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.

HiTIPS: High-Throughput Image Processing Software for the Study of Nuclear Architecture and Gene Expression.

High-throughput imaging (HTI) generates complex imaging datasets from a large number of experimental perturbations. Commercial HTI software for image analysis workflows does not allow full customization and adoption of new image processing algorithms in the analysis modules. While open-source HTI analysis platforms provide individual modules in the workflow, like nuclei segmentation, spot detection, or cell tracking, they are often limited in integrating novel analysis modules or algorithms. Here, we introduce the High-Throughput Image Processing Software (HiTIPS) to expand the range and customization of existing HTI analysis capabilities. HiTIPS incorporates advanced image processing and machine learning algorithms for automated cell and nuclei segmentation, spot signal detection, nucleus tracking, spot tracking, and quantification of spot signal intensity. Furthermore, HiTIPS features a graphical user interface that is open to integration of new algorithms for existing analysis pipelines and to adding new analysis pipelines through separate plugins. To demonstrate the utility of HiTIPS, we present three examples of image analysis workflows for high-throughput DNA FISH, immunofluorescence (IF), and live-cell imaging of transcription in single cells. Altogether, we demonstrate that HiTIPS is a user-friendly, flexible, and open-source HTI analysis platform for a variety of cell biology applications.

Synthetic analysis of chromatin tracing and live-cell imaging indicates pervasive spatial coupling between genes.

The role of the spatial organization of chromosomes in directing transcription remains an outstanding question in gene regulation. Here, we analyze two recent single-cell imaging methodologies applied across hundreds of genes to systematically analyze the contribution of chromosome conformation to transcriptional regulation. Those methodologies are (1) single-cell chromatin tracing with super-resolution imaging in fixed cells; and (2) high-throughput labeling and imaging of nascent RNA in living cells. Specifically, we determine the contribution of physical distance to the coordination of transcriptional bursts. We find that individual genes adopt a constrained conformation and reposition toward the centroid of the surrounding chromatin upon activation. Leveraging the variability in distance inherent in single-cell imaging, we show that physical distance - but not genomic distance - between genes on individual chromosomes is the major factor driving co-bursting. By combining this analysis with live-cell imaging, we arrive at a corrected transcriptional correlation of [Formula: see text] for genes separated by < 400 nm. We propose that this surprisingly large correlation represents a physical property of human chromosomes and establishes a benchmark for future experimental studies.

Post-transcriptional polyadenylation site cleavage maintains 3'-end processing upon DNA damage.

The recognition of polyadenylation signals (PAS) in eukaryotic pre-mRNAs is usually coupled to transcription termination, occurring while pre-mRNA is chromatin-bound. However, for some pre-mRNAs, this 3'-end processing occurs post-transcriptionally, i.e., through a co-transcriptional cleavage (CoTC) event downstream of the PAS, leading to chromatin release and subsequent PAS cleavage in the nucleoplasm. While DNA-damaging agents trigger the shutdown of co-transcriptional chromatin-associated 3'-end processing, specific compensatory mechanisms exist to ensure efficient 3'-end processing for certain pre-mRNAs, including those that encode proteins involved in the DNA damage response, such as the tumor suppressor p53. We show that cleavage at the p53 polyadenylation site occurs in part post-transcriptionally following a co-transcriptional cleavage event. Cells with an engineered deletion of the p53 CoTC site exhibit impaired p53 3'-end processing, decreased mRNA and protein levels of p53 and its transcriptional target p21, and altered cell cycle progression upon UV-induced DNA damage. Using a transcriptome-wide analysis of PAS cleavage, we identify additional pre-mRNAs whose PAS cleavage is maintained in response to UV irradiation and occurring post-transcriptionally. These findings indicate that CoTC-type cleavage of pre-mRNAs, followed by PAS cleavage in the nucleoplasm, allows certain pre-mRNAs to escape 3'-end processing inhibition in response to UV-induced DNA damage.

Regulating gene expression through control of transcription factor multivalent interactions.

Chong et al. (2022) show how the propensity of transcription factors (TFs) to associate into hubs must be finely regulated for optimal transcription.

MYC amplifies gene expression through global changes in transcription factor dynamics.

The MYC oncogene has been studied for decades, yet there is still intense debate over how this transcription factor controls gene expression. Here, we seek to answer these questions with an in vivo readout of discrete events of gene expression in single cells. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursting and transcription factor binding dynamics in human cells. We find that the immediate consequence of MYC overexpression is an increase in the duration rather than in the frequency of bursts, a functional role that is different from the majority of human transcription factors. We further propose that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA polymerase II complex assembly and productive elongation.

Towards a 'Spot On' Understanding of Transcription in the Nucleus.

Regulation of transcription by RNA Polymerase II (RNAPII) is a rapidly evolving area of research. Technological developments in microscopy have revealed insight into the dynamics, structure, and localization of transcription components within single cells. A frequent observation in many studies is the appearance of 'spots' in cell nuclei associated with the transcription process. In this review we highlight studies that characterize the temporal and spatial characteristics of these spots, examine possible pitfalls in interpreting these kind of imaging data, and outline directions where single-cell imaging may advance in ways to further our understanding of transcription regulation.

Dynamic imaging of nascent RNA reveals general principles of transcription dynamics and stochastic splice site selection.

The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.

