Biology subjects

Suter, D. M.

Publications and source records attributed to Suter, D. M..

17 recordsLinked to original sources

bioRxiv2026

Fluorescent timer-based screening of protein turnover modulators in human neurons

Disturbances in protein homeostasis are a defining feature of aging and neurodegenerative diseases. However, current proteomics approaches do not enable screening-scale pharmacological interrogation of protein turnover in post-mitotic human neurons. Here we establish a Mammalian Cell-optimized Fluorescent Timer (MCFT)-based live-cell imaging platform to quantify global protein synthesis and degradation rates in human embryonic stem cell-derived neurons at screening scale. Among 5,897 tested compounds, 199 increased protein synthesis and degradation rates, and three selected compounds upregulated translation-associated genes in human neurons. We then tested their ability to suppress the accumulation of pathogenic -synuclein aggregates in models of Lewy body-like pathology. All three compounds induced efficient clearance of -synuclein aggregates in mouse primary neurons, and one compound demonstrated similar efficacy in human dopaminergic neurons. This platform provides a broadly applicable strategy for identifying biomedically-relevant compounds that enhance protein turnover across pluripotent stem cell-derived cellular models.

cell biology↗

bioRxiv2026

The role of charge, hydrophobicity, and cooperativity in target search of SOX2 and ESRRB

Transcription factors (TFs) locate and bind specific DNA sequences within a crowded nuclear environment, yet how their biophysical properties and interactions influence their search remains unclear. Here, we modulate hydrophobicity and charge of the pluripotency TFs SOX2 and ESRRB, and combine single-molecule imaging with genomic analyses to dissect their target search dynamics. We show that increased hydrophobicity impairs specific binding while promoting confined intranuclear interactions, whereas additional negative charges decrease sampling of weak binding sites. While SOX2 retains effective target site recognition despite decreased search efficiency, impairment of ESRRB search results in a global loss of genome occupancy and redirection to SOX2-bound regions, indicating a strong reliance of ESRRB on TF-TF interactions for locating its binding sites. Single molecule imaging analysis of ESRRB search upon SOX2 depletion demonstrated that SOX2 reduces ESRRB search time by restricting its nuclear diffusion and facilitating stable DNA binding. Together, our findings highlight how TF biophysical features and cooperativity jointly shape the efficiency and specificity of target search in the nucleus.

molecular biology↗

bioRxiv2025

Endogenous variability in transcription factor concentrations shapes their genome-wide occupancy

The control of transcription factor (TF) concentrations is crucial for the precise regulation of gene expression and cell fate decisions during development. Yet, TF concentrations can display substantial temporal fluctuations and intercellular variations. How TF levels quantitatively shape genome-wide occupancy patterns remains largely unexplored. Here, we systematically investigate how physiological fluctuations in the concentrations of the pluripotency TFs OCT4, SOX2, and NANOG impact their genomic binding profiles in mouse embryonic stem cells. We uncover distinct concentration-dependent binding behaviors for each TF. NANOG occupancy increased monotonously with its concentration, even though a common set of regions made accessible by OCT4 and SOX2 are highly and uniformly bound at all NANOG concentrations. In contrast, SOX2 occupancy does not increase with its concentrations, while OCT4 displayed an intermediate behavior. Our results challenge the notion that TF binding follows simple mass-action dynamics and reveal a core set of pluripotency regions that are bound by all three TFs at all TF concentrations, revealing how pluripotency can be maintained despite marked fluctuations in core pluripotency TFs.

genomics↗

bioRxiv2025

Electrostatic properties of disordered regions control transcription factor search and pioneer activity

Transcription factors (TFs) recognize specific target sites to control gene expression. While prokaryotic TFs combine 1D and 3D diffusion to efficiently locate their binding sites, the target search of eukaryotic TFs within the chromatinized genome is poorly characterized. Here we combine in vivo and in vitro single-molecule imaging to dissect the role of DBD-flanking regions (DFRs) of the mammalian Sox2 and Sox17 TFs in searching for their binding sites. We demonstrate that the DFRSox2 dramatically enhances the search efficiency of Sox2 as compared to the negatively-charged DFRSox17. An enhanced search on naked DNA is primarily mediated by an increase in target recognition rate during 1D sliding. In contrast, target site recognition within nucleosomal DNA is mainly enhanced by increased nonspecific interactions with nucleosomes, facilitating binding to closed chromatin and reinforcing pioneering activity. These findings provide critical insights into the biophysical mechanisms governing TF target search on the chromatinized genome.

biophysics↗

bioRxiv2024

Coordination of protein synthesis and decay in mammalian cells

The maintenance of cellular homeostasis requires tight regulation of proteome concentration and composition. To achieve this, protein production and elimination must be robustly coordinated. However, the mechanistic basis of this coordination remains unclear. Here, we address this question using quantitative live cell imaging, computational modeling, transcriptomics, and proteomics approaches. We found that protein decay rates systematically adapt to global alterations of protein synthesis rates. This adaptation is driven by a core passive mechanism supplemented by facultative changes in mTOR signaling. Passive adaptation hinges on changes in the production rate of the machinery governing protein decay and allows partial maintenance of the cellular proteome. Sustained changes in mTOR signaling provide an additional layer of adaptation unique to naive pluripotent stem cells, allowing near-perfect maintenance of proteome composition. Our work unravels the mechanisms protecting the integrity of mammalian proteomes upon variations in protein synthesis rates.

cell biology↗

bioRxiv2024

Mechanistic basis of gene regulation by SRCAP and H2A.Z

Discriminating regulatory functions of chromatin composition from those of chromatin-modifying complexes is a central problem in gene regulation. This question remains unexplored in the context of histone variants and their dedicated chromatin remodelers. Here we dissect the distinct and cell cycle-dependent functions of Snf2 Related CREBBP Activator Protein (SRCAP) and H2A.Z in gene regulation of pluripotent stem cells. We uncover dynamic changes of H2A.Z occupancy and continuous requirement of SRCAP over the cell cycle. We discover that SRCAP exhibits essential H2A.Z-independent functions in inhibiting DNA binding of dozens of pioneer transcription factors at enhancers by steric hindrance. In contrast, H2A.Z acts mainly as a transcriptional repressor gatekeeping the expression of lineage-specific genes. Our study establishes the catalytic-independent role of a chromatin remodeler in broadly regulating transcription factor binding, and demonstrates how a chromatin remodeler-histone variant pair orchestrates transcription to maintain self-renewal and plasticity of pluripotent stem cells.

molecular biology↗

bioRxiv2023

Orchestration of pluripotent stem cell genome reactivation during mitotic exit

The maintenance of cell identity faces many challenges during mitosis, as most DNA-binding proteins are evicted from DNA and transcription is virtually abolished. How cells maintain their identity through cell division and faithfully re-initiate gene expression during mitotic exit is unclear. Here, we developed a novel reporter system enabling cell cycle synchronization-free separation of pluripotent stem cells in temporal bins of < 30 minutes during mitotic exit. This allowed us to quantify genome-wide reactivation of transcription, sequential changes in chromatin accessibility, and re-binding of the pluripotency transcription factors OCT4, SOX2, and NANOG (OSN). We found that transcriptional activity progressively ramped up after mitosis and that OSN rapidly reoccupied the genome during the anaphase-telophase transition. We also demonstrate transcription factor-specific, dynamic relocation patterns and a hierarchical reorganization of the OSN binding landscape governed by OCT4 and SOX2. Our study sheds light on the dynamic orchestration of transcriptional reactivation after mitosis.

molecular biology↗

bioRxiv2023

ERK signalling orchestrates metachronous transition from naive to formative pluripotency

Naive epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, precipitating breakdown of the naive state gene regulatory network. Cell variability in pERK dynamics results in metachronous down-regulation of Nanog and naive state exit. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This failure is attributable to loss of expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naive state and preserves pluripotency. These results illustrate that a single signalling pathway can both drive exit from a developmental state and safeguard progression to the successor state.

developmental biology↗

bioRxiv2022

Flux analysis reveals specific regulatory modalities of gene expression

The level of a given protein is determined by rates of mRNA and protein synthesis and degradation. The reliable quantification of these rates is thus essential to understand how protein levels are regulated. While several studies have quantified the contribution of different steps of gene expression in regulating protein levels, these are limited by the use of equilibrium approximations when studying out-of-equilibrium biological systems. Here we introduce flux analysis to quantify the contribution of gene expression steps to regulate the dynamics of the expression level for specific proteins. We use flux analysis to analyze a published RNA-seq and proteomics dataset of mouse bone marrow-derived DCs stimulated with LPS. Our analysis revealed six regulatory modalities shared by sets of genes with clear functional signatures. We also find that protein degradation plays a stronger role than expected in the adaptation of protein levels to LPS stimulation. These findings suggest that shared regulatory strategies can lead to versatile responses at the protein level and highlight the importance of going beyond steady-state approximations to uncover the dynamic contribution of different steps of gene expression to protein levels.

systems biology↗

bioRxiv2022

Atypical peripheral actin band formation via overactivation of RhoA and Non-muscle myosin II in Mitofusin 2 deficient cells

Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophillike cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After the initial spreading period, the wild-type cells polarize and migrate, whereas the Mfn2-/- cells maintain a circular shape. Increased cytosolic Ca2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and ER to close vicinity. Elevated cytosolic Ca2+ activates Ca2+/calmodulin-dependent protein kinase II, RhoA, and Myosin light-chain kinase, causing an over-activation of non-muscle myosin II and a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The formation of the peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases.

cell biology↗

bioRxiv2022

Barcode-free prediction of cell lineages from scRNA-seq datasets

The integration of lineage tracing with scRNA-seq has transformed our understanding of gene expression heritability during development, regeneration, and disease. However, lineage tracing is technically demanding and most existing scRNA-seq datasets are devoid of lineage information. Here we introduce Gene Expression Memory-based Lineage Inference (GEMLI), a computational pipeline allowing to predict cell lineages over several cell divisions solely from scRNA-seq datasets. GEMLI leverages genes displaying conserved expression levels over cell divisions, and allows i.a. identifying cell lineages in a broad range of cultured cell types, in intestinal organoids, and in crypts from adult mice. GEMLI recovers GO-terms enriched for heritable gene expression, allows to discriminate symmetric and asymmetric cell fate decisions and to reconstruct individual cellular structures from pooled scRNA-seq datasets. GEMLI considerably extends the pool of datasets from which lineage information can be obtained, thereby facilitating the study of gene expression heritability in a broad range of contexts. GEMLI is available at (https://github.com/UPSUTER/GEMLI).

developmental biology↗

bioRxiv2022

Individual activator and repressor transcription factors induce global changes in chromatin mobility

The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine High-resolution Diffusion mapping (Hi-D) and Dense Flow reConstruction and Correlation (DFCC) in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) increases local chromatin mobility nucleus-wide, yet induces opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of CDX2 leads to changes in local chromatin interactions and compartmentalization. These results thus document a close relation between chromatin dynamics on the microscale and changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II have almost no impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 alters chromatin structures independently from transcription. Our biophysical analysis shows that sequence-specific TFs mobilize long-range chromatin structure on multiple levels, providing evidence that local chromatin changes brought about by TFs can alter both the dynamics and the long-range organization of chromatin in living cells. Significance statementIn eukaryotes, DNA is embedded into a higher-order structure called chromatin that varies between a closed state that is inaccessible to DNA-binding proteins, and an open state that allows the assembly of the transcriptional machinery on DNA. The state of chromatin is dynamic and locally controlled by sequence-specific transcription factors (TFs). How local chromatin opening and closing initiated by TFs alter long-range dynamics of chromatin structures is unknown. Here we combine two nucleus-wide live-imaging techniques, Hi-D and DFCC, along with Hi-C (genomic analysis technique) to quantify both local and global chromatin dynamics, then to correlate these dynamics to structural changes. Our quantitative analysis reveals a differential impact of TFs in shaping and mobilizing long-range chromatin structures in living cells.

biophysics↗

bioRxiv2019

Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.

genomics↗

bioRxiv2018

Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells

The transduction of extracellular signals through signaling pathways that culminate in a transcriptional response is central to many biological processes. However, quantitative relationships between activities of signaling pathway components and transcriptional output of target genes remain poorly explored. Here we developed a dual bioluminescence imaging strategy allowing simultaneous monitoring of nuclear translocation of the SMAD4 and SMAD2 transcriptional activators upon TGF-{beta} stimulation, and the transcriptional response of the endogenous connective tissue growth factor (ctgf) gene. Using cell lines allowing to vary exogenous SMAD4/2 expression levels, we performed quantitative measurements of the temporal profiles of SMAD4/2 translocation and ctgf transcription kinetics in hundreds of individual cells at high temporal resolution. We found that while nuclear translocation efficiency had little impact on initial ctgf transcriptional activation, high total cellular SMAD4 but not SMAD2 levels increased the probability of cells to exhibit a sustained ctgf transcriptional response. The approach we present here allows time-resolved single cell quantification of transcription factor dynamics and transcriptional responses and thereby sheds light on the quantitative relationship between SMADs and target gene responses.

molecular biology↗

bioRxiv2018

Gene-specific transcriptional memory in mammalian cell lineages

Phenotypically identical mammalian cells often display considerable variability in transcript levels of individual genes. How transcriptional activity propagates in cell lineages, and how this varies across genes is poorly understood. Here we combined live-cell imaging of short- lived transcriptional reporters in mouse embryonic stem cells with mathematical modelling to quantify the propagation of transcriptional activity over time and across cell generations. In sister cells we found mean transcriptional activity to be strongly correlated and transcriptional dynamics tended to be synchronous; both features control how quickly sister cells diverge in a gene-specific manner. Mean transcriptional activity was also highly correlated between mother and daughter cells, leading to multi-generational transcriptional memory whose duration scaled with the spread of transcriptional activities in the population. The resulting family-specific transcriptional levels suggest a potential role of transcriptional memory in patterning tissue gene expression.

cell biology↗

bioRxiv2018

Mitotic chromosome binding predicts transcription factor properties in interphase

Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.

genomics↗

bioRxiv2018

Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions

SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. However, how temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cell towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

developmental biology↗