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1.
bioRxiv ; 2024 May 07.
Article in English | MEDLINE | ID: mdl-38352321

ABSTRACT

Transcript buffering entails the reciprocal modulation of mRNA synthesis and degradation rates to maintain stable RNA levels under varying cellular conditions. Current research supports a global, non-sequence-specific connection between mRNA synthesis and degradation, but the underlying mechanisms are still unclear. In this study, we investigated changes in RNA metabolism following acute depletion of TIP60/KAT5, the acetyltransferase subunit of the NuA4 transcriptional coactivator complex, in mouse embryonic stem cells. By combining RNA sequencing of nuclear, cytoplasmic, and newly synthesised transcript fractions with biophysical modelling, we demonstrate that TIP60 predominantly enhances transcription of numerous genes, while a smaller set of genes undergoes TIP60-dependent transcriptional repression. Surprisingly, transcription changes caused by TIP60 depletion were offset by corresponding changes in RNA nuclear export and cytoplasmic stability, indicating gene-specific buffering mechanisms. Similarly, disruption of the unrelated ATAC coactivator complex also resulted in gene-specific transcript buffering. These findings reveal that transcript buffering functions at a gene-specific level and suggest that cells dynamically adjust RNA splicing, export, and degradation in response to individual RNA synthesis alterations, thereby sustaining cellular homeostasis.

2.
Res Sq ; 2023 Aug 16.
Article in English | MEDLINE | ID: mdl-37645793

ABSTRACT

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the spatiotemporal arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find a close link between chromatin mobility and transcriptional status: active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

3.
Cell Rep Methods ; 3(7): 100512, 2023 07 24.
Article in English | MEDLINE | ID: mdl-37533652

ABSTRACT

Time-specific modulation of gene expression during differentiation by transcription factors promotes cell diversity. However, estimating their dynamic regulatory activity at the single-cell level and in a high-throughput manner remains challenging. We present FateCompass, an integrative approach that utilizes single-cell transcriptomics data to identify lineage-specific transcription factors throughout differentiation. By combining a probabilistic framework with RNA velocities or differentiation potential, we estimate transition probabilities, while a linear model of gene regulation is employed to compute transcription factor activities. Considering dynamic changes and correlations of expression and activities, FateCompass identifies lineage-specific regulators. Our validation using in silico data and application to pancreatic endocrine cell differentiation datasets highlight both known and potentially novel lineage-specific regulators. Notably, we uncovered undescribed transcription factors of an enterochromaffin-like population during in vitro differentiation toward ß-like cells. FateCompass provides a valuable framework for hypothesis generation, advancing our understanding of the gene regulatory networks driving cell-fate decisions.


Subject(s)
Gene Expression Regulation , Transcription Factors , Transcription Factors/genetics , Cell Differentiation/genetics , Gene Regulatory Networks , Gene Expression Profiling
4.
bioRxiv ; 2023 Apr 26.
Article in English | MEDLINE | ID: mdl-37162887

ABSTRACT

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

5.
Nat Commun ; 13(1): 2865, 2022 05 23.
Article in English | MEDLINE | ID: mdl-35606383

ABSTRACT

Despite the fact that the cell cycle is a fundamental process of life, a detailed quantitative understanding of gene regulation dynamics throughout the cell cycle is far from complete. Single-cell RNA-sequencing (scRNA-seq) technology gives access to these dynamics without externally perturbing the cell. Here, by generating scRNA-seq libraries in different cell systems, we observe cycling patterns in the unspliced-spliced RNA space of cell cycle-related genes. Since existing methods to analyze scRNA-seq are not efficient to measure cycling gene dynamics, we propose a deep learning approach (DeepCycle) to fit these patterns and build a high-resolution map of the entire cell cycle transcriptome. Characterizing the cell cycle in embryonic and somatic cells, we identify major waves of transcription during the G1 phase and systematically study the stages of the cell cycle. Our work will facilitate the study of the cell cycle in multiple cellular models and different biological contexts.


