Transcriptional Bursting Research Articles

Noise Contributions in an Inducible Genetic Switch: A Whole-Cell Simulation Study

Stochastic expression of genes produces heterogeneity in clonal populations of bacteria under identical conditions. We analyze and compare the behavior of the inducible lac genetic switch using well-stirred and spatially resolved simulations for Escherichia coli cells modeled under fast and slow-growth conditions. Our new kinetic model describing the switching of the lac operon from one phenotype to the other incorporates parameters obtained from recently published in vivo single-molecule fluorescence experiments along with in vitro rate constants. For the well-stirred system, investigation of the intrinsic noise in the circuit as a function of the inducer concentration and in the presence/absence of the feedback mechanism reveals that the noise peaks near the switching threshold. Applying maximum likelihood estimation, we show that the analytic two-state model of gene expression can be used to extract stochastic rates from the simulation data. The simulations also provide mRNA–protein probability landscapes, which demonstrate that switching is the result of crossing both mRNA and protein thresholds. Using cryoelectron tomography of an E. coli cell and data from proteomics studies, we construct spatial in vivo models of cells and quantify the noise contributions and effects on repressor rebinding due to cell structure and crowding in the cytoplasm. Compared to systems without spatial heterogeneity, the model for the fast-growth cells predicts a slight decrease in the overall noise and an increase in the repressors rebinding rate due to anomalous subdiffusion. The tomograms for E. coli grown under slow-growth conditions identify the positions of the ribosomes and the condensed nucleoid. The smaller slow-growth cells have increased mRNA localization and a larger internal inducer concentration, leading to a significant decrease in the lifetime of the repressor–operator complex and an increase in the frequency of transcriptional bursts.

Transcription Activation via Transcriptional Bursting

Transcription factors (TFs) often regulate their own expression; monitoring TF production in real time will help elucidate autoregulatory mechanisms. However, many TFs are expressed at low levels and are difficult to detect. Further, labeling TFs with fluorescent proteins risks disrupting their regulatory functions. Here, we report a novel strategy, Co-Translational Activation by Cleavage (CoTrAC), to monitor autoregulation of a transcription factor, λ repressor cI, in live E. coli cells. CI becomes fully functional upon co-translational cleavage from a membrane-targeted fluorescent reporter, which can be counted individually. Using this strategy, we discovered that cI activates its own expression via transcriptional bursting_the production of multiple mRNA molecules in brief, well-separated periods of time. CI increases not only the frequency but also the size of transcriptional bursts. Negative autoregulation by cI decreases the burst frequency, but not the burst size. These results link activation of gene expression by a TF to burst-like transcription.

Two Types of Transcriptional Repression in Living Cells of Bacillus Subtilis Characterized by Number and Brightness Analysis

Number and brightness analysis (N&B) is a useful technique for characterizing the concentration and molecular brightness of fluorescent molecules in vivo. Here we investigate stochastic gene expression in Catabolite Repression in Bacillus subtilis. In particular the transcriptional activity of promoters implicated in the switch between glycolysis and gluconeogenesis was investigated. Promoter activity was measured using green fluorescent protein (GFP) promoter fusions. Two photon laser scanning microscopy and true N&B analysis allowed determination of the absolute concentration of GFP molecules inside the bacterial cells. We collected data on hundreds of B. subtilis cells expressing GFP under control of the promoters of interest and grown under glycolytic or gluconeogenic conditions. Results showed no regulation of the promoter expressing the gluconeogenic repressor, strong repression of the gluconeogenic enzyme promoters and weak auto-repression of the glycolytic promoter, with a highly asymmetric distribution when repressed. All promoters showed strong evidence for transcriptional bursting. Analysis of the data using stochastic models of gene expression is currently underway. The figure shows number maps of bacterial cells grown on glucose(G) or Malate(M). Each change in color represents 10 molecules up to 180.View Large Image | View Hi-Res Image | Download PowerPoint Slide

A transgenic mouse for in vivo detection of endogenous labeled mRNA

Live-cell single mRNA imaging is a powerful tool, but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line in which an MS2 binding site (MBS) cassette was targeted to the 3′UTR of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we were able to uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real-time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We performed tracking of single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS also provided a means for high sensitivity Fluorescence In Situ Hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.

