many leukocytes use fast and flexible amoeboid migration strategies to move autonomously throughout tissues. Here, we show that the movement of mast cells (MCs), leukocytes with important roles during allergies and anaphylaxis, fundamentally differs from this rapid adhesion-free leukocyte migration. We identify a crucial role for integrin-dependent adhesion in controlling slow MC movement, which shapes the positioning and network-like tissue distribution of this long-lived immune cell type. In contrast to other immune and non-immune cells, MCs cannot compensate for the lack of integrin function by switching to another migration mode. Single-cell RNA-sequencing revealed a special role for integrins in defining a mature MC phenotype in the periarteriolar tissue space where several stromal cell types provide an anatomical niche rich in Kit ligand, the major MC growth and survival factor. Collectively, this study highlights substrate-dependent haptokinesis as an important mechanism for MC network formation and the tissue organization of resident immune cells.

cell biology↗

bioRxiv2022

Resident T cells orchestrate adipose tissue remodeling in a site peripheral to infection

Infection with helminth parasites can affect adiposity, but underlying mechanisms that regulate this process are unclear. We found that fat content of mesenteric adipose tissue (mAT) declined in mice during infection with gut-restricted parasitic worms. This was associated with the accumulation of metabolically activated, immunostimulatory cytokine- and extracellular matrix-secreting multipotent stromal cells, which had potential to differentiate into preadipocytes. Concomitantly, mAT became infiltrated with Th2 lymphocytes that took up long-term residence and responded to signals from stromal cells by producing stromal cell-stimulating cytokines, including Amphiregulin. Signals delivered by Amphiregulin to stromal cells were required for immunity to infection. Our findings reveal intricate intercellular communication between Th2 cells and adipocyte progenitors and link immunity to intestinal infection to T cell-dependent effects on the adipocyte lineage.

immunology↗

bioRxiv2021

Single-cell transcriptomic resolution of stem cells and their developmental trajectories in the hippocampus reveals epigenetic control of cell state perseverance

Despite conceptual research on hippocampus development and the application of single-cell-resolved technologies, the nature and maturation of its diverse progenitor populations are unexplored. The chromatin modifier DOT1L balances progenitor proliferation and differentiation, and conditional loss-of-function mice featured impaired hippocampus development. We applied single-cell RNA sequencing on DOT1L-mutant mice and explored cell trajectories in the E16.5 hippocampus. We resolved in our data five distinct neural stem cell populations with the developmental repertoire to specifically generate the cornu ammonis (CA) 1 field and the dentate gyrus (DG). Within the two developing CA1- and CA3-fields, we identified two distinct maturation states and we thus propose CA1- and CA3-differentiation along the radial axis. In the developing hippocampus, DOT1L is primarily involved in the proper development of CA3 and the DG, and it serves as a state-preserving epigenetic factor that orchestrates the expression of several important transcription factors that impact neuronal differentiation and maturation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/452775v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@d611caorg.highwire.dtl.DTLVardef@2a0e3corg.highwire.dtl.DTLVardef@10c9082org.highwire.dtl.DTLVardef@1e358c8_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIThe developing hippocampus contains distinct and spatially separated NSC populations that differ in expression of a specific set of firstly described marker genes. C_LIO_LICA pyramidal neurons mature along the radial axis and pass through distinct maturation states. C_LIO_LIDOT1L preserves the dentate granule cell lineage in the developing hippocampus and limits maturation in the CA1- and CA3-fields development. C_LIO_LIDOT1L gates cell maturation as upstream regulator of transcription factor expression that confer instrumental roles in hippocampus development. C_LI

neuroscience↗

bioRxiv2019

A Human Liver Cell Atlas: Revealing Cell Type Heterogeneity and Adult Liver Progenitors by Single-Cell RNA-sequencing

