Transcriptional Bursting Research Articles

Novel insights into RNA polymerase II transcription regulation: transcription factors, phase separation, and their roles in cardiovascular diseases.

Transcription factors (TFs) are specialized proteins that bind DNA in a sequence-specific manner and modulate RNA polymerase II (Pol II) in multiple steps of the transcription process. Phase separation is a spontaneous or driven process that can form membrane-less organelles called condensates. By creating different liquid phases at active transcription sites, the formation of transcription condensates can reduce the water content of the condensate and lower the dielectric constant in biological systems, which in turn alters the structure and function of proteins and nucleic acids in the condensate. In RNA Pol II transcription, phase separation formation shortens the time at which TFs bind to target DNA sites and promotes transcriptional bursting. RNA Pol II transcription is engaged in developing several diseases, such as cardiovascular disease, by regulating different TFs and mediating the occurrence of phase separation. This review aims to summarize the advances in the molecular mechanisms of RNA Pol II transcriptional regulation, in particular the effect of TFs and phase separation. The role of RNA Pol II transcriptional regulation in cardiovascular disease will be elucidated, providing potential therapeutic targets for the management and treatment of cardiovascular disease.

Optogenetic dissection of transcriptional repression in a multicellular organism

Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive, particularly in the endogenous context of developing animals. Here, we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 min) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

Conserved helical motifs in the IKZF1 disordered region mediate NuRD interaction and transcriptional repression

The transcription factor IKZF1 is essential for B cell development, and recurrently mutated in human B-ALL. IKZF1 has been ascribed both activating and repressive functions via interactions with coactivator and corepressor complexes, but the relative abundance of IKZF1-associated coregulators and their contribution to IKZF1-mediated gene regulation are not well understood. To address this, we performed an unbiased identification of IKZF1-interacting proteins in pre-B cells and found that IKZF1 interacts overwhelmingly with corepressors and heterochromatin-associated proteins. Time-resolved analysis of transcription and chromatin state identified transcriptional repression as the immediate response to IKZF1 induction. Transcriptional repression preceded transcriptional activation by several hours, manifesting as a decrease in the fraction of transcriptional bursts at the single molecule level. Repression was accompanied by a rapid loss of chromatin accessibility and reduced levels of H3K27ac particularly at enhancers. We identified highly conserved helical motifs within the intrinsically disordered region in IKZF1 that mediate its association with the NuRD corepressor complex through critical “KRK” residues that bind the NuRD subunit RBBP4, a mechanism shared with the TFs FOG1, BCL11A, and SALL4. Functional characterization reveals this region is necessary for to the efficient silencing of target genes and antiproliferative functions of IKZF1 in B-ALL.

MONITTR allows real-time imaging of transcription and endogenous proteins in C. elegans.

Maximizing cell survival under stress requires rapid and transient adjustments of RNA and protein synthesis. However, capturing these dynamic changes at both single-cell level and across an organism has been challenging. Here, we developed a system named MONITTR (MS2-embedded mCherry-based monitoring of transcription) for real-time simultaneous measurement of nascent transcripts and endogenous protein levels in C. elegans. Utilizing this system, we monitored the transcriptional bursting of fasting-induced genes and found that the epidermis responds to fasting by modulating the proportion of actively transcribing nuclei and transcriptional kinetics of individual alleles. Additionally, our findings revealed the essential roles of the transcription factors NHR-49 and HLH-30 in governing the transcriptional kinetics of fasting-induced genes under fasting. Furthermore, we tracked transcriptional dynamics during heat-shock response and ER unfolded protein response and observed rapid changes in the level of nascent transcripts under stress conditions. Collectively, our study provides a foundation for quantitatively investigating how animals spatiotemporally modulate transcription in various physiological and pathological conditions.

Energy Aware Technology Mapping of Genetic Logic Circuits.