Subject(s)
Deep Learning , Single-Cell Analysis , Gene Expression Profiling/methods , Genes, cdc , RNA/genetics , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Transcriptome
6.
Nat Commun ; 12(1): 6184, 2021 10 26.
Article in English | MEDLINE | ID: mdl-34702821

ABSTRACT

The spatiotemporal organization of chromatin influences many nuclear processes: from chromosome segregation to transcriptional regulation. To get a deeper understanding of these processes, it is essential to go beyond static viewpoints of chromosome structures, to accurately characterize chromatin's diffusion properties. We present GP-FBM: a computational framework based on Gaussian processes and fractional Brownian motion to extract diffusion properties from stochastic trajectories of labeled chromatin loci. GP-FBM uses higher-order temporal correlations present in the data, therefore, outperforming existing methods. Furthermore, GP-FBM allows to interpolate incomplete trajectories and account for substrate movement when two or more particles are present. Using our method, we show that average chromatin diffusion properties are surprisingly similar in interphase and mitosis in mouse embryonic stem cells. We observe surprising heterogeneity in local chromatin dynamics, correlating with potential regulatory activity. We also present GP-Tool, a user-friendly graphical interface to facilitate usage of GP-FBM by the research community.


Subject(s)
Chromatin/physiology , Models, Biological , Animals , Chromatin Assembly and Disassembly , Computational Biology , Homeodomain Proteins/genetics , Interphase , Mice , Mitosis , Motion , Mouse Embryonic Stem Cells , Normal Distribution
7.
PLoS Comput Biol ; 17(10): e1009354, 2021 10.
Article in English | MEDLINE | ID: mdl-34606497

ABSTRACT

Proliferating cells experience a global reduction of transcription during mitosis, yet their cell identity is maintained and regulatory information is propagated from mother to daughter cells. Mitotic bookmarking by transcription factors has been proposed as a potential mechanism to ensure the reactivation of transcription at the proper set of genes exiting mitosis. Recently, mitotic transcription and waves of transcription reactivation have been observed in synchronized populations of human hepatoma cells. However, the study did not consider that mitotic-arrested cell populations progressively desynchronize leading to measurements of gene expression on a mixture of cells at different internal cell-cycle times. Moreover, it is not well understood yet what is the precise role of mitotic bookmarking on mitotic transcription as well as on the transcription reactivation waves. Ultimately, the core gene regulatory network driving the precise transcription reactivation dynamics remains to be identified. To address these questions, we developed a mathematical model to correct for the progressive desynchronization of cells and estimate gene expression dynamics with respect to a cell-cycle pseudotime. Furthermore, we used a multiple linear regression model to infer transcription factor activity dynamics. Our analysis allows us to characterize waves of transcription factor activities exiting mitosis and predict a core gene regulatory network responsible of the transcription reactivation dynamics. Moreover, we identified more than 60 transcription factors that are highly active during mitosis and represent new candidates of mitotic bookmarking factors which could be relevant therapeutic targets to control cell proliferation.


Subject(s)
Computational Biology/methods , Mitosis/genetics , Transcription, Genetic/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation/genetics , Humans , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome/genetics
8.
Mol Cell ; 81(2): 255-267.e6, 2021 01 21.
Article in English | MEDLINE | ID: mdl-33290745

ABSTRACT

Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements (CREs). Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we present a DNA footprinting method that detects individual molecular interactions of transcription factors and nucleosomes with DNA in vivo. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules at mouse CREs. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that high DNA co-occupancy occurs for most types of transcription factors, in the absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy in binding cooperativity. Our results reveal the interactions regulating CREs at molecular resolution and identify DNA co-occupancy as a widespread cooperativity mechanism used by transcription factors to remodel chromatin.


Subject(s)
DNA Footprinting/methods , DNA/genetics , Nucleosomes/chemistry , Regulatory Elements, Transcriptional , Transcription Factors/genetics , Animals , Binding Sites , DNA/chemistry , DNA/metabolism , Male , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Nucleosomes/metabolism , Protein Binding , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, Genetic
9.
iScience ; 23(9): 101529, 2020 Sep 25.
Article in English | MEDLINE | ID: mdl-33083759