The Stochastic Genome and Its Role in Gene Expression.

Mammalian genomes have distinct levels of spatial organization and structure that have been hypothesized to play important roles in transcription regulation. Although much has been learned about these architectural features with ensemble techniques, single-cell studies are showing a new universal trend: Genomes are stochastic and dynamic at every level of organization. Stochastic gene expression, on the other hand, has been studied for years. In this review, we probe whether there is a causative link between the two phenomena. We specifically discuss the functionality of chromatin state, topologically associating domains (TADs), and enhancer biology in light of their stochastic nature and their specific roles in stochastic gene expression. We highlight persistent fundamental questions in this area of research.

Enhancers, gene regulation, and genome organization.

How transcriptional enhancers function to activate distant genes has been the subject of lively investigation for decades. "Enhancers, gene regulation, and genome organization" was the subject of a virtual meeting held November 16-17, 2020, under sponsorship of the National Cancer Institute (NCI), the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH). The goal of the meeting was to advance an understanding of how transcriptional enhancers function within the framework of the folded genome as we understand it, emphasizing how levels of organization may influence each other and may contribute to the spatiotemporal specification of transcription. Here we focus on broad questions about enhancer function that remain unsettled and that we anticipate will be central to work in this field going forward. Perforce, we cover contributions of only some speakers and apologize to other contributors in vital areas that we could not include because of space constraints.

Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation.

U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3' splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.

Transcriptional Decoding of Morphogen Gradients during Development.

The ability to visualize transcription in real time in living organisms has enabled a new generation of gene expression studies in development. In this issue of Developmental Cell, Hoppe et al. show that the bone morphogenetic protein gradient is decoded through frequency modulation encoded by enhancers.

Transcription in Living Cells: Molecular Mechanisms of Bursting.

Transcription in several organisms from certain bacteria to humans has been observed to be stochastic in nature: toggling between active and inactive states. Periods of active nascent RNA synthesis known as bursts represent individual gene activation events in which multiple polymerases are initiated. Therefore, bursting is the single locus illustration of both gene activation and repression. Although transcriptional bursting was originally observed decades ago, only recently have technological advances enabled the field to begin elucidating gene regulation at the single-locus level. In this review, we focus on how biochemical, genomic, and single-cell data describe the regulatory steps of transcriptional bursts.

Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting.

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.

Structure and Function in Drosophila Chromosomes: Visualizing Topological Domains.

Cardozo Gizzi et al. (2019) develop a new sequential imaging methodology (Hi-M) for observing chromosome structure in the Drosophila blastoderm and find that topological domains in single nuclei change in response to transcriptional activation.

The splicing factor U2AF1 contributes to cancer progression through a noncanonical role in translation regulation.

Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias, and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid-binding domain, recognition of the 3' splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we uncovered a noncanonical function of U2AF1, showing that it directly binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysome profiling indicates that the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8, which contributes to metastasis, inflammation, and cancer progression in mice and humans.

Intrinsic Dynamics of a Human Gene Reveal the Basis of Expression Heterogeneity.

Transcriptional regulation in metazoans occurs through long-range genomic contacts between enhancers and promoters, and most genes are transcribed in episodic "bursts" of RNA synthesis. To understand the relationship between these two phenomena and the dynamic regulation of genes in response to upstream signals, we describe the use of live-cell RNA imaging coupled with Hi-C measurements and dissect the endogenous regulation of the estrogen-responsive TFF1 gene. Although TFF1 is highly induced, we observe short active periods and variable inactive periods ranging from minutes to days. The heterogeneity in inactive times gives rise to the widely observed "noise" in human gene expression and explains the distribution of protein levels in human tissue. We derive a mathematical model of regulation that relates transcription, chromosome structure, and the cell's ability to sense changes in estrogen and predicts that hypervariability is largely dynamic and does not reflect a stable biological state.

A Muscle-Specific Enhancer RNA Mediates Cohesin Recruitment and Regulates Transcription In trans.

The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA (CEeRNA) regulates transcription of the adjacent MyoD gene, whereas DRReRNA affects expression of Myogenin in trans. We found that DRReRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRReRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressing DRReRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRReRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRReRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression.

Splicing heterogeneity: separating signal from noise.

Single-cell analyses have revealed a tremendous variety among cells in the abundance and chemical composition of RNA. Much of this heterogeneity is due to alternative splicing by the spliceosome. Little is known about how many of the resulting isoforms are biologically functional or just provide noise with little to no impact. The dynamic nature of the spliceosome provides numerous opportunities for regulation but is also the source of stochastic fluctuations. We discuss possible origins of splicing stochasticity, the experimental approaches for studying heterogeneity in isoforms, and the potential biological significance of noisy splicing in development and disease.

Single-cell systems biology: probing the basic unit of information flow.

Gene expression varies across cells in a population or a tissue. This heterogeneity has come into sharp focus in recent years through developments in new imaging and sequencing technologies. However, our ability to measure variation has outpaced our ability to interpret it. Much of the variability may arise from random effects occurring in the processes of gene expression (transcription, RNA processing and decay, translation). The molecular basis of these effects is largely unknown. Likewise, a functional role of this variability in growth, differentiation and disease has only been elucidated in a few cases. In this review, we highlight recent experimental and theoretical advances for measuring and analyzing stochastic variation.