The method in the madness: Transcriptional control from stochastic action at the single-molecule scale.

Cell states result from the ordered activation of gene expression by transcription factors. Transcription factors face opposing design constraints: they need to be dynamic to trigger rapid cell state transitions, but also stable enough to maintain terminal cell identities indefinitely. Recent progress in live-cell single-molecule microscopy has helped define the biophysical principles underlying this paradox. Beyond transcription factor activity, single-molecule experiments have revealed that at nearly every level of transcription regulation, control emerges from multiple short-lived stochastic interactions, rather than deterministic, stable interactions typical of other biochemical pathways. This architecture generates consistent outcomes that can be rapidly choreographed. Here, we highlight recent results that demonstrate how order in transcription regulation emerges from the apparent molecular-scale chaos and discuss remaining conceptual challenges.

Identifying in vivo genetic dependencies of melanocyte and melanoma development.

The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. Validating the function of these candidates in vivo is challenging, due to low efficiency and low throughput of most model systems. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked-in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. We used this system to identify both cell-autonomous and non-cell autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our high-efficiency genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.

Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription.

Numerous components of the transcription machinery, including RNA polymerase II (Pol II), accumulate in regions of high local concentration known as clusters, which are thought to facilitate transcription. Using the histone locus of Drosophila nurse cells as a model, we find that Pol II forms long-lived, transcriptionally poised clusters distinct from liquid droplets, which contain unbound and paused Pol II. Depletion of the Integrator complex endonuclease module, but not its phosphatase module or Pol II pausing factors disperses these Pol II clusters. Consequently, histone transcription fails to reach peak levels during S-phase and aberrantly continues throughout the cell cycle. We propose that Pol II clustering is a regulatory step occurring near promoters that limits rapid gene activation to defined times. One Sentence Summary: Using the Drosophila histone locus as a model, we show that clustered RNA polymerase II is poised for synchronous activation.

Connecting Chromatin Structures to Gene Regulation Using Dynamic Polymer Simulations.

The transfer of regulatory information between distal loci on chromatin is thought to involve physical proximity, but key biophysical features of these contacts remain unclear. For instance, it is unknown how close and for how long two loci need to be in order to productively interact. The main challenge is that it is currently impossible to measure chromatin dynamics with high spatiotemporal resolution at scale. Polymer simulations provide an accessible and rigorous way to test biophysical models of chromatin regulation, yet there is a lack of simple and general methods for extracting the values of model parameters. Here we adapt the Nelder-Mead simplex optimization algorithm to select the best polymer model matching a given Hi-C dataset, using the MYC locus as an example. The model's biophysical parameters predict a compartmental rearrangement of the MYC locus in leukemia, which we validate with single-cell measurements. Leveraging trajectories predicted by the model, we find that loci with similar Hi-C contact frequencies can exhibit widely different contact dynamics. Interestingly, the frequency of productive interactions between loci exhibits a non-linear relationship with their Hi-C contact frequency when we enforce a specific capture radius and contact duration. These observations are consistent with recent experimental observations and suggest that the dynamic ensemble of chromatin configurations, rather than average contact matrices, is required to fully predict long-range chromatin interactions.

A universal deep-learning model for zinc finger design enables transcription factor reprogramming.

Cys2His2 zinc finger (ZF) domains engineered to bind specific target sequences in the genome provide an effective strategy for programmable regulation of gene expression, with many potential therapeutic applications. However, the structurally intricate engagement of ZF domains with DNA has made their design challenging. Here we describe the screening of 49 billion protein-DNA interactions and the development of a deep-learning model, ZFDesign, that solves ZF design for any genomic target. ZFDesign is a modern machine learning method that models global and target-specific differences induced by a range of library environments and specifically takes into account compatibility of neighboring fingers using a novel hierarchical transformer architecture. We demonstrate the versatility of designed ZFs as nucleases as well as activators and repressors by seamless reprogramming of human transcription factors. These factors could be used to upregulate an allele of haploinsufficiency, downregulate a gain-of-function mutation or test the consequence of regulation of a single gene as opposed to the many genes that a transcription factor would normally influence.

RS-FISH: precise, interactive, fast, and scalable FISH spot detection.

Fluorescent in-situ hybridization (FISH)-based methods extract spatially resolved genetic and epigenetic information from biological samples by detecting fluorescent spots in microscopy images, an often challenging task. We present Radial Symmetry-FISH (RS-FISH), an accurate, fast, and user-friendly software for spot detection in two- and three-dimensional images. RS-FISH offers interactive parameter tuning and readily scales to large datasets and image volumes of cleared or expanded samples using distributed processing on workstations, clusters, or the cloud. RS-FISH maintains high detection accuracy and low localization error across a wide range of signal-to-noise ratios, a key feature for single-molecule FISH, spatial transcriptomics, or spatial genomics applications.

Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation.

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.

Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.

Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.

Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.

Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin.

Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending 'tug-of-war' that controls a temporally shifting window of accessibility for the transcription initiation machinery.

Transcription Factor Dynamics.

To predict transcription, one needs a mechanistic understanding of how the numerous required transcription factors (TFs) explore the nuclear space to find their target genes, assemble, cooperate, and compete with one another. Advances in fluorescence microscopy have made it possible to visualize real-time TF dynamics in living cells, leading to two intriguing observations: first, most TFs contact chromatin only transiently; and second, TFs can assemble into clusters through their intrinsically disordered regions. These findings suggest that highly dynamic events and spatially structured nuclear microenvironments might play key roles in transcription regulation that are not yet fully understood. The emerging model is that while some promoters directly convert TF-binding events into on/off cycles of transcription, many others apply complex regulatory layers that ultimately lead to diverse phenotypic outputs. Cracking this kinetic code is an ongoing and challenging task that is made possible by combining innovative imaging approaches with biophysical models.

Single-molecule tracking of transcription protein dynamics in living cells: seeing is believing, but what are we seeing?

A universe of transcription factors (TFs), cofactors, as well as chromatin remodeling and modifying enzymes combine or compete on chromatin to control transcription. Measuring quantitatively how these proteins dynamically interact is required in order to formulate models with predictive ability to elucidate transcription control mechanisms. Single molecule tracking (SMT) provides a powerful tool towards this goal: it is a fluorescence microscopy approach that measures the location and mobility of individual TF molecules, as well as their rates of association with and dissociation from chromatin in the physiological context of the living cell. Here we review SMT principles, and discuss key TF properties uncovered by live-cell SMT, such as fast turnover (seconds), and formation of clusters that locally increase activity.

Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction.

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

Three-dimensional chromatin landscapes in T cell acute lymphoblastic leukemia.

Differences in three-dimensional (3D) chromatin architecture can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer-promoter interactions, impacting gene expression and leading to human disease. Here we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) by using primary human leukemia specimens and examine the dynamic responses of this architecture to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP-seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD 'fusion' event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small-molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia.

Histone H3K27 acetylation precedes active transcription during zebrafish zygotic genome activation as revealed by live-cell analysis.

Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.

Imaging the Life and Death of mRNAs in Single Cells.

RNA plays a central role in gene expression from its transcription in the nucleus through translation and degradation in the cytoplasm. Technological advances in fluorescent microscopy and labeling methodologies have made it possible to detect single molecules of RNA in both fixed and living cells. Here, we focus on the recent developments in RNA imaging that have allowed quantitatively measuring the lives of individual transcripts from birth to death and all the events in between in single cells and tissues. Direct observation of RNAs within their native cellular environment has revealed a complex layer of spatial and temporal regulation that has profoundly impacted our understanding of RNA biology.

Single-Molecule Sensitivity RNA FISH Analysis of Influenza Virus Genome Trafficking.

Influenza A virus is an enveloped virus with a segmented genome consisting of eight negative-sense, single-stranded RNAs. Accumulating evidence has revealed that influenza viruses selectively package their genomes. However, less is known about how different viral RNA segments are selected for incorporation into progeny virions. Understanding the trafficking routes and assembly process of various viral RNA segments during infection will shed light on the mechanisms of selective genome packaging for influenza A viruses. This chapter describes the single-molecule sensitivity RNA fluorescence in situ hybridization assay (smFISH) for influenza viral RNAs, a method used to analyze the distributions and trafficking of viral RNAs in infected cells with segment specificity. Hybridization using 20 or more short fluorescently labeled DNA probes allows the detection of viral RNAs with single-molecule sensitivity. The following imaging analyses provide information regarding quantitative measurements of vRNA abundance and the relative positions of the different viral RNA segments in cells. This chapter also includes a protocol for combining immunofluorescence techniques with smFISH, which is useful to analyze the positions of viral RNAs relative to viral/cellular proteins in infected cells.

Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments.

The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging.

mRNA quantification using single-molecule FISH in Drosophila embryos.

Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured illumination microscopy (iSIM, a super-resolution imaging approach) and a spot-detection algorithm. Fixed Drosophila embryos are hybridized in solution with a mixture of smFISH probes, mounted onto coverslips and imaged in 3D. Individual fluorescently labeled mRNAs are then localized within tissues and counted using spot-detection software to generate quantitative, spatially resolved gene expression data sets. With minimum guidance, a graduate student can successfully implement this protocol. The smFISH procedure described here can be completed in 4-5 d.

Quantitative mRNA imaging throughout the entire Drosophila brain.

We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).