ABSTRACT

Nuclear factor (NF)-κB controls the transcriptional response to inflammatory signals by translocating into the nucleus, but we lack a single-cell characterization of the resulting transcription dynamics. Here we show that upon tumor necrosis factor (TNF)-α transcription of NF-κB target genes is heterogeneous in individual cells but results in an average nascent transcription profile that is prompt (i.e., occurs almost immediately) and sharp (i.e., increases and decreases rapidly) compared with NF-κB nuclear localization. Using an NF-κB-controlled MS2 reporter we show that the single-cell nascent transcription is more heterogeneous than NF-κB translocation dynamics, with a fraction of synchronized "first responders" that shape the average transcriptional profile and are more prone to respond to multiple TNF-α stimulations. A mathematical model combining NF-κB-mediated gene activation and a gene refractory state is able to reproduce these features. Our work shows how the expression of target genes induced by transcriptional activators can be heterogeneous across single cells and yet time resolved on average.

10.
EMBO J ; 39(12): e104486, 2020 06 17.
Article in English | MEDLINE | ID: mdl-32162708

ABSTRACT

Immune cells provide defense against non-self and have recently been shown to also play key roles in diverse processes such as development, metabolism, and tumor progression. The heterogeneity of Drosophila immune cells (hemocytes) remains an open question. Using bulk RNA sequencing, we find that the hemocytes display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single-cell RNA sequencing, we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct processes, e.g., proliferation, phagocytosis, metabolic homeostasis, and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation, which triggers the differentiation of a novel hemocyte type, the lamellocyte. This first molecular atlas of hemocytes provides insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions.


Subject(s)
Drosophila Proteins/immunology , Hemocytes/immunology , Animals , Drosophila Proteins/genetics , Drosophila melanogaster , Larva/genetics , Larva/immunology
11.
Mol Metab ; 29: 24-39, 2019 11.
Article in English | MEDLINE | ID: mdl-31668390

ABSTRACT

OBJECTIVE: Enteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell-Riley syndrome, characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function; however, the role of Rfx6 in EECs differentiation remained to be elucidated. METHODS: We examined the molecular, cellular, and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes. RESULTS: Rfx6 is expressed in the gut endoderm; later, it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, the differentiation programs of which are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1-, or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine. CONCLUSION: These studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell-Riley syndrome.


Subject(s)
Cell Differentiation , Regulatory Factor X Transcription Factors/metabolism , Serotonin/metabolism , Animals , Cell Lineage , Diarrhea/metabolism , Diarrhea/pathology , Energy Metabolism , Enterochromaffin Cells/cytology , Enterochromaffin Cells/metabolism , Enteroendocrine Cells/cytology , Enteroendocrine Cells/metabolism , Female , Gene Expression Regulation , Homeodomain Proteins/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , LIM-Homeodomain Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Regulatory Factor X Transcription Factors/deficiency , Regulatory Factor X Transcription Factors/genetics , Single-Cell Analysis , Transcription Factors/metabolism
12.
Methods Mol Biol ; 2038: 209-221, 2019.
Article in English | MEDLINE | ID: mdl-31407287

ABSTRACT

In this chapter, we describe an antibody electroporation-based imaging approach that allows for precise imaging and quantification of endogenous transcription factor (i.e., RNA Polymerase II) distributions in single cells using 3D structured illumination microscopy (3D-SIM). The labeling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies into living cells and the specific binding of these antibodies to the targeted factors. Our step-by-step protocol describes the procedure of the labeling of the specific antibodies, their electroporation into living cells, the sample preparation and 3D-SIM imaging as well as the postimaging analyses of the labeled endogenous transcription factors to obtain information about their nuclear distribution as well as their function. This protocol can be applied to a plethora of endogenous nuclear factors by using target specific noninhibiting antibodies.


Subject(s)
Antibodies/metabolism , Electroporation , Microscopy, Fluorescence , Molecular Imaging/methods , RNA Polymerase II/metabolism , Single-Cell Analysis/methods , Transcription Factors/metabolism , Antibodies/immunology , Cell Line, Tumor , Fluorescent Dyes/chemistry , Humans , RNA Polymerase II/genetics , Transcription Factors/genetics
13.
Elife ; 72018 07 04.
Article in English | MEDLINE | ID: mdl-29972352

ABSTRACT

Coordination of cell growth with division is essential for proper cell function. In budding yeast, although some molecular mechanisms responsible for cell size control during G1 have been elucidated, the mechanism by which cell size homeostasis is established remains to be discovered. Here, we developed a new technique based on quantification of histone levels to monitor cell cycle progression in individual cells with unprecedented accuracy. Our analysis establishes the existence of a mechanism controlling bud size in G2/M that prevents premature onset of anaphase, and controls the overall size variability. While most G1 mutants do not display impaired size homeostasis, mutants in which cyclin B-Cdk regulation is altered display large size variability. Our study thus demonstrates that size homeostasis is not controlled by a G1-specific mechanism alone but is likely to be an emergent property resulting from the integration of several mechanisms that coordinate cell and bud growth with division.