A Strand-Specific Burst in Transcription of Pericentric Satellites Is Required for Chromocenter Formation and Early Mouse Development

At the time of fertilization, the paternal genome lacks the typical configuration and marks characteristic of pericentric heterochromatin. It is thus essential to understand the dynamics of this region during early development, its importance during that time period and how a somatic configuration is attained. Here, we show that pericentric satellites undergo a transient peak in expression precisely at the time of chromocenter formation. This transcription is regulated in a strand-specific manner in time and space and is strongly biased by the parental asymmetry. The transcriptional upregulation follows a developmental clock, yet when replication is blocked chromocenter formation is impeded. Furthermore, interference with major satellite transcripts using locked nucleic acid (LNA)-DNA gapmers results in developmental arrest before completion of chromocenter formation. We conclude that the exquisite strand-specific expression dynamics at major satellites during the 2-cell stage, with both up and downregulation, are necessary events for proper chromocenter organization and developmental progression.

HIV promoter integration site primarily modulates transcriptional burst size rather than frequency.

Mammalian gene expression patterns, and their variability across populations of cells, are regulated by factors specific to each gene in concert with its surrounding cellular and genomic environment. Lentiviruses such as HIV integrate their genomes into semi-random genomic locations in the cells they infect, and the resulting viral gene expression provides a natural system to dissect the contributions of genomic environment to transcriptional regulation. Previously, we showed that expression heterogeneity and its modulation by specific host factors at HIV integration sites are key determinants of infected-cell fate and a possible source of latent infections. Here, we assess the integration context dependence of expression heterogeneity from diverse single integrations of a HIV-promoter/GFP-reporter cassette in Jurkat T-cells. Systematically fitting a stochastic model of gene expression to our data reveals an underlying transcriptional dynamic, by which multiple transcripts are produced during short, infrequent bursts, that quantitatively accounts for the wide, highly skewed protein expression distributions observed in each of our clonal cell populations. Interestingly, we find that the size of transcriptional bursts is the primary systematic covariate over integration sites, varying from a few to tens of transcripts across integration sites, and correlating well with mean expression. In contrast, burst frequencies are scattered about a typical value of several per cell-division time and demonstrate little correlation with the clonal means. This pattern of modulation generates consistently noisy distributions over the sampled integration positions, with large expression variability relative to the mean maintained even for the most productive integrations, and could contribute to specifying heterogeneous, integration-site-dependent viral production patterns in HIV-infected cells. Genomic environment thus emerges as a significant control parameter for gene expression variation that may contribute to structuring mammalian genomes, as well as be exploited for survival by integrating viruses.

Genomic deletion of the whole IgH 3′ regulatory region (hs3a, hs1,2, hs3b, and hs4) dramatically affects class switch recombination and Ig secretion to all isotypes

AbstractThe immunoglobulin heavy chain locus (IgH) undergoes multiple changes along B-cell differentiation. In progenitor B cells, V(D)J assembly allows expression of µ heavy chains. In mature B cells, class switch recombination may replace the expressed constant (C)µ gene with a downstream CH gene. Finally, plasma cell differentiation strongly boosts IgH transcription. How the multiple IgH transcriptional enhancers tune these changes is unclear. Here we demonstrate that deletion of the whole IgH 3′ regulatory region (3′RR) allows normal maturation until the stage of IgM/IgD expressing lymphocytes, but nearly abrogates class switch recombination to all CH genes. Although plasma cell numbers are unaffected, we reveal the role of the 3′RR into the transcriptional burst normally associated with plasma cell differentiation. Our study shows that transcriptional changes and recombinations occurring after antigen-encounter appear mainly controlled by the 3′RR working as a single functional unit.

