ABSTRACT
Sulfur mustard (SM) is a highly reactive organic chemical has been used as a chemical warfare agent and terrorist threat since World War I. The cornea is highly sensitive to SM toxicity and exposure to low vapor doses can cause incapacitating acute injuries. Exposure to higher doses can elicit persistent secondary keratopathies that cause reduced quality of life and impaired or lost vision. Despite a century of research, there are no specific treatments for acute or persistent ocular SM injuries. SM cytotoxicity emerges, in part, through DNA alkylation and double-strand breaks (DSBs). Because DSBs can naturally be repaired by DNA damage response pathways with low efficiency, we hypothesized that enhancing the homologous recombination pathway could pose a novel approach to mitigate SM injury. Here, we demonstrate that a dilithium salt of adenosine diphosphoribose (INV-102) increases protein levels of p53 and Sirtuin 6, upregulates transcription of BRCA1/2, enhances γH2AX focus formation, and promotes assembly of repair complexes at DSBs. Based on in vitro evidence showing INV-102 enhancement of DNA damage response through both p53-dependent and p53-independent pathways, we next tested INV-102 in a rabbit preclinical model of corneal injury. In vivo studies demonstrate a marked reduction in the incidence and severity of secondary keratopathies in INV-102-treated eyes compared with vehicle-treated eyes when treatment was started 24 hours after SM vapor exposure. These results suggest DNA repair mechanisms are a viable therapeutic target for SM injury and suggest topical treatment with INV-102 is a promising approach for SM as well as other conditions associated with DSBs. SIGNIFICANCE STATEMENT: Sulfur mustard gas corneal injury currently has no therapeutic treatment. This study aims to show the therapeutic potential of activating the body's natural DNA damage response to activate tissue repair.
Subject(s)
Chemical Warfare Agents , Corneal Injuries , Mustard Gas , Animals , Rabbits , Mustard Gas/toxicity , BRCA1 Protein , Tumor Suppressor Protein p53 , Quality of Life , BRCA2 Protein , Corneal Injuries/chemically induced , Corneal Injuries/drug therapy , Chemical Warfare Agents/toxicity , DNA Repair , DNA DamageABSTRACT
Biophysical profiling of primary tumors has revealed that individual tumor cells fall along a highly heterogeneous continuum of mechanical phenotypes. One idea is that a subset of tumor cells is "softer" to facilitate detachment and escape from the primary site, a step required to initiate metastasis. However, it has also been postulated that cells must be able to deform and generate sufficient force to exit into distant sites. Here, we aimed to dissect the mechanical changes that occur during extravasation and organ colonization. Using multiplexed methods of intravital microscopy and optical tweezer based active microrheology, we obtained longitudinal images and mechanical profiles of cells during organ colonization in vivo. We determined that cells were softer, more liquid like upon exit of the vasculature but stiffened and became more solid like once in the new organ microenvironment. We also determined that a YAP mediated mechanogenotype influenced the global dissemination in our in vivo and in vitro models and that reducing mechanical heterogeneity could reduce extravasation. Moreover, our high throughput analysis of mechanical phenotypes of patient samples revealed that this mechanics was in part regulated by the external hydrodynamic forces that the cancer cells experienced within capillary mimetics. Our findings indicate that disseminated cancer cells can keep mutating with a continuum landscape of mechano-phenotypes, governed by the YAP-mediated mechanosensing of hydrodynamic flow.