Subject(s)
Cell Cycle , Homeostasis , Saccharomyces cerevisiae/cytology , Anaphase , Cell Cycle/genetics , Cyclin B/metabolism , Fluorescence , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/metabolism , Histones/biosynthesis , Hydroxyurea/pharmacology , Metaphase , Microbial Viability , Microfluidics , Models, Biological , Mutation/genetics , Saccharomyces cerevisiae/genetics , Time-Lapse Imaging
14.
J Cell Biol ; 217(4): 1537-1552, 2018 04 02.
Article in English | MEDLINE | ID: mdl-29440513

ABSTRACT

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.


Subject(s)
Cell Nucleus/metabolism , Fluorescent Antibody Technique, Direct , Histones/metabolism , Microscopy, Confocal , Single-Cell Analysis/methods , Transcription Factors/metabolism , Animals , Apoptosis , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Nucleus/pathology , Cell Proliferation , Fibroblasts/metabolism , Humans , Kinetics , Mice , Mouse Embryonic Stem Cells/metabolism , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/pathology , Phosphorylation , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , TATA-Binding Protein Associated Factors/genetics , TATA-Binding Protein Associated Factors/metabolism , TATA-Box Binding Protein/genetics , TATA-Box Binding Protein/metabolism , Transcription Factor TFIID/genetics , Transcription Factor TFIID/metabolism , Transcription Factors/genetics
15.
Proc Natl Acad Sci U S A ; 115(8): E1916-E1925, 2018 02 20.
Article in English | MEDLINE | ID: mdl-29432155

ABSTRACT

The mammalian circadian clock coordinates physiology with environmental cycles through the regulation of daily oscillations of gene expression. Thousands of transcripts exhibit rhythmic accumulations across mouse tissues, as determined by the balance of their synthesis and degradation. While diurnally rhythmic transcription regulation is well studied and often thought to be the main factor generating rhythmic mRNA accumulation, the extent of rhythmic posttranscriptional regulation is debated, and the kinetic parameters (e.g., half-lives), as well as the underlying regulators (e.g., mRNA-binding proteins) are relatively unexplored. Here, we developed a quantitative model for cyclic accumulations of pre-mRNA and mRNA from total RNA-seq data, and applied it to mouse liver. This allowed us to identify that about 20% of mRNA rhythms were driven by rhythmic mRNA degradation, and another 15% of mRNAs regulated by both rhythmic transcription and mRNA degradation. The method could also estimate mRNA half-lives and processing times in intact mouse liver. We then showed that, depending on mRNA half-life, rhythmic mRNA degradation can either amplify or tune phases of mRNA rhythms. By comparing mRNA rhythms in wild-type and Bmal1-/- animals, we found that the rhythmic degradation of many transcripts did not depend on a functional BMAL1. Interestingly clock-dependent and -independent degradation rhythms peaked at distinct times of day. We further predicted mRNA-binding proteins (mRBPs) that were implicated in the posttranscriptional regulation of mRNAs, either through stabilizing or destabilizing activities. Together, our results demonstrate how posttranscriptional regulation temporally shapes rhythmic mRNA accumulation in mouse liver.


Subject(s)
Circadian Clocks , Gene Expression Regulation , Liver/metabolism , Mice/genetics , RNA, Messenger/genetics , Animals , Male , Mice/metabolism , Mice, Inbred C57BL , Promoter Regions, Genetic , RNA, Messenger/metabolism , Transcription, Genetic
16.
Genes Dev ; 30(17): 2005-17, 2016 09 01.
Article in English | MEDLINE | ID: mdl-27633015

ABSTRACT

In mammals, body temperature fluctuates diurnally around a mean value of 36°C-37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells' circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide "approach to steady-state" kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression.


