Monitoring dynamics of single-cell gene expression over multiple cell cycles

Recent progress in reconstructing gene regulatory networks has established a framework for a quantitative description of the dynamics of many important cellular processes. Such a description will require novel experimental techniques that enable the generation of time-series data for the governing regulatory proteins in a large number of individual living cells. Here, we utilize microfabrication to construct a Tesla microchemostat that permits single-cell fluorescence imaging of gene expression over many cellular generations. The device is used to capture and constrain asymmetrically dividing or motile cells within a trapping region and to deliver nutrients and regulate the cellular population within this region. We illustrate the operation of the microchemostat with Saccharomyces cerevisiae and explore the evolution of single-cell gene expression and cycle time as a function of generation. Our findings highlight the importance of novel assays for quantifying the dynamics of gene expression and cellular growth, and establish a methodology for exploring the effects of gene expression on long-term processes such as cellular aging

Coordination of frontline defense mechanisms under severe oxidative stress

Inference of an environmental and gene regulatory influence network (EGRINOS) by integrating transcriptional responses to H2O2 and paraquat (PQ) has revealed a multi-tiered oxidative stress (OS)-management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids and gas vesicles, metal trafficking, and various other aspects of metabolism.ChIP-chip, microarray, and survival assays have validated important architectural aspects of this network, identified novel defense mechanisms (including two evolutionarily distant peroxidase enxymes), and showed that general transcription factors of the transcription factor B family have an important function in coordinating the OS response (OSR) despite their inability to directly sense ROS.A comparison of transcriptional responses to sub-lethal doses of H2O2 and PQ with predictions of these responses made by an EGRIN model generated earlier from responses to other environmental factors has confirmed that a significant fraction of the OSR is made up of a generalized component that is also observed in response to other stressors.Analysis of active regulons within environment and gene regulatory influence network for OS (EGRINOS) across diverse environmental conditions has identified the specialized component of oxidative stress response (OSR) that is triggered by sub-lethal OS, but not by other stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of γ rays

Prevalence of transcription promoters within archaeal operons and coding sequences

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements

Quantitative analysis of genetic expression responses to dynamic microenvironmental perturbation

Dynamic environments are commonplace in the natural world, from fluctuations in nutrient sources that control metabolic rates, to radiative cycling that drives circadian rhythms, to mechanical stresses that reform vasculature. So, intuitively one would assume that the regulatory systems that control cellular behavior are acutely adapted to respond to such variable conditions in a robust and appropriate fashion. Yet, despite their potential to provide increased quantitative detail and insight to the natural behavior of cells, highly dynamic perturbations are rarely utilized in the analysis of cellular gene expression and regulation. Part of this stems from the lack of technologies that enable such studies. However, recent advances in microfluidic devices designed to address biologically relevant questions promise to fill this void. Moreover, recently discovered knowledge that the galactose metabolism in S. cerevisiae and possibly similar pathways, are in fact rudimentary memory systems, strengthens the need for the ability to examine gene regulation under complex and dynamic stimulation. In this project, microfluidic technology was developed specifically for isolating, observing, and dynamically probing colonies of model host microbes. The devices created not only sustain cells under ideal growth conditions, but do so in a way that allows for long duration acquisition of highly resolved time evolved gene expression within single cells. Furthermore, these imaging capabilities were coupled to a novel microfluidic system that was able to produce precise and continuous concentration waveforms. The microfluidic platform was then utilized to explore the dynamic response profile of the galactose utilization pathway in S. cerevisiae under fluctuating nutrient conditions. Using experimental data, this study revealed that the pathway kinetics lead to low- pass information filtration. Further experimental investigation coupled with computational model simulations uncovered coupling to glucose metabolism that provides a globally robust response, despite galactose utilization impairment. These results emphasize both the utility of microfluidic device platforms in quantitative biological studies, and the importance of studies conducted in more natural environments for gaining a more detailed understanding of how gene systems result in complex behavio

Quantitative analysis of genetic expression responses to dynamic microenvironmental perturbation

Dynamic environments are commonplace in the natural world, from fluctuations in nutrient sources that control metabolic rates, to radiative cycling that drives circadian rhythms, to mechanical stresses that reform vasculature. So, intuitively one would assume that the regulatory systems that control cellular behavior are acutely adapted to respond to such variable conditions in a robust and appropriate fashion. Yet, despite their potential to provide increased quantitative detail and insight to the natural behavior of cells, highly dynamic perturbations are rarely utilized in the analysis of cellular gene expression and regulation. Part of this stems from the lack of technologies that enable such studies. However, recent advances in microfluidic devices designed to address biologically relevant questions promise to fill this void. Moreover, recently discovered knowledge that the galactose metabolism in S. cerevisiae and possibly similar pathways, are in fact rudimentary memory systems, strengthens the need for the ability to examine gene regulation under complex and dynamic stimulation. In this project, microfluidic technology was developed specifically for isolating, observing, and dynamically probing colonies of model host microbes. The devices created not only sustain cells under ideal growth conditions, but do so in a way that allows for long duration acquisition of highly resolved time evolved gene expression within single cells. Furthermore, these imaging capabilities were coupled to a novel microfluidic system that was able to produce precise and continuous concentration waveforms. The microfluidic platform was then utilized to explore the dynamic response profile of the galactose utilization pathway in S. cerevisiae under fluctuating nutrient conditions. Using experimental data, this study revealed that the pathway kinetics lead to low- pass information filtration. Further experimental investigation coupled with computational model simulations uncovered coupling to glucose metabolism that provides a globally robust response, despite galactose utilization impairment. These results emphasize both the utility of microfluidic device platforms in quantitative biological studies, and the importance of studies conducted in more natural environments for gaining a more detailed understanding of how gene systems result in complex behavio

Prevalence of transcription promoters within archaeal operons and coding sequences.

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of approximately 64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes-events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements