Construction and Characterization of Light-Responsive Transcriptional Systems.

Optogenetic tools provide a means for controlling cellular processes that is rapid, noninvasive, and spatially and temporally precise. With the increase in available optogenetic systems, quantitative comparisons of their performances become important to guide experiments. In this chapter, we first discuss how photoreceptors can be repurposed for light-mediated control of transcription. Then, we provide a detailed protocol for characterizing light-regulated transcriptional systems in budding yeast using fluorescence time-lapse microscopy and mathematical modeling, expanding on our recent publication (Gligorovski et al., Nat Commun 14:3810, 2023).

A bistable autoregulatory module in the developing embryo commits cells to binary expression fates.

Bistable autoactivation has been proposed as a mechanism for cells to adopt binary fates during embryonic development. However, it is unclear whether the autoactivating modules found within developmental gene regulatory networks are bistable, unless their parameters are quantitatively determined. Here, we combine in vivo live imaging with mathematical modeling to dissect the binary cell fate dynamics of the fruit fly pair-rule gene fushi tarazu (ftz), which is regulated by two known enhancers: the early (non-autoregulating) element and the autoregulatory element. Live imaging of transcription and protein concentration in the blastoderm revealed that binary Ftz fates are achieved as Ftz expression rapidly transitions from being dictated by the early element to the autoregulatory element. Moreover, we discovered that Ftz concentration alone is insufficient to activate the autoregulatory element, and that this element only becomes responsive to Ftz at a prescribed developmental time. Based on these observations, we developed a dynamical systems model and quantitated its kinetic parameters directly from experimental measurements. Our model demonstrated that the ftz autoregulatory module is indeed bistable and that the early element transiently establishes the content of the binary cell fate decision to which the autoregulatory module then commits. Further in silico analysis revealed that the autoregulatory element locks the Ftz fate quickly, within 35 min of exposure to the transient signal of the early element. Overall, our work confirms the widely held hypothesis that autoregulation can establish developmental fates through bistability and, most importantly, provides a framework for the quantitative dissection of cellular decision-making.

Division of developmental phases of freshwater leech Whitmania pigra and key genes related to neurogenesis revealed by whole genome and transcriptome analysis.

The freshwater leech Whitmania pigra (W. pigra) Whitman (Annelida phylum) is a model organism for neurodevelopmental studies. However, molecular biology research on its embryonic development is still scarce. Here, we described a series of developmental stages of the W. pigra embryos and defined five broad stages of embryogenesis: cleavage stages, blastocyst stage, gastrula stage, organogenesis and refinement, juvenile. We obtained a total of 239.64 Gb transcriptome data of eight representative developmental phases of embryos (from blastocyst stage to maturity), which was then assembled into 21,482 unigenes according to our reference genome sequenced by single-molecule real-time (SMRT) long-read sequencing. We found 3114 genes differentially expressed during the eight phases with phase-specific expression pattern. Using a comprehensive transcriptome dataset, we demonstrated that 57, 49 and 77 DEGs were respectively related to morphogenesis, signal pathways and neurogenesis. 49 DEGs related to signal pathways included 30 wnt genes, 14 notch genes, and 5 hedgehog genes. In particular, we found a cluster consisting of 7 genes related to signal pathways as well as synapses, which were essential for regulating embryonic development. Eight genes cooperatively participated in regulating neurogenesis. Our results reveal the whole picture of W. pigra development mechanism from the perspective of transcriptome and provide new clues for organogenesis and neurodevelopmental studies of Annelida species.

Competing constraints shape the nonequilibrium limits of cellular decision-making.