Subject(s)
Gene Expression Regulation , Protein Splicing/physiology , RNA-Binding Proteins/metabolism , Temperature , Animals , Body Temperature , Cold Temperature , Genome-Wide Association Study , Liver/metabolism , Mice , NIH 3T3 Cells , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA Stability/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism
17.
Biophys J ; 110(7): 1574-1581, 2016 Apr 12.
Article in English | MEDLINE | ID: mdl-27074682

ABSTRACT

Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways.


Subject(s)
Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Models, Molecular , Pore Forming Cytotoxic Proteins/chemistry , Pore Forming Cytotoxic Proteins/metabolism , Single-Cell Analysis/methods , Erythrocytes/cytology , Humans , Kinetics , Peptide Fragments/metabolism
18.
Article in English | MEDLINE | ID: mdl-26382436

ABSTRACT

Gene expression is an inherently stochastic process that depends on the structure of the biochemical regulatory network in which the gene is embedded. Here we study the dynamical consequences of the interplay between stochastic gene switching and the widespread negative feedback regulatory loop in a simple model of a biochemical regulatory network. Using a simplified hybrid simulation approach, in which only the gene activation is modeled stochastically, we find that stochasticity in gene switching by itself can induce pulses in the system, providing also analytical insights into their origin. Furthermore, we find that this simple network is able to reproduce both exponential and peaked distributions of gene active and inactive times similar to those that have been observed experimentally. This simplified hybrid simulation approach also allows us to link these patterns to the dynamics of the system for each gene state.


Subject(s)
Feedback, Physiological/physiology , Gene Expression/physiology , Models, Biological , Models, Genetic , Computer Simulation , Stochastic Processes
19.
Mol Syst Biol ; 11(7): 823, 2015 Jul 27.
Article in English | MEDLINE | ID: mdl-26215071

ABSTRACT

Mammalian transcription occurs stochastically in short bursts interspersed by silent intervals showing a refractory period. However, the underlying processes and consequences on fluctuations in gene products are poorly understood. Here, we use single allele time-lapse recordings in mouse cells to identify minimal models of promoter cycles, which inform on the number and durations of rate-limiting steps responsible for refractory periods. The structure of promoter cycles is gene specific and independent of genomic location. Typically, five rate-limiting steps underlie the silent periods of endogenous promoters, while minimal synthetic promoters exhibit only one. Strikingly, endogenous or synthetic promoters with TATA boxes show simplified two-state promoter cycles. Since transcriptional bursting constrains intrinsic noise depending on the number of promoter steps, this explains why TATA box genes display increased intrinsic noise genome-wide in mammals, as revealed by single-cell RNA-seq. These findings have implications for basic transcription biology and shed light on interpreting single-cell RNA-counting experiments.


Subject(s)
Promoter Regions, Genetic , Time-Lapse Imaging/methods , Transcription, Genetic , Animals , Markov Chains , Mice , Mouse Embryonic Stem Cells/physiology , NIH 3T3 Cells , TATA Box
20.
Proc Natl Acad Sci U S A ; 110(51): 20563-8, 2013 Dec 17.
Article in English | MEDLINE | ID: mdl-24297917

ABSTRACT

Mammalian genes are often transcribed discontinuously as short bursts of RNA synthesis followed by longer silent periods. However, how these "on" and "off" transitions, together with the burst sizes, are modulated in single cells to increase gene expression upon stimulation is poorly characterized. By combining single-cell time-lapse luminescence imaging with stochastic modeling of the time traces, we quantified the transcriptional responses of the endogenous connective tissue growth factor gene to different physiological stimuli: serum and TGF-ß1. Both stimuli caused a rapid and acute increase in burst sizes. Whereas TGF-ß1 showed prolonged transcriptional activation mediated by an increase of transcription rate, serum stimulation resulted in a large and temporally tight first transcriptional burst, followed by a refractory period in the range of hours. Our study thus reveals how different physiological stimuli can trigger kinetically distinct transcriptional responses of the same gene.


Subject(s)
Models, Biological , Transcription, Genetic/physiology , Transforming Growth Factor beta1/metabolism , Animals , Mice , NIH 3T3 Cells , Stochastic Processes
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