[Show abstract][Hide abstract] ABSTRACT: Studies of individual living cells have revealed that many transcription factors activate in dynamic, and often stochastic, pulses within the same cell. However, it has remained unclear whether cells might exploit the dynamic interaction of these pulses to control gene expression. Here, using quantitative single-cell time-lapse imaging of Saccharomyces cerevisiae, we show that the pulsatile transcription factors Msn2 and Mig1 combinatorially regulate their target genes through modulation of their relative pulse timing. The activator Msn2 and repressor Mig1 showed pulsed activation in either a temporally overlapping or non-overlapping manner during their transient response to different inputs, with only the non-overlapping dynamics efficiently activating target gene expression. Similarly, under constant environmental conditions, where Msn2 and Mig1 exhibit sporadic pulsing, glucose concentration modulated the temporal overlap between pulses of the two factors. Together, these results reveal a time-based mode of combinatorial gene regulation. Regulation through relative signal timing is common in engineering and neurobiology, and these results suggest that it could also function broadly within the signalling and regulatory systems of the cell.
[Show abstract][Hide abstract] ABSTRACT: Like life itself, synthetic biology began with unicellular
organisms. Early synthetic biologists built genetic circuits
in model prokaryotes and yeast because of their relative
biological simplicity and ease of genetic manipulation. With
superior genetic tools, faster generation times, and betterunderstood endogenous gene expression machinery, prokaryotes and yeast were (and remain) appealing hosts for the engineering of synthetic systems. Now in its second decade, synthetic biology in unicellular organisms has produced myriad synthetic genetic circuits, a number of industrial applications, and fundamental new biological insights unlikely to have emerged from nonsynthetic approaches.
Preview · Article · Dec 2014 · ACS Synthetic Biology
[Show abstract][Hide abstract] ABSTRACT: The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation. - See more at: http://elifesciences.org/content/3/e02950
[Show abstract][Hide abstract] ABSTRACT: The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation. DOI: http://dx.doi.org/10.7554/eLife.02950.001
[Show abstract][Hide abstract] ABSTRACT: The activation of transcription factors in response to environmental conditions is fundamental to cellular regulation. Recent work has revealed that some transcription factors are activated in stochastic pulses of nuclear localization, rather than at a constant level, even in a constant environment [1-12]. In such cases, signals control the mean activity of the transcription factor by modulating the frequency, duration, or amplitude of these pulses. Although specific pulsatile transcription factors have been identified in diverse cell types, it has remained unclear how prevalent pulsing is within the cell, how variable pulsing behaviors are between genes, and whether pulsing is specific to transcriptional regulators or is employed more broadly. To address these issues, we performed a proteome-wide movie-based screen to systematically identify localization-based pulsing behaviors in Saccharomyces cerevisiae. The screen examined all genes in a previously developed fluorescent protein fusion library of 4,159 strains  in multiple media conditions. This approach revealed stochastic pulsing in ten proteins, all transcription factors. In each case, pulse dynamics were heterogeneous and unsynchronized among cells in clonal populations. Pulsing is the only dynamic localization behavior that we observed, and it tends to occur in pairs of paralogous and redundant proteins. Taken together, these results suggest that pulsatile dynamics play a pervasive role in yeast and may be similarly prevalent in other eukaryotic species.