Stochastic bursts in the kinetics of gene expression with regulation by long non-coding RNAs

One of the main recent breakthroughs in cellular biology is a discovery of numerous non-coding RNAs (ncR-NAs). We outline abilities of long ncRNAs and articulate that the corresponding kinetics may frequently exhibit stochastic bursts. For example, we scrutinize one of the generic cases when the gene transcription is regulated by competitive attachment of ncRNA and protein to a regulatory site. Our Monte Carlo simulations show that in this case one can observe huge long transcriptional bursts consisting of short bursts.

Applications of Little’s Law to stochastic models of gene expression

The intrinsic stochasticity of gene expression can lead to large variations in protein levels across a population of cells. To explain this variability, different sources of messenger RNA (mRNA) fluctuations ("Poisson" and "telegraph" processes) have been proposed in stochastic models of gene expression. Both Poisson and telegraph scenario models explain experimental observations of noise in protein levels in terms of "bursts" of protein expression. Correspondingly, there is considerable interest in establishing relations between burst and steady-state protein distributions for general stochastic models of gene expression. In this work, we address this issue by considering a mapping between stochastic models of gene expression and problems of interest in queueing theory. By applying a general theorem from queueing theory, Little's Law, we derive exact relations which connect burst and steady-state distribution means for models with arbitrary waiting-time distributions for arrival and degradation of mRNAs and proteins. The derived relations have implications for approaches to quantify the degree of transcriptional bursting and hence to discriminate between different sources of intrinsic noise in gene expression. To illustrate this, we consider a model for regulation of protein expression bursts by small RNAs. For a broad range of parameters, we derive analytical expressions (validated by stochastic simulations) for the mean protein levels as the levels of regulatory small RNAs are varied. The results obtained show that the degree of transcriptional bursting can, in principle, be determined from changes in mean steady-state protein levels for general stochastic models of gene expression.

Single-allele analysis of transcription kinetics in living mammalian cells

We generated a system for in vivo visualization and analysis of mammalian mRNA transcriptional kinetics of single alleles in real time, using single-gene integrations. We obtained high-resolution transcription measurements of a single cyclin D1 allele under endogenous or viral promoter control, including quantification of temporal kinetics of transcriptional bursting, promoter firing, nascent mRNA numbers and transcription rates during the cell cycle, and in relation to DNA replication.

Transcriptional Bursting from the HIV-1 Promoter Is a Significant Source of Stochastic Noise in HIV-1 Gene Expression

Analysis of noise in gene expression has proven a powerful approach for analyzing gene regulatory architecture. To probe the regulatory mechanisms controlling expression of HIV-1, we analyze noise in gene-expression from HIV-1's long terminal repeat (LTR) promoter at different HIV-1 integration sites across the human genome. Flow cytometry analysis of GFP expression from the HIV-1 LTR shows high variability (noise) at each integration site. Notably, the measured noise levels are inconsistent with constitutive gene expression models. Instead, quantification of expression noise indicates that HIV-1 gene expression occurs through randomly timed bursts of activity from the LTR and that each burst generates an average of 2–10 mRNA transcripts before the promoter returns to an inactive state. These data indicate that transcriptional bursting can generate high variability in HIV-1 early gene products, which may critically influence the viral fate-decision between active replication and proviral latency.

Transcriptional Memory: Mothers SET the Table for Daughters

Eukaryotic gene transcription within individual cells of a population is often associated with heterogeneous pulses of gene activity. A recent study, however, shows that mothers and their daughters share similar transcriptional frequencies, and inheritance of mother's transcriptional tendencies requires methylation of histone H3 by a Set1 methyltransferase.

214 Distinct pathological characteristics of lobular carcinoma

Bursts of nascent mRNA have been shown to lead to substantial cell-cell variation in unicellular organisms, facilitating diverse responses to environmental challenges. It is unknown whether similar bursts and gene-expression noise occur in mammalian tissues. To address this, we combine single molecule transcript counting with dual-color labeling and quantification of nascent mRNA to characterize promoter states, transcription rates, and transcript lifetimes in the intact mouse liver. We find that liver gene expression is highly bursty, with promoters stochastically switching between transcriptionally active and inactive states. Promoters of genes with short mRNA lifetimes are active longer, facilitating rapid response while reducing burst-associated noise. Moreover, polyploid hepatocytes exhibit less noise than diploid hepatocytes, suggesting a possible benefit to liver polyploidy. Thus, temporal averaging and liver polyploidy dampen the intrinsic variability associated with transcriptional bursts. Our approach can be used to study transcriptional bursting in diverse mammalian tissues.