The human liver is an essential multifunctional organ, and liver diseases are rising with limited treatment options. However, the cellular complexity and heterogeneity of the liver remain poorly understood. Here, we performed single-cell RNA-sequencing of ~5,000 cells from normal liver tissue of 6 human donors to construct the first human liver cell atlas. Our analysis revealed previously unknown sub-types among endothelial cells, Kupffer cells, and hepatocytes with transcriptome-wide zonation of these populations. We show that the EPCAM+ population is highly heterogeneous and consists of hepatocyte progenitors, cholangiocytes and a MUC6+ stem cell population with a specific potential to form liver organoids. As proof-of-principle, we applied our atlas to unravel phenotypic changes in cells from hepatocellular carcinoma tissue and to investigate cellular phenotypes of human hepatocytes and liver endothelial cells engrafted into a humanized FAH-/- mouse liver. Our human liver cell atlas provides a powerful and innovative resource enabling the discovery of previously unknown cell types in the normal and diseased liver.

genomics↗

bioRxiv2019

Benchmarking Single-Cell RNA Sequencing Protocols for Cell Atlas Projects

Single-cell RNA sequencing (scRNA-seq) is the leading technique for charting the molecular properties of individual cells. The latest methods are scalable to thousands of cells, enabling in-depth characterization of sample composition without prior knowledge. However, there are important differences between scRNA-seq techniques, and it remains unclear which are the most suitable protocols for drawing cell atlases of tissues, organs and organisms. We have generated benchmark datasets to systematically evaluate techniques in terms of their power to comprehensively describe cell types and states. We performed a multi-center study comparing 13 commonly used single-cell and single-nucleus RNA-seq protocols using a highly heterogeneous reference sample resource. Comparative and integrative analysis at cell type and state level revealed marked differences in protocol performance, highlighting a series of key features for cell atlas projects. These should be considered when defining guidelines and standards for international consortia, such as the Human Cell Atlas project.

genomics↗

bioRxiv2018

Deciphering the Regulatory Landscape of γδ T Cell Development by Single-Cell RNA-Sequencing

Recent studies have established{gamma}{delta} T cells as critical players in a broad range of infections, antitumor surveillance, autoimmune diseases and tissue homeostasis. However, differentiation of{gamma}{delta} T cells in the adult thymus remains poorly understood, due to the rare frequency of this lineage. Here, we infer high-resolution developmental trajectories of this rare population by single-cell RNA-sequencing. We reveal previously unknown subtypes and identify the transcription factor c-MAF as a novel key regulator of IL-17-producing{gamma}{delta} T cell ({gamma}{delta}T17) differentiation. c-MAF knockout mice exhibit a complete block in{gamma}{delta} T17 differentiation, absence of these cells from peripheral organs, and protection from an autoimmune phenotype in a psoriasis model. Single-cell RNA-sequencing of Sox13 and Rorc knockout mice pinpoints c-MAF as an essential missing link between these lineage-specifying factors. These findings significantly enhance our understanding of{gamma}{delta} T cell ontogeny. Our experimental strategy provides a blueprint for deciphering differentiation of rare cell types.

immunology↗

bioRxiv2017

The Polycomb-dependent epigenome controls β-cell dysfunction, dedifferentiation and diabetes

Chromatin is the physical template that stabilizes and specifies transcriptional programs. To date, it remains largely unclear to what extent chromatin machinery contributes to the susceptibility and progression of complex diseases. Here, we combined deep epigenome mapping with single cell transcriptomics to mine for evidence of chromatin dysregulation in type-2 diabetes. We identify two chromatin-state signatures that track the trajectory of {beta}-cell dysfunction in mice and humans: ectopic activation of bivalent Polycomb-domains and a loss of expression at a subclass of highly active domains containing key lineage-defining genes. {beta}-cell specific deletion of Polycomb (Eed/PRC2) triggers parallel transcriptional signatures. Intriguingly, these {beta}-cell Eed-knockouts also exhibit highly penetrant hyperglycemia-independent dedifferentiation indicating that Polycomb dysregulation sensitizes the {beta}-cell for dedifferentiation. These findings provide novel resources for exploring transcriptional and epigenetic control of {beta}-cell (dys)function. They identify PRC2 as necessary for long-term maintenance of {beta}-cell identity. The data suggest a two-hit model for loss of {beta}-cell identity in diabetes and highlight epigenetic therapeutic potential to block dedifferentiation.

genomics↗