[Show abstract][Hide abstract] ABSTRACT: Hematopoietic stem and progenitor cells employ circuits of regulatory genes to integrate developmental signals and stabilize fate choices. These progenitors show considerable cell-to-cell heterogeneity in their fate choices; thus, to better understand how regulatory genes control fate decisions, we tracked their levels in single progenitors over time using timelapse live-cell imaging. We examined two hematopoietic fate transitions: 1) macrophage differentiation, driven by the up-regulation of the myeloid transcription factor PU.1; and 2) T-cell fate commitment, controlled by the activation of the T-cell specific transcription factor Bcl11b. In our study of macrophage differentiation, we found that cell cycle length acts as critical mediator in the positive feedback circuit controlling differentiation (Kueh et al. 2013). By following PU.1 regulation in single cells containing a knock-in PU.1-GFP reporter, we found that developing macrophages lengthen their cell cycles to promote stable PU.1 accumulation, and that PU.1 itself promotes cell cycle lengthening, completing a positive feedback loop that stabilizes its own expression. Mathematical modeling furthered showed that this cell cycle feedback circuit robustly stabilizes a slow-dividing differentiated state. In our studies on T-cell fate commitment, we found that Notch signaling – the primary driver of development – enhances the frequency of all-or-none Bcl11b gene activation to promote commitment. By analyzing progenitors from mice containing a knock-in Bcl11b-YFP reporter, we found that uncommitted (Bcl11b-DN2A) progenitors can activate Bcl11b transcription and undergo fate commitment in the absence of Notch signaling, and that Notch signaling does not modulate the level of Bcl11b transcription, but instead increases the rate at which progenitors switch Bcl11b to an actively expressing state. These results reveal insights into how signaling pathways activate regulatory gene expression to instruct cell fate.
No preview · Article · Aug 2014 · Experimental Hematology
[Show abstract][Hide abstract] ABSTRACT: Cell populations can be strikingly heterogeneous, composed of multiple cellular states, each exhibiting stochastic noise in its gene expression. A major challenge is to disentangle these two types of variability and to understand the dynamic processes and mechanisms that control them. Embryonic stem cells (ESCs) provide an ideal model system to address this issue because they exhibit heterogeneous and dynamic expression of functionally important regulatory factors. We analyzed gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. These data discriminated stochastic switching between two coherent (correlated) gene expression states and burst-like transcriptional noise. We further showed that the "2i" signaling pathway inhibitors modulate both types of variation. Finally, we found that DNA methylation plays a key role in maintaining these metastable states. Together, these results show how ESC gene expression states and dynamics arise from a combination of intrinsic noise, coherent cellular states, and epigenetic regulation.
[Show abstract][Hide abstract] ABSTRACT: AU-rich element mRNA-binding proteins (AUBPs) are key regulators of development, but how they are controlled and what functional roles they play depends on cellular context. Here, we show that Brf1 (zfp36l1), an AUBP from the Zfp36 protein family, operates downstream of FGF/Erk MAP kinase signaling to regulate pluripotency and cell fate decision making in mouse embryonic stem cells (mESCs). FGF/Erk MAP kinase signaling up-regulates Brf1, which disrupts the expression of core pluripotency-associated genes and attenuates mESC self-renewal without inducing differentiation. These regulatory effects are mediated by rapid and direct destabilization of Brf1 targets, such as Nanog mRNA. Enhancing Brf1 expression does not compromise mESC pluripotency but does preferentially regulate mesendoderm commitment during differentiation, accelerating the expression of primitive streak markers. Together, these studies demonstrate that FGF signals use targeted mRNA degradation by Brf1 to enable rapid posttranscriptional control of gene expression in mESCs.
Preview · Article · Apr 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Synthetic biology, despite still being in its infancy, is increasingly
providing valuable information for applications in the clinic, the biotechnology
industry and in basic molecular research. Both its unique potential and the
challenges it presents have brought together the expertise of an eclectic group of
scientists, from cell biologists to engineers. In this Viewpoint article, five experts
discuss their views on the future of synthetic biology, on its main achievements in
basic and applied science, and on the bioethical issues that are associated with the
design of new biological systems.
No preview · Article · Mar 2014 · Nature Reviews Molecular Cell Biology
[Show abstract][Hide abstract] ABSTRACT: A fundamental problem in biology is to understand how genetic circuits implement core cellular functions. Time-lapse microscopy techniques are beginning to provide a direct view of circuit dynamics in individual living cells. Unexpectedly, we are discovering that key transcription and regulatory factors pulse on and off repeatedly, and often stochastically, even when cells are maintained in constant conditions. This type of spontaneous dynamic behavior is pervasive, appearing in diverse cell types from microbes to mammalian cells. Here, we review recent work showing how pulsing is generated and controlled by underlying regulatory circuits and how it provides critical capabilities to cells in stress response, signaling, and development. A major theme is the ability of pulsing to enable time-based regulation analogous to strategies used in engineered systems. Thus, pulsatile dynamics is emerging as a central, and still largely unexplored, layer of temporal organization in the cell.