Gene regulation is central to cellular function. Yet, despite decades of work, we lack quantitative models that can predict how transcriptional control emerges from molecular interactions at the gene locus. Thermodynamic models of transcription, which assume that gene circuits operate at equilibrium, have previously been employed with considerable success in the context of bacterial systems. However, the presence of ATP-dependent processes within the eukaryotic transcriptional cycle suggests that equilibrium models may be insufficient to capture how eukaryotic gene circuits sense and respond to input transcription factor concentrations. Here, we employ simple kinetic models of transcription to investigate how energy dissipation within the transcriptional cycle impacts the rate at which genes transmit information and drive cellular decisions. We find that biologically plausible levels of energy input can lead to significant gains in how rapidly gene loci transmit information but discover that the regulatory mechanisms underlying these gains change depending on the level of interference from noncognate activator binding. When interference is low, information is maximized by harnessing energy to push the sensitivity of the transcriptional response to input transcription factors beyond its equilibrium limits. Conversely, when interference is high, conditions favor genes that harness energy to increase transcriptional specificity by proofreading activator identity. Our analysis further reveals that equilibrium gene regulatory mechanisms break down as transcriptional interference increases, suggesting that energy dissipation may be indispensable in systems where noncognate factor interference is sufficiently large.

Promoter-pervasive transcription causes RNA polymerase II pausing to boost DOG1 expression in response to salt.

Eukaryotic genomes are pervasively transcribed by RNA polymerase II. Yet, the molecular and biological implications of such a phenomenon are still largely puzzling. Here, we describe noncoding RNA transcription upstream of the Arabidopsis thaliana DOG1 gene, which governs salt stress responses and is a key regulator of seed dormancy. We find that expression of the DOG1 gene is induced by salt stress, thereby causing a delay in seed germination. We uncover extensive transcriptional activity on the promoter of the DOG1 gene, which produces a variety of lncRNAs. These lncRNAs, named PUPPIES, are co-directionally transcribed and extend into the DOG1 coding region. We show that PUPPIES RNAs respond to salt stress and boost DOG1 expression, resulting in delayed germination. This positive role of pervasive PUPPIES transcription on DOG1 gene expression is associated with augmented pausing of RNA polymerase II, slower transcription and higher transcriptional burst size. These findings highlight the positive role of upstream co-directional transcription in controlling transcriptional dynamics of downstream genes.

The dynamic response to hypo-osmotic stress reveals distinct stages of freshwater acclimation by a euryhaline diatom.

The salinity gradient separating marine and freshwater environments is a major ecological divide, and the mechanisms by which most organisms adapt to new salinity environments are poorly understood. Diatoms are a lineage of ancestrally marine microalgae that have repeatedly colonized and diversified in freshwaters. Cyclotella cryptica is a euryhaline diatom found in salinities ranging from fully freshwater to fully marine, thus providing a powerful system for understanding the genomic mechanisms for mitigating and acclimating to low salinity. To understand how diatoms mitigate acute hypo-osmotic stress, we abruptly shifted C. cryptica from seawater to freshwater and performed transcriptional profiling during the first 10 h. Freshwater shock dramatically remodelled the transcriptome, with ~50% of the genome differentially expressed in at least one time point. The peak response occurred within 1 h, with strong repression of genes involved in cell growth and osmolyte production, and strong induction of specific stress defence genes. Transcripts largely returned to baseline levels within 4-10 h, with growth resuming shortly thereafter, suggesting that gene expression dynamics may be useful for predicting acclimation. Moreover, comparison to a transcriptomics study of C. cryptica following months-long acclimation to freshwater revealed little overlap between the genes and processes differentially expressed in cells exposed to acute stress versus fully acclimated conditions. Altogether, this study highlights the power of time-resolved transcriptomics to reveal fundamental insights into how cells dynamically respond to an acute environmental shift and provides new insights into how diatoms mitigate natural salinity fluctuations and have successfully diversified across freshwater habitats worldwide.

Supercoiling-mediated feedback rapidly couples and tunes transcription.

Transcription induces a wave of DNA supercoiling, altering the binding affinity of RNA polymerases and reshaping the biochemical landscape of gene regulation. As supercoiling rapidly diffuses, transcription dynamically reshapes the regulation of proximal genes, forming a complex feedback loop. However, a theoretical framework is needed to integrate biophysical regulation with biochemical transcriptional regulation. To investigate the role of supercoiling-mediated feedback within multi-gene systems, we model transcriptional regulation under the influence of supercoiling-mediated polymerase dynamics, allowing us to identify patterns of expression that result from physical inter-gene coupling. We find that gene syntax-the relative ordering and orientation of genes-defines the expression profiles, variance, burst dynamics, and inter-gene correlation of two-gene systems. Furthermore, supercoiling can enhance or weaken biochemical regulation. Our results suggest that supercoiling couples behavior between neighboring genes, providing a regulatory mechanism that tunes transcriptional variance in engineered gene networks and explains the behavior of co-localized native circuits.