Energy and its dissipation are fundamental to all living systems, including cells. Insufficient abundance of energy carriers─as caused by the additional burden of artificial genetic circuits─shifts a cell's priority to survival, also impairing the functionality of the genetic circuit. Moreover, recent works have shown the importance of energy expenditure in information transmission. Despite living organisms being non-equilibrium systems, non-equilibrium models capable of accounting for energy dissipation and non-equilibrium response curves are not yet employed in genetic design automation (GDA) software. To this end, we introduce Energy Aware Technology Mapping, the automated design of genetic logic circuits with respect to energy efficiency and functionality. The basis for this is an energy aware non-equilibrium steady state model of gene expression, capturing characteristics like energy dissipation─which we link to the entropy production rate─and transcriptional bursting, relevant to eukaryotes as well as prokaryotes. Our evaluation shows that a genetic logic circuit's functional performance and energy efficiency are disjoint optimization goals. For our benchmark, energy efficiency improves by 37.2% on average when comparing to functionally optimized variants. We discover a linear increase in energy expenditure and overall protein expression with the circuit size, where Energy Aware Technology Mapping allows for designing genetic logic circuits with the energetic costs of circuits that are one to two gates smaller. Structural variants improve this further, while results show the Pareto dominance among structures of a single Boolean function. By incorporating energy demand into the design, Energy Aware Technology Mapping enables energy efficiency by design. This extends current GDA tools and complements approaches coping with burden in vivo.

Energy Aware Technology Mapping of Genetic Logic Circuits.

Energy and its dissipation are fundamental to all living systems, including cells. Insufficient abundance of energy carriers -as caused by the additional burden of artificial genetic circuits- shifts a cell's priority to survival, also impairing the functionality of the genetic circuit. Moreover, recent works have shown the importance of energy expenditure in information transmission. Despite living organisms being non-equilibrium systems, non-equilibrium models capable of accounting for energy dissipation and non-equilibrium response curves are not yet employed in genetic design automation (GDA) software. To this end, we introduce Energy Aware Technology Mapping, the automated design of genetic logic circuits with respect to energy efficiency and functionality. The basis for this is an energy aware non-equilibrium steady state (NESS) model of gene expression, capturing characteristics like energy dissipation -which we link to the entropy production rate- and transcriptional bursting, relevant to eukaryotes as well as prokaryotes. Our evaluation shows that a genetic logic circuit's functional performance and energy efficiency are disjoint optimization goals. For our benchmark, energy efficiency improves by 37.2% on average when comparing to functionally optimized variants. We discover a linear increase in energy expenditure and overall protein expression with the circuit size, where Energy Aware Technology Mapping allows for designing genetic logic circuits with the energy efficiency of circuits that are one to two gates smaller. Structural variants improve this further, while results show the Pareto dominance among structures of a single Boolean function. By incorporating energy demand into the design, Energy Aware Technology Mapping enables energy efficiency by design. This extends current GDA tools and complements approaches coping with burden in vivo.

Deciphering transcriptional bursts and enhancer dynamics: Advancing cancer therapeutics through single‐cell global run‐on sequencing

Deciphering transcriptional bursts and enhancer dynamics: Advancing cancer therapeutics through single‐cell global run‐on sequencing

Transcriptional bursting: from fundamentals to novel insights.

Transcription occurs as irregular bursts in a very wide range of systems, including numerous different species and many genes within these. In this review, we examine the underlying theories, discuss how these relate to experimental measurements, and explore some of the discrepancies that have emerged among various studies. Finally, we consider more recent works that integrate novel concepts, such as the involvement of biomolecular condensates in enhancer-promoter interactions and their effects on the dynamics of transcriptional bursting.

Single-cell new RNA sequencing reveals principles of transcription at the resolution of individual bursts

Analyses of transcriptional bursting from single-cell RNA-sequencing data have revealed patterns of variation and regulation in the kinetic parameters that could be inferred. Here we profiled newly transcribed (4-thiouridine-labelled) RNA across 10,000 individual primary mouse fibroblasts to more broadly infer bursting kinetics and coordination. We demonstrate that inference from new RNA profiles could separate the kinetic parameters that together specify the burst size, and that the synthesis rate (and not the transcriptional off rate) controls the burst size. Importantly, transcriptome-wide inference of transcriptional on and off rates provided conclusive evidence that RNA polymerase II transcribes genes in bursts. Recent reports identified examples of transcriptional co-bursting, yet no global analyses have been performed. The deep new RNA profiles we generated with allelic resolution demonstrated that co-bursting rarely appears more frequently than expected by chance, except for certain gene pairs, notably paralogues located in close genomic proximity. Altogether, new RNA single-cell profiling critically improves the inference of transcriptional bursting and provides strong evidence for independent transcriptional bursting of mammalian genes.