Transcript counting in single cells reveals dynamics of rDNA transcription

Most eukaryotes contain many tandem repeats of ribosomal RNA genes of which only a subset is transcribed at any given time. Current biochemical methods allow for the determination of the fraction of transcribing repeats (ON) versus non-transcribing repeats (OFF) but do not provide any dynamical information and obscure any transcription activity at the single-cell level. Here, we use a fluorescence in situ hybridization (FISH) technique that allows the detection of single-RNA molecules in individual yeast cells. We use this method complemented with theoretical modeling to determine the rate of switching from OFF to ON (activation rate) and the average number of RNA molecules produced during each transcriptional burst (burst size). We explore how these two variables change in mutants and different growth conditions, and show that this method resolves changes in these two variables even when the average rDNA expression is unaltered. These phenotypic changes could not have been detected by traditional biochemical assays.

Attenuation of transcriptional bursting in mRNA transport

Because of the stochastic nature of biochemical processes, the copy number of any given type of molecule inside a living cell often exhibits large temporal fluctuations. Here, we develop analytic methods to investigate how the noise arising from a bursting input is reshaped by a transport reaction which is either linear or of the Michaelis–Menten type. A slow transport rate smoothes out fluctuations at the output end and minimizes the impact of bursting on the downstream cellular activities. In the context of gene expression in eukaryotic cells, our results indicate that transcriptional bursting can be substantially attenuated by the transport of mRNA from nucleus to cytoplasm. Saturation of the transport mediators or nuclear pores contributes further to the noise reduction. We suggest that the mRNA transport should be taken into account in the interpretation of relevant experimental data on transcriptional bursting.

Model of gene transcription including the return of a RNA polymerase to the beginning of a transcriptional cycle

The gene transcription occurs via the RNA polymerase (RNAP) recruitment on the DNA promoter sequence, formation of a locally open DNA chain, promoter escape, steps of the RNA synthesis, and RNA and RNAP release after reading the final DNA base. Just after the end of the RNA synthesis, RNAP surrounds the closed DNA chain and may diffuse along DNA, desorb, or reach the promoter and start the RNA-synthesis cycle again. We present a generic kinetic model taking the latter steps into account and show analytically and by Monte Carlo simulations that it predicts transcriptional bursts even in the absence of explicit regulation of the transcription by master proteins.

Visualization of Individual Scr mRNAs during Drosophila Embryogenesis Yields Evidence for Transcriptional Bursting

The detection and counting of transcripts within single cells via fluorescent in situ hybridization (FISH) has allowed researchers to ask quantitative questions about gene expression at the level of individual cells. This method is often preferable to quantitative RT-PCR, because it does not necessitate destruction of the cells being probed and maintains spatial information that may be of interest. Until now, studies using FISH at single-molecule resolution have only been rigorously carried out in isolated cells (e.g., yeast cells or mammalian cell culture). Here, we describe the detection and counting of transcripts within single cells of fixed, whole-mount Drosophila embryos via a combination of FISH, immunohistochemistry, and image segmentation. Our method takes advantage of inexpensive, long RNA probes detected with antibodies, and we present novel evidence to show that we can robustly detect single mRNA molecules. We use this method to characterize transcription at the endogenous locus of the Hox gene Sex combs reduced (Scr), by comparing a stably expressing group of cells to a group that only transiently expresses the gene. Our data provide evidence for transcriptional bursting, as well for divergent "accumulation" and "maintenance" phases of gene activity at the Scr locus.