Transcriptomic Analysis Reveals the Correlation between End-of-Day Far Red Light and Chilling Stress in Setaria viridis.

Low temperature and end-of-day far-red (EOD-FR) light signaling are two key factors limiting plant production and geographical location worldwide. However, the transcriptional dynamics of EOD-FR light conditions during chilling stress remain poorly understood. Here, we performed a comparative RNA-Seq-based approach to identify differentially expressed genes (DEGs) related to EOD-FR and chilling stress in Setaria viridis. A total of 7911, 324, and 13431 DEGs that responded to low temperature, EOD-FR and these two stresses were detected, respectively. Further DEGs analysis revealed that EOD-FR may enhance cold tolerance in plants by regulating the expression of genes related to cold tolerance. The result of weighted gene coexpression network analysis (WGCNA) using 13431 nonredundant DEGs exhibited 15 different gene network modules. Interestingly, a CO-like transcription factor named BBX2 was highly expressed under EOD-FR or chilling conditions. Furthermore, we could detect more expression levels when EOD-FR and chilling stress co-existed. Our dataset provides a valuable resource for the regulatory network involved in EOD-FR signaling and chilling tolerance in C4 plants.

Image-Based Single-Molecule Analysis of Notch-Dependent Transcription in Its Natural Context.

Notch signaling is crucial to animal development and homeostasis. Notch triggers the transcription of its target genes, which produce diverse outcomes depending on context. The high resolution and spatially precise assessment of Notch-dependent transcription is essential for understanding how Notch operates normally in its native context in vivo and how Notch defects lead to pathogenesis. Here we present biological and computational methods to assess Notch-dependent transcriptional activation in stem cells within their niche, focusing on germline stem cells in the nematode Caenorhabditis elegans. Specifically, we describe visualization of single RNAs in fixed gonads using single-molecule RNA fluorescence in situ hybridization (smFISH), live imaging of transcriptional bursting in the intact organism using the MS2 system, and custom-made MATLAB codes, implementing new image processing algorithms to capture the spatiotemporal patterns of Notch-dependent transcriptional activation. These methods allow a powerful analysis of in vivo transcriptional activation and its dynamics in a whole tissue. Our methods can be adapted to essentially any tissue or cell type for any transcript.

Comparison of Transcriptional Signatures of Three Staphylococcal Superantigenic Toxins in Human Melanocytes.

Staphylococcus aureus, a gram-positive bacterium, causes toxic shock through the production of superantigenic toxins (sAgs) known as Staphylococcal enterotoxins (SE), serotypes A-J (SEA, SEB, etc.), and toxic shock syndrome toxin-1 (TSST-1). The chronology of host transcriptomic events that characterizes the response to the pathogenesis of superantigenic toxicity remains uncertain. The focus of this study was to elucidate time-resolved host responses to three toxins of the superantigenic family, namely SEA, SEB, and TSST-1. Due to the evolving critical role of melanocytes in the host's immune response against environmental harmful elements, we investigated herein the transcriptomic responses of melanocytes after treatment with 200 ng/mL of SEA, SEB, or TSST-1 for 0.5, 2, 6, 12, 24, or 48 h. Functional analysis indicated that each of these three toxins induced a specific transcriptional pattern. In particular, the time-resolved transcriptional modulations due to SEB exposure were very distinct from those induced by SEA and TSST-1. The three superantigens share some similarities in the mechanisms underlying apoptosis, innate immunity, and other biological processes. Superantigen-specific signatures were determined for the functional dynamics related to necrosis, cytokine production, and acute-phase response. These differentially regulated networks can be targeted for therapeutic intervention and marked as the distinguishing factors for the three sAgs.