[Show abstract][Hide abstract] ABSTRACT: Regulatory gene circuits with positive feedback loops control stem cell differentiation, but several mechanisms can contribute to positive feedback. Here, we dissect feedback mechanisms through which the transcription factor PU.1 controls lymphoid and myeloid differentiation. Quantitative live-cell imaging revealed that developing B cells decrease PU.1 levels by reducing PU.1 transcription, whereas developing macrophages increase PU.1 levels by lengthening their cell cycles, which causes stable PU.1 accumulation. Exogenous PU.1 expression in progenitors increases endogenous PU.1 levels by inducing cell-cycle lengthening, implying positive feedback between a regulatory factor and the cell cycle. Mathematical modeling showed that this cell-cycle coupled feedback architecture effectively stabilizes a slow-dividing differentiated state. These results show that cell-cycle duration functions as an integral part of a positive auto-regulatory circuit to control cell fate.
[Show abstract][Hide abstract] ABSTRACT: Cells use general stress response pathways to activate diverse target genes in response to a variety of stresses. However, general stress responses coexist with more specific pathways that are activated by individual stresses, provoking the fundamental question of whether and how cells control the generality or specificity of their response to a particular stress. Here we address this issue using quantitative time-lapse microscopy of the Bacillus subtilis environmental stress response, mediated by σ. We analyzed σ activation in response to stresses such as salt and ethanol imposed at varying rates of increase. Dynamically, σ responded to these stresses with a single adaptive activity pulse, whose amplitude depended on the rate at which the stress increased. This rate-responsive behavior can be understood from mathematical modeling of a key negative feedback loop in the underlying regulatory circuit. Using RNAseq we analyzed the effects of both rapid and gradual increases of ethanol and salt stress across the genome. Because of the rate responsiveness of σ activation, salt and ethanol regulons overlap under rapid, but not gradual, increases in stress. Thus, the cell responds specifically to individual stresses that appear gradually, while using σ to broaden the cellular response under more rapidly deteriorating conditions. Such dynamic control of specificity could be a critical function of other general stress response pathways.
Preview · Article · Mar 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Pulsing is abolished in the constitutively active Spo0A mutant Spo0Asad67. Typical time traces of Pspo0F-yfp mean fluorescence (left) and promoter activity (center), along with cell length (right) in a typical cell lineage (strain JL065). The promoter activity exhibited fluctuations but lacked the characteristic cell cycle phased pulses present in the wild type.
[Show abstract][Hide abstract] ABSTRACT: Simplified one-dimensional models capture qualitative circuit deferral behaviors. One-dimensional models are described in Text S1. (A) Dynamic traces of models tuned to cross a threshold of X = 100, starting from X = 1, with a five cell cycle deferral. X is plotted on both linear (left) and logarithmic (right) scales to illustrate exponential behavior. (B) Deferral time dependence on feedback strength for each model. For comparison, b of each model is plotted normalized to the minimal value b0 needed to reach the threshold of X = 100. Open loop: b0 = 100; instantaneous: b0 = 1; polyphasic: b0 = e−1. (C) One-dimensional models were compared for their ability to generate multi-cell-cycle deferral times, as with the two-component model of the main text. For each circuit, feedback strength b was tuned to produce different deferral times (x-axis). The sensitivity of deferral time to feedback strength was calculated as in the two-component model. The three circuits differ systematically in both the magnitude and rate of increase of sensitivity with deferral time. The open loop circuit is the most sensitive, followed by the instantaneous feedback, with the polyphasic feedback showing the least sensitivity.
[Show abstract][Hide abstract] ABSTRACT: The trpE promoter fluctuates but does not pulse. Typical time traces of PtrpE-mCherry mean fluorescence (left) and promoter activity (center), along with cell length (right) in a typical cell lineage (strain JL024). Promoter activity, while fluctuating, has a lower dynamic range and less temporal structure than Spo0AP regulated promoters.