Single cell expression and chromatin accessibility of the Toxoplasma gondii lytic cycle identifies AP2XII-8 as an essential ribosome regulon driver

Sequential lytic cycles driven by cascading transcriptional waves underlie pathogenesis in the apicomplexan parasite Toxoplasma gondii. This parasite’s unique division by internal budding, short cell cycle, and jumbled up classically defined cell cycle stages have restrained in-depth transcriptional program analysis. Here, unbiased transcriptome and chromatin accessibility maps throughout the lytic cell cycle are established at the single-cell level. Correlated pseudo-timeline assemblies of expression and chromatin profiles maps transcriptional versus chromatin level transition points promoting the cell division cycle. Sequential clustering analysis identifies functionally related gene groups promoting cell cycle progression. Promoter DNA motif mapping reveals patterns of combinatorial regulation. Pseudo-time trajectory analysis reveals transcriptional bursts at different cell cycle points. The dominant burst in G1 is driven largely by transcription factor AP2XII-8, which engages a conserved DNA motif, and promotes the expression of 44 ribosomal proteins encoding regulon. Overall, the study provides integrated, multi-level insights into apicomplexan transcriptional regulation.

How Transcription Factors Binding Stimulates Transcriptional Bursting.

Transcription is a fundamental biological process of transferring genetic information which often occurs in stochastic bursts when periods of intense activity alternate with quiescent phases. Recent experiments identified strong correlations between the association of transcription factors (TFs) to gene promoters on DNA and transcriptional activity. However, the underlying molecular mechanisms of this phenomenon remain not well understood. Here, we present a theoretical framework that allowed us to investigate how binding dynamics of TF influences transcriptional bursting. Our minimal theoretical model incorporates the most relevant physical-chemical features, including TF exchange among multiple binding sites at gene promoters and TF association/dissociation dynamics. Using analytical calculations supported by Monte Carlo computer simulations, it is demonstrated that transcriptional bursting dynamics depends on the strength of TF binding and the number of binding sites. Stronger TF binding affinity prolongs burst duration but reduces variability, while an optimal number of binding sites maximizes transcriptional noise, facilitating cellular adaptation. Our theoretical method explains available experimental observations quantitatively, confirming the model's predictive accuracy. This study provides important insights into molecular mechanisms of gene expression and regulation, offering a new theoretical tool for understanding complex biological processes.

3D chromatin architecture, BRD4, and Mediator have distinct roles in regulating genome-wide transcriptional bursting and gene network.

Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Despite comparable effects on RNA levels among these perturbations, acute depletion of MED26 had the most profound impact on the entire gene regulatory network, acting downstream of chromatin spatial architecture and without affecting TATA box-binding protein (TBP) recruitment. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells.

Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single-cell RNA-seq

We describe a time-resolved nascent single-cell RNA sequencing (RNA-seq) approach that measures gene-specific transcriptional noise and the fraction of active genes in S.cerevisiae. Most genes are expressed with near-constitutive behavior, while a subset of genes show high mRNA variance suggestive of transcription bursting. Transcriptional noise is highest in the cofactor/coactivator-redundant (CR) gene class (dependent on both SAGA and TFIID) and strongest in TATA-containing CR genes. Using this approach, we also find that histone gene transcription switches from a low-level, low-noise constitutive mode during M and M/G1 to an activated state in S phase that shows both an increase in the fraction of active promoters and a switch to a noisy and bursty transcription mode. Rapid depletion of cofactors SAGA and MED Tail indicates that both factors play an important role in stimulating the fraction of active promoters at CR genes, with a more modest role in transcriptional noise.

How Transcription Factor Clusters Shape the Transcriptional Landscape.