Suppression and Activation of Intracellular Immune Response in Initial Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most important emerging pathogen worldwide, but its early transcriptional dynamics and host immune response remain unclear. Herein, the expression profiles of viral interactions with different types of hosts were comprehensively dissected to shed light on the early infection strategy of SARS-CoV-2 and the host immune response against infection. SARS-CoV-2 was found to exhibit a two-stage transcriptional strategy within the first 24 h of infection, comprising a lag phase that ends with the virus being paused and a log phase that starts when the viral load increases rapidly. Interestingly, the host innate immune response was found not to be activated (latent period) until the virus entered the log stage. Noteworthy, when intracellular immunity is suppressed, SARS-CoV-2 shows a correlation with dysregulation of metal ion homeostasis. Herein, the inhibitory activity of copper ions against SARS-CoV-2 was further validated in in vitro experiments. Coronavirus disease 2019-related genes (including CD38, PTX3, and TCN1) were also identified, which may serve as candidate host-restricted factors for interventional therapy. Collectively, these results confirm that the two-stage strategy of SARS-CoV-2 effectively aids its survival in early infection by regulating the host intracellular immunity, highlighting the key role of interferon in viral infection and potential therapeutic candidates for further investigations on antiviral strategies.

Investigating Transcriptional Dynamics Changes and Time-Dependent Marker Gene Expression in the Early Period After Skeletal Muscle Injury in Rats.

Following skeletal muscle injury (SMI), from post-injury reaction to repair consists of a complex series of dynamic changes. However, there is a paucity of research on detailed transcriptional dynamics and time-dependent marker gene expression in the early stages after SMI. In this study, skeletal muscle tissue in rats was taken at 4 to 48 h after injury for next-generation sequencing. We examined the transcriptional kinetics characteristics during above time periods after injury. STEM and maSigPro were used to screen time-correlated genes. Integrating 188 time-correlated genes with 161 genes in each time-related gene module by WGCNA, we finally identified 18 network-node regulatory genes after SMI. Histological staining analyses confirmed the mechanisms underlying changes in the tissue damage to repair process. Our research linked a variety of dynamic biological processes with specific time periods and provided insight into the characteristics of transcriptional dynamics, as well as screened time-related biological indicators with biological significance in the early stages after SMI.

Integrative Single-Cell Transcriptomic Analysis of Human Fetal Thymocyte Development.

Intrathymic differentiation of T lymphocytes begins as early as intrauterine stage, yet the T cell lineage decisions of human fetal thymocytes at different gestational ages are not currently understood. Here, we performed integrative single-cell analyses of thymocytes across gestational ages. We identified conserved candidates underlying the selection of T cell receptor (TCR) lineages in different human fetal stages. The trajectory of early thymocyte commitment during fetal growth was also characterized. Comparisons with mouse data revealed conserved and species-specific transcriptional dynamics of thymocyte proliferation, apoptosis and selection. Genome-wide association study (GWAS) data associated with multiple autoimmune disorders were analyzed to characterize susceptibility genes that are highly expressed at specific stages during fetal thymocyte development. In summary, our integrative map describes previously underappreciated aspects of human thymocyte development, and provides a comprehensive reference for understanding T cell lymphopoiesis in a self-tolerant and functional adaptive immune system.

Temporal integration of inductive cues on the way to gastrulation.

Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila, a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein (hkb) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.

Capturing and Understanding the Dynamics and Heterogeneity of Gene Expression in the Living Cell.

The regulation of gene expression is a fundamental process enabling cells to respond to internal and external stimuli or to execute developmental programs. Changes in gene expression are highly dynamic and depend on many intrinsic and extrinsic factors. In this review, we highlight the dynamic nature of transient gene expression changes to better understand cell physiology and development in general. We will start by comparing recent in vivo procedures to capture gene expression in real time. Intrinsic factors modulating gene expression dynamics will then be discussed, focusing on chromatin modifications. Furthermore, we will dissect how cell physiology or age impacts on dynamic gene regulation and especially discuss molecular insights into acquired transcriptional memory. Finally, this review will give an update on the mechanisms of heterogeneous gene expression among genetically identical individual cells. We will mainly focus on state-of-the-art developments in the yeast model but also cover higher eukaryotic systems.

