Multifaceted effects on even-skipped transcriptional dynamics upon Krüppel dosage changes.

Although fluctuations in transcription factor (TF) dosage are often well tolerated, TF dosage modulation can change the target gene expression dynamics and result in significant non-lethal developmental phenotypes. Using MS2/MCP-mediated quantitative live imaging in early Drosophila embryos, we analyzed how changing levels of the gap gene Krüppel (Kr) affects transcriptional dynamics of the pair-rule gene even-skipped (eve). Halving the Kr dosage leads to a transient posterior expansion of the eve stripe 2 and an anterior shift of stripe 5. Surprisingly, the most significant changes are observed in eve stripes 3 and 4, the enhancers of which do not contain Kr-binding sites. In Kr heterozygous embryos, both stripes 3 and 4 display narrower widths, anteriorly shifted boundaries and reduced mRNA production levels. We show that Kr dosage indirectly affects stripe 3 and 4 dynamics by modulating other gap gene dynamics. We quantitatively correlate moderate body segment phenotypes of Kr heterozygotes with spatiotemporal changes in eve expression. Our results indicate that nonlinear relationships between TF dosage and phenotypes underlie direct TF-DNA and indirect TF-TF interactions.

Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos.

It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. In this study, we utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, we found that a mutation in any one of these binding sites causes a drastic reduction in transcriptional amplitude, resulting in a reduction in mRNA production of the target gene. We provide evidence of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. We propose that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates.

Regulation of the dynamic RNA Pol II elongation rate in Drosophila embryos.

An increasing number of studies have shown the key role that RNA polymerase II (RNA Pol II) elongation plays in gene regulation. We systematically examine how various enhancers, promoters, and gene body composition influence the RNA Pol II elongation rate through a single-cell-resolution live imaging assay. By using reporter constructs containing 5' MS2 and 3' PP7 repeating stem loops, we quantify the rate of RNA Pol II elongation in live Drosophila embryos. We find that promoters and exonic gene lengths have no effect on elongation rate, while enhancers and the presence of long introns may significantly change how quickly RNA Pol II moves across a gene. Furthermore, we observe in multiple constructs that the RNA Pol II elongation rate accelerates after the transcriptional onset of nuclear cycle 14 in Drosophila embryos. Our study provides a single-cell view of various mechanisms that affect the dynamic RNA Pol II elongation rate, ultimately affecting the rate of mRNA production.

Transcriptional bursting: stochasticity in deterministic development.

The transcription of DNA by RNA polymerase occurs as a discontinuous process described as transcriptional bursting. This bursting behavior is observed across species and has been quantified using various stochastic modeling approaches. There is a large body of evidence that suggests the bursts are actively modulated by transcriptional machinery and play a role in regulating developmental processes. Under a commonly used two-state model of transcription, various enhancer-, promoter- and chromatin microenvironment-associated features are found to differentially influence the size and frequency of bursting events - key parameters of the two-state model. Advancement of modeling and analysis tools has revealed that the simple two-state model and associated parameters may not sufficiently characterize the complex relationship between these features. The majority of experimental and modeling findings support the view of bursting as an evolutionarily conserved transcriptional control feature rather than an unintended byproduct of the transcription process. Stochastic transcriptional patterns contribute to enhanced cellular fitness and execution of proper development programs, which posit this mode of transcription as an important feature in developmental gene regulation. In this Review, we present compelling examples of the role of transcriptional bursting in development and explore the question of how stochastic transcription leads to deterministic organism development.

Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos.

It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. In this study, we utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, we found that a mutation in any one of these binding sites causes a drastic reduction in transcriptional amplitude, resulting in a reduction in total mRNA production of the target gene. We provide evidence of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. We propose that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates.

Unveiling dynamic enhancer-promoter interactions in Drosophila melanogaster.

Proper enhancer-promoter interactions are essential to maintaining specific transcriptional patterns and preventing ectopic gene expression. Drosophila is an ideal model organism to study transcriptional regulation due to extensively characterized regulatory regions and the ease of implementing new genetic and molecular techniques for quantitative analysis. The mechanisms of enhancer-promoter interactions have been investigated over a range of length scales. At a DNA level, compositions of both enhancer and promoter sequences affect transcriptional dynamics, including duration, amplitude, and frequency of transcriptional bursting. 3D chromatin topology is also important for proper enhancer-promoter contacts. By working competitively or cooperatively with one another, multiple, simultaneous enhancer-enhancer, enhancer-promoter, and promoter-promoter interactions often occur to maintain appropriate levels of mRNAs. For some long-range enhancer-promoter interactions, extra regulatory elements like insulators and tethering elements are required to promote proper interactions while blocking aberrant ones. This review provides an overview of our current understanding of the mechanism of enhancer-promoter interactions and how perturbations of such interactions affect transcription and subsequent physiological outcomes.