In eukaryotic cells, gene transcription typically occurs in discrete periods of promoter activity, interspersed with intervals of inactivity. This pattern deviates from simple stochastic events and warrants a closer examination of the molecular interactions that activate the promoter. Recent studies have identified transcription factor (TF) clusters as key precursors to transcriptional bursting. Often, these TF clusters form at chromatin segments that are physically distant from the promoter, making changes in chromatin conformation crucial for promoter-TF cluster interactions. In this review, I explore the formation and constituents of TF clusters, examining how the dynamic interplay between chromatin architecture and TF clustering influences transcriptional bursting. Additionally, I discuss techniques for visualizing TF clusters and provide an outlook on understanding the remaining gaps in this field.

Stochastic Modeling of Biophysical Responses to Perturbation.

Recent advances in high-throughput, multi-condition experiments allow for genome-wide investigation of how perturbations affect transcription and translation in the cell across multiple biological entities or modalities, from chromatin and mRNA information to protein production and spatial morphology. This presents an unprecedented opportunity to unravel how the processes of DNA and RNA regulation direct cell fate determination and disease response. Most methods designed for analyzing large-scale perturbation data focus on the observational outcomes, e.g., expression; however, many potential transcriptional mechanisms, such as transcriptional bursting or splicing dynamics, can underlie these complex and noisy observations. In this analysis, we demonstrate how a stochastic biophysical modeling approach to interpreting high-throughout perturbation data enables deeper investigation of the 'how' behind such molecular measurements. Our approach takes advantage of modalities already present in data produced with current technologies, such as nascent and mature mRNA measurements, to illuminate transcriptional dynamics induced by perturbation, predict kinetic behaviors in new perturbation settings, and uncover novel populations of cells with distinct kinetic responses to perturbation.

O-135 The consequences of paternal pre-conceptional health for offspring health and pregnancy outcomes: involvement of the germ-cell epigenome and non-germ cell factors

Abstract Study question Paternal health at conception is important for offspring health. The susceptibility windows within the male reproductive tract and the molecular mechanisms are not completely understood. Summary answer Using mouse genetics, (epi)genomics and in-depth phenotyping, we show that the germ-cell epigenome and components of the seminal fluid are critical for paternal intergenerational effects. What is known already Paternal intergenerational effects are highly conserved across mammals and lower organisms including flies and nematodes. In mammals, the sperm epigenome, as well as the composition of the seminal fluid are sensitive to environmental challenges and influence offspring development and adult phenotypic trajectories. The best known of these mechanisms are DNA methylation and small non-coding RNAs in spermatozoa and the balance between inflammatory and tolerogenic cytokines in the seminal fluid. Small non-coding RNAs are mostly acquired by maturing spermatozoa during the epididymal transit from extracellular vesicle of epididymal epithelial origin, and have the potential to influence embryonic development and offspring health. Study design, size, duration We use two paternal paradigms of pre-conceptional challenge in wild-type C57BL6 mice: high-fat diet feeding for 2 weeks and circadian disruption for 4 weeks before conception. The duration of the environmental challenges has been chosen to meet timing of sperm maturation in the mouse (high-fat diet) or to be the minimum sufficient to induce circadian disruption without overt side effects. Exposed males and their offspring have undergone deep metabolic and molecular phenotyping. Participants/materials, setting, methods We used both bulk and single cell (epi)genomics on exposed males as well as male and female offspring. The molecular phenotyping has been instructed by the results of the metabolic phenotyping performed in collaboration with the German Mouse Clinic, the biggest mouse clinic in Europe, using highly standardized and robust procedures. Importantly, we have used a family-based approach for the data analysis, according to which the experimental n is the exposed father. Main results and the role of chance Paternal circadian disruption achieved by four weeks of day-restricted feeding (food only during the light phase), leads to hyperphagia, hyperglycemia, hypercorticosteronemia and partial circadian disruption in unexposed male offspring. The phenotypes are gender-specific and the females feature partial protection from the paternal effects. Mechanistically, mouse seminal fluid contains corticosterone, whose appearance dynamic follows the organismal circadian rhythm and is blunted upon circadian disruption. Offspring phenotypes are sensitive to the levels of corticosterone in the seminal fluid and consequential to impaired placentation, reduced placental efficiency and fetal growth restriction. Paternal exposure to an acute high-fat diet challenge, instead, leads to a 30% penetrant glucose intolerance phenotype exclusively in male offspring. Mechanistically, this is linked to diet-induced mitochondrial dysfunction in somatic and germ cells and a compensatory burst of mitochondrial DNA transcription in spermatozoa. Mitochondrial DNA encoded small non-coding RNAs accumulate in spermatozoa, are inherited at fertilization, and reprogram early embryo transcription towards premature activation of oxidative metabolism, a predisposing factor to adult-onset glucose intolerance. Importantly, phenotypes and mechanisms are conserved across highly characterized human cohorts. These studies strengthen the importance of paternal health at conception for pregnancy and offspring health and identify germ-cell and non-germ cell factors as potential mechanistic mediators. Limitations, reasons for caution Although we have shown for the first time that stress hormones are present in the seminal fluid and relay paternal stress to the offspring and provided the first example of epigenetic inheritance of sperm small non-coding RNAs, mechanisms are still to be completely uncovered. Complementary mechanisms cannot be excluded. Wider implications of the findings Our findings strengthen the importance of paternal health at conception for pregnancy and offspring health. They also provide potential male tract biomarkers and novel strategies to monitor paternal health at conception and preconception lifestyle interventions aimed at improving pregnancy outcomes and preventing the spread of metabolic disorders through epigenetic inheritance. Trial registration number not applicable