A matter of time: Using dynamics and theory to uncover mechanisms of transcriptional bursting.

Eukaryotic transcription generally occurs in bursts of activity lasting minutes to hours; however, state-of-the-art measurements have revealed that many of the molecular processes that underlie bursting, such as transcription factor binding to DNA, unfold on timescales of seconds. This temporal disconnect lies at the heart of a broader challenge in physical biology of predicting transcriptional outcomes and cellular decision-making from the dynamics of underlying molecular processes. Here, we review how new dynamical information about the processes underlying transcriptional control can be combined with theoretical models that predict not only averaged transcriptional dynamics, but also their variability, to formulate testable hypotheses about the molecular mechanisms underlying transcriptional bursting and control.

Variation of Human Neural Stem Cells Generating Organizer States In Vitro before Committing to Cortical Excitatory or Inhibitory Neuronal Fates.

Better understanding of the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here, we use RNA sequencing, cell imaging, and lineage tracing of mouse and human in vitro NSCs and monkey brain sections to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs develop from pluripotency in vitro, they transition through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines vary in these early patterning states, leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analyses of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon.

Understanding Quantitative Circadian Regulations Are Crucial Towards Advancing Chronotherapy.

Circadian rhythms have a deep impact on most aspects of physiology. In most organisms, especially mammals, the biological rhythms are maintained by the indigenous circadian clockwork around geophysical time (~24-h). These rhythms originate inside cells. Several core components are interconnected through transcriptional/translational feedback loops to generate molecular oscillations. They are tightly controlled over time. Also, they exert temporal controls over many fundamental physiological activities. This helps in coordinating the body's internal time with the external environments. The mammalian circadian clockwork is composed of a hierarchy of oscillators, which play roles at molecular, cellular, and higher levels. The master oscillation has been found to be developed at the hypothalamic suprachiasmatic nucleus in the brain. It acts as the core pacemaker and drives the transmission of the oscillation signals. These signals are distributed across different peripheral tissues through humoral and neural connections. The synchronization among the master oscillator and tissue-specific oscillators offer overall temporal stability to mammals. Recent technological advancements help us to study the circadian rhythms at dynamic scale and systems level. Here, we outline the current understanding of circadian clockwork in terms of molecular mechanisms and interdisciplinary concepts. We have also focused on the importance of the integrative approach to decode several crucial intricacies. This review indicates the emergence of such a comprehensive approach. It will essentially accelerate the circadian research with more innovative strategies, such as developing evidence-based chronotherapeutics to restore de-synchronized circadian rhythms.

Watching gene expression in color.

A combination of two fluorescent proteins with different half-lives allows gene expression to be followed with improved time resolution.

Transcriptomic Analysis of Leaf Sheath Maturation in Maize.

The morphological development of the leaf greatly influences plant architecture and crop yields. The maize leaf is composed of a leaf blade, ligule and sheath. Although extensive transcriptional profiling of the tissues along the longitudinal axis of the developing maize leaf blade has been conducted, little is known about the transcriptional dynamics in sheath tissues, which play important roles in supporting the leaf blade. Using a comprehensive transcriptome dataset, we demonstrated that the leaf sheath transcriptome dynamically changes during maturation, with the construction of basic cellular structures at the earliest stages of sheath maturation with a transition to cell wall biosynthesis and modifications. The transcriptome again changes with photosynthesis and lignin biosynthesis at the last stage of sheath tissue maturation. The different tissues of the maize leaf are highly specialized in their biological functions and we identified 15 genes expressed at significantly higher levels in the leaf sheath compared with their expression in the leaf blade, including the BOP2 homologs GRMZM2G026556 and GRMZM2G022606, DOGT1 (GRMZM2G403740) and transcription factors from the B3 domain, C2H2 zinc finger and homeobox gene families, implicating these genes in sheath maturation and organ specialization.