Shared Transcriptional Machinery at Homologous Alleles Leads to Reduced Transcription in Early Drosophila Embryos.

The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. We explored the possibility that interallelic interactions between two homologous alleles might affect gene regulation. Using an MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcripts of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the corresponding hemizygous allele, suggesting the possibility of allelic competition in homozygotes. However, the competition was not observed when the enhancer-promoter interaction was weakened by placing the reporter construct in a different chromosome location or by moving the enhancer further away from the promoter. Moreover, the reporter gene showed reduced transcriptional activity when a partial transcription unit (either an enhancer or reporter gene only) was in the homologous position. We propose that the transcriptional machinery that binds both the enhancer and promoter regions, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. We showed that the degree of allelic interference increased over developmental time as more Pol II was needed to activate zygotic genes. Such allelic competition was observed for an endogenous gene as well. Our study provides new insights into the role of 3D interallelic interactions in gene regulation.

Epigenome editing and epigenetic gene regulation in disease phenotypes.

Proper gene control across space and time is crucial for the seamless execution of various cellular functions. Rapid advancements in genome-wide studies revealed that in addition to genetic mutations, epigenetic modifications also play an important role in cellular processes and disease development. Epigenetic modifications, including DNA methylation and post-translational modifications on histones via methylation, acetylation, phosphorylation, etc., do not alter DNA sequences. Yet, disruptions of the epigenome can still induce gene malfunction, aberrant cell differentiation, proliferation, and apoptosis, resulting in various diseases such as cancer, neurological disorders, and autoimmune diseases. This review describes the association between epigenetic modifications and disease phenotypes, current techniques to perturb epigenome and analyze changes in gene expression, and perspectives on future epigenetic research.

Temperature-responsive optogenetic probes of cell signaling.

We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and phosphatidyl inositol-3-kinase signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.

Diverse heterochromatin-associated proteins repress distinct classes of genes and repetitive elements.

Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3. Here, we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many common genes and repeat elements. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that the enhancer of rudimentary homologue (ERH) is conserved from Schizosaccharomyces pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.

The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities.

Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, we directly assayed the behavior of several previously identified N/C ratio-dependent genes using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene that we examined, we found that nascent RNA output per cycle is delayed in haploid embryos. Moreover, we found that the N/C ratio influences transcription through three overlapping modes of action. For some genes (knirps, fushi tarazu, and snail), the effect of ploidy can be primarily attributed to changes in cell cycle duration. However, additional N/C ratio-mediated mechanisms contribute significantly to transcription delays for other genes. For giant and bottleneck, the kinetics of transcription activation are significantly disrupted in haploids, while for frühstart and Krüppel, the N/C ratio controls the probability of transcription initiation. Our data demonstrate that the regulatory elements of N/C ratio-dependent genes respond directly to the N/C ratio through multiple modes of regulation.

Enhancer-promoter communication: hubs or loops?

There has been a sea change in our view of transcription regulation during the past decade (Fukaya et al., 2016, Lim et al., 2018, Hnisz et al., 2017 [3], Liu et al., 2018 [4], Kato et al., 2012). Classical models of enhancer-promoter interactions and the stepwise assembly of individual RNA Polymerase II (Pol II) complexes have given way to the realization that active transcription foci contain clusters-hubs-of transcriptional activators and Pol II. Here we summarize recent findings pointing to the occurrence of transcription hubs and the implications of such hubs on the regulation of gene activity.

Regulation of spatiotemporal limits of developmental gene expression via enhancer grammar.

The regulatory specificity of a gene is determined by the structure of its enhancers, which contain multiple transcription factor binding sites. A unique combination of transcription factor binding sites in an enhancer determines the boundary of target gene expression, and their disruption often leads to developmental defects. Despite extensive characterization of binding motifs in an enhancer, it is still unclear how each binding site contributes to overall transcriptional activity. Using live imaging, quantitative analysis, and mathematical modeling, we measured the contribution of individual binding sites in transcriptional regulation. We show that binding site arrangement within the Rho-GTPase component t48 enhancer mediates the expression boundary by mainly regulating the timing of transcriptional activation along the dorsoventral axis of Drosophila embryos. By tuning the binding affinity of the Dorsal (Dl) and Zelda (Zld) sites, we show that single site modulations are sufficient to induce significant changes in transcription. Yet, no one site seems to have a dominant role; rather, multiple sites synergistically drive increases in transcriptional activity. Interestingly, Dl and Zld demonstrate distinct roles in transcriptional regulation. Dl site modulations change spatial boundaries of t48, mostly by affecting the timing of activation and bursting frequency rather than transcriptional amplitude or bursting duration. However, modulating the binding site for the pioneer factor Zld affects both the timing of activation and amplitude, suggesting that Zld may potentiate higher Dl recruitment to target DNAs. We propose that such fine-tuning of dynamic gene control via enhancer structure may play an important role in ensuring normal development.