A potent enhancer element in the 5′-UTR intron is crucial for transcriptional regulation of the human ubiquitin C gene

Ubiquitin (Ub) plays a crucial role in almost every aspect of cellular functions. It is encoded by four genes, of which UbC is known to meet cell demand for ubiquitin in both basal and stressful conditions. To understand the molecular mechanisms regulating UbC gene expression, we performed a functional characterization of the UbC promoter. Deletion analyses on the 5′ end of the − 916/+ 878 promoter region, excluded the functional importance of nt − 916/− 371 in the transcriptional regulation of the gene, while 3′ deletions revealed that intron removal (nt+ 65/+ 876) resulted in a marked reduction of promoter activity in all the reporter constructs, regardless of the cell types. Intron substitution with a heterologous chimeric intron failed to restore promoter activity, thus allowing to exclude that the splicing event, per se, can be responsible for the intron-mediated burst of transcription. Gel shift assays demonstrated nuclear factor binding with the + 137/+ 766 intron region. Reporter constructs carrying partial intron deletions confirmed that this sequence is, indeed, required for maximal transcriptional activity. Computer-based analysis found potential Sp1 binding motifs within the intron sequence and electrophoretic mobility shift and supershift assays demonstrated that both Sp1 and Sp3 transcription factors interact, in vitro, with the UbC intron, at multiple binding sites. Moreover, ectopic expression of Sp1 and Sp3 revealed that both transcription factors positively regulate UbC promoter activity. Collectively, our data highlight the very new evidence that the 5′-UTR intron is crucial in regulating UbC gene expression and provide insights into the pivotal role of Sp1/Sp3 binding to the intronic enhancer in the regulation of UbC transcription.

Random dynamics of gene transcription activation in single cells

The recent measurements of gene transcription activity at single cell resolution revealed that genes are often transcribed randomly and discontinuously. In order to elucidate how the environmental signals contribute to the stochasticity of gene transcription, a random transition model was recently proposed [M. Tang, The mean and noise of stochastic gene transcription, J. Theor. Biol. 253 (2008) 271–280; M. Tang, The mean frequency of transcriptional bursting and its variation in single cells, J. Math. Biol. (2009) doi:10.1007/s00285-009-0258-7, in press; published online: March 10, 2009]. In this model it is assumed that the transcription system transits randomly between three different functional states, quantifying the timing and strength of gene transcription by a sequence of probability functions P n x ( t ) , coupled in an infinite differential system of master equations. Here n ⩾ 1 are integers and x specifies each of the three functional states. In this work we further study this model aiming to understand the stochastic dynamics of gene transcription. When n ⩽ 3 , the exact form of P n x ( t ) is found analytically by solving the system of master equations. For larger n however, it is unfeasible to find P n x ( t ) explicitly, so we explore the properties of probability functions by analyzing the master operator L that transforms P ( n − 1 ) x ( t ) to P n x ( t ) . We prove that L “mollifies” the behavior of P ( n − 1 ) x ( t ) by increasing its order of differentiability and by flattening its growth globally. We also show that the n-th cycle of transcription activity condenses at distinct peak instants, with a decreasing peak strength with respect to n. The timings of these peak instants are estimated and several further open questions toward a general theory are discussed.

Recruitment of a chromatin remodelling complex by the Hog1 MAP kinase to stress genes

For efficient transcription, RNA PolII must overcome the presence of nucleosomes. The p38-related MAPK Hog1 is an important regulator of transcription upon osmostress in yeast and thereby it is involved in initiation and elongation. However, the role of this protein kinase in elongation has remained unclear. Here, we show that during stress there is a dramatic change in the nucleosome organization of stress-responsive loci that depends on Hog1 and the RSC chromatin remodelling complex. Upon stress, the MAPK Hog1 physically interacts with RSC to direct its association with the ORF of osmo-responsive genes. In RSC mutants, PolII accumulates on stress promoters but not in coding regions. RSC mutants also display reduced stress gene expression and enhanced sensitivity to osmostress. Cell survival under acute osmostress might thus depend on a burst of transcription that in turn could occur only with efficient nucleosome eviction. Our results suggest that the selective targeting of the RSC complex by Hog1 provides the necessary mechanistic basis for this event.