Unified bursting strategies in ectopic and endogenous even-skipped expression patterns.

Transcription often occurs in bursts as gene promoters switch stochastically between active and inactive states. Enhancers can dictate transcriptional activity in animal development through the modulation of burst frequency, duration, or amplitude. Previous studies observed that different enhancers can achieve a wide range of transcriptional outputs through the same strategies of bursting control. For example, despite responding to different transcription factors, all even-skipped enhancers increase transcription by upregulating burst frequency and amplitude while burst duration remains largely constant. These shared bursting strategies suggest that a unified molecular mechanism constraints how enhancers modulate transcriptional output. Alternatively, different enhancers could have converged on the same bursting control strategy because of natural selection favoring one of these particular strategies. To distinguish between these two scenarios, we compared transcriptional bursting between endogenous and ectopic gene expression patterns. Because enhancers act under different regulatory inputs in ectopic patterns, dissimilar bursting control strategies between endogenous and ectopic patterns would suggest that enhancers adapted their bursting strategies to their trans-regulatory environment. Here, we generated ectopic even-skipped transcription patterns in fruit fly embryos and discovered that bursting strategies remain consistent in endogenous and ectopic even-skipped expression. These results provide evidence for a unified molecular mechanism shaping even-skipped bursting strategies and serve as a starting point to uncover the realm of strategies employed by other enhancers.

Click chemistry–based RNA sequencing captures bursts of transcription

Click chemistry–based RNA sequencing captures bursts of transcription

Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli.

Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.

Identification of molecular determinants of gene-specific bursting patterns by high-throughput imaging screens.

Stochastic transcriptional bursting is a universal property of active genes. While different genes exhibit distinct bursting patterns, the molecular mechanisms for gene-specific stochastic bursting are largely unknown. We have developed and applied a high-throughput-imaging based screening strategy to identify cellular factors and molecular mechanisms that determine the bursting behavior of human genes. Focusing on epigenetic regulators, we find that protein acetylation is a strong acute modulator of burst frequency, burst size and heterogeneity of bursting. Acetylation globally affects the Off-time of genes but has gene-specific effects on the On-time. Yet, these effects are not strongly linked to promoter acetylation, which do not correlate with bursting properties, and forced promoter acetylation has variable effects on bursting. Instead, we demonstrate acetylation of the Integrator complex as a key determinant of gene bursting. Specifically, we find that elevated Integrator acetylation decreases bursting frequency. Taken together our results suggest a prominent role of non-histone proteins in determining gene bursting properties, and they identify histone-independent acetylation of a transcription cofactor as an allosteric modulator of bursting via a far-downstream bursting checkpoint.