Temporal dynamics of pair-rule stripes in living Drosophila embryos.

Traditional studies of gene regulation in the Drosophila embryo centered primarily on the analysis of fixed tissues. These methods provided considerable insight into the spatial control of gene activity, such as the borders of eve stripe 2, but yielded only limited information about temporal dynamics. The advent of quantitative live-imaging and genome-editing methods permits the detailed examination of the temporal control of endogenous gene activity. Here, we present evidence that the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) undergo dynamic shifts in gene expression. We observe sequential anterior shifting of the stripes along the anterior to posterior axis, with stripe 1 exhibiting movement before stripe 2 and the more posterior stripes. Conversely, posterior stripes shift over greater distances (two or three nuclei) than anterior stripes (one or two nuclei). Shifting of the ftz and eve stripes are slightly offset, with ftz moving faster than eve This observation is consistent with previous genetic studies, suggesting that eve is epistatic to ftz The precision of pair-rule temporal dynamics might depend on enhancer-enhancer interactions within the eve locus, since removal of the endogenous eve stripe 1 enhancer via CRISPR/Cas9 genome editing led to precocious and expanded expression of eve stripe 2. These observations raise the possibility of an added layer of complexity in the positional information encoded by the segmentation gene regulatory network.

Visualization of Transvection in Living Drosophila Embryos.

How remote enhancers interact with appropriate target genes persists as a central mystery in gene regulation. Here, we exploit the properties of transvection to explore enhancer-promoter communication between homologous chromosomes in living Drosophila embryos. We successfully visualized the activation of an MS2-tagged reporter gene by a defined developmental enhancer located in trans on the other homolog. This trans-homolog activation depends on insulator DNAs, which increase the stability-but not the frequency-of homolog pairing. A pair of heterotypic insulators failed to mediate transvection, raising the possibility that insulator specificity underlies the formation of chromosomal loop domains. Moreover, we found that a shared enhancer co-activates separate PP7 and MS2 reporter genes incis and intrans. Transvecting alleles weakly compete with one another, raising the possibility that they share a common pool of the transcription machinery. We propose that transvecting alleles form a trans-homolog "hub," which serves as a scaffold for the accumulation of transcription complexes.

Imaging transcriptional dynamics.

Recent advances in imaging techniques have enabled visualizations of nascent transcripts or individual protein molecules at high spatiotemporal resolution, revealing the complex nature of transcriptional regulation. Here, we highlight recent studies that have provided comprehensive insights to transcriptional dynamics using such quantitative imaging techniques. Specifically, they demonstrated that transcriptional activity is stochastic, and such transcriptional bursting is modulated by multiple components like chromatin environments, concentration of transcription factors, and enhancer-promoter interactions. Moreover, recent studies suggested that regulation of transcriptional activity is more complex than previously thought, by showing that transcription factors and RNA polymerases also move within the cell with distinct kinetics and sometimes form dynamic clusters to mediate transcriptional initiation.

Synthesizing developmental trajectories.

Dynamical processes in biology are studied using an ever-increasing number of techniques, each of which brings out unique features of the system. One of the current challenges is to develop systematic approaches for fusing heterogeneous datasets into an integrated view of multivariable dynamics. We demonstrate that heterogeneous data fusion can be successfully implemented within a semi-supervised learning framework that exploits the intrinsic geometry of high-dimensional datasets. We illustrate our approach using a dataset from studies of pattern formation in Drosophila. The result is a continuous trajectory that reveals the joint dynamics of gene expression, subcellular protein localization, protein phosphorylation, and tissue morphogenesis. Our approach can be readily adapted to other imaging modalities and forms a starting point for further steps of data analytics and modeling of biological dynamics.

A novel function for the IκB inhibitor Cactus in promoting Dorsal nuclear localization and activity in the Drosophila embryo.

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals.

Rapid Rates of Pol II Elongation in the Drosophila Embryo.

Elongation of RNA polymerase II (Pol II) is thought to be an important mechanism for regulating gene expression [1]. We measured the first wave of de novo transcription in living Drosophila embryos using dual-fluorescence detection of nascent transcripts containing 5' MS2 and 3' PP7 RNA stem loops. Pol II elongation rates of 2.4-3.0 kb/min were observed, approximately twice as fast as earlier estimates [2-6]. The revised rates permit substantial levels of zygotic gene activity prior to the mid-blastula transition. We also provide evidence that variable rates of elongation are not a significant source of differential gene activity, suggesting that transcription initiation and Pol II release are the key determinants of gene control in development.