ABSTRACT
Entrainment to an external stimulus enables a synchronized oscillatory response across a population of cells, increasing coherent responses by reducing cell-to-cell heterogeneity. It is unclear whether the property of entrainability extends to systems where responses are intrinsic to the individual cell, rather than dependent on coherence across a population of cells. Using a combination of mathematical modeling, time-lapse fluorescence microscopy, and single-cell tracking, we demonstrated that p53 oscillations triggered by DNA double-strand breaks (DSBs) can be entrained with a periodic damage stimulus, despite such synchrony not known to function in effective DNA damage responses. Surprisingly, p53 oscillations were experimentally entrained over a wider range of DSB frequencies than predicted by an established computational model for the system. We determined that recapitulating the increased range of entrainment frequencies required, non-intuitively, a less robust oscillator and wider steady-state valley on the energy landscape. Further, we show that p53 entrainment can lead to altered expression dynamics of downstream targets responsible for cell fate in a manner dependent on target mRNA stability. Overall, this study demonstrates that entrainment can occur in a biological oscillator despite the apparent lack of an evolutionary advantage conferred through synchronized responses and highlights the potential of externally entraining p53 dynamics to reduce cellular variability and synchronize cell-fate responses for therapeutic outcomes.
ABSTRACT
Energy landscapes can provide intuitive depictions of population heterogeneity and dynamics. However, it is unclear whether individual cell behavior, hypothesized to be determined by initial position and noise, is faithfully recapitulated. Using the p21-/Cdk2-dependent quiescence-proliferation decision in breast cancer dormancy as a testbed, we examined single-cell dynamics on the landscape when perturbed by hypoxia, a dormancy-inducing stress. Combining trajectory-based energy landscape generation with single-cell time-lapse microscopy, we found that initial position on a p21/Cdk2 landscape did not fully explain the observed cell-fate heterogeneity under hypoxia. Instead, cells with higher cell state velocities prior to hypoxia, influenced by epigenetic parameters, tended to remain proliferative under hypoxia. Thus, the fate decision on this landscape is significantly influenced by "inertia", a velocity-dependent ability to resist directional changes despite reshaping of the underlying landscape, superseding positional effects. Such inertial effects may markedly influence cell-fate trajectories in tumors and other dynamically changing microenvironments.
ABSTRACT
In response to different cellular stresses, the transcription factor p53 undergoes different dynamics. p53 dynamics, in turn, control cell fate. However, distinct stresses can generate the same p53 dynamics but different cell fate outcomes, suggesting integration of dynamic information from other pathways is important for cell fate regulation. To determine how MAPK activities affect p53-mediated responses to DNA breaks and oxidative stress, we simultaneously tracked p53 and either ERK, JNK, or p38 activities in single cells. While p53 dynamics were comparable between the stresses, cell fate outcomes were distinct. Combining MAPK dynamics with p53 dynamics was important for distinguishing between the stresses and for generating temporal ordering of cell fate pathways. Furthermore, cross-talk between MAPKs and p53 controlled the balance between proliferation and cell death. These findings provide insight into how cells integrate signaling pathways with distinct temporal patterns of activity to encode stress specificity and drive different cell fate decisions.
Subject(s)
DNA Damage , Tumor Suppressor Protein p53 , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Oxidative Stress , Signal Transduction , Gene Expression Regulation , ApoptosisABSTRACT
The size of the satellite cell pool is reduced in estradiol (E2)-deficient female mice and humans. Here, we use a combination of in vivo and in vitro approaches to identify mechanisms, whereby E2 deficiency impairs satellite cell maintenance. By measuring satellite cell numbers in mice at several early time points postovariectomy (Ovx), we determine that satellite cell numbers decline by 33% between 10 and 14 days post-Ovx in tibialis anterior and gastrocnemius muscles. At 14 days post-Ovx, we demonstrate that satellite cells have a reduced propensity to transition from G0/G1 to S and G2/M phases, compared with cells from ovary-intact mice, associated with changes in two key satellite cell cycle regulators, ccna2 and p16INK4a. Further, freshly isolated satellite cells treated with E2 in vitro have 62% greater cell proliferation and require less time to complete the first division. Using clonal and differentiation assays, we measured 69% larger satellite cell colonies and enhanced satellite cell-derived myoblast differentiation with E2 treatment compared with vehicle-treated cells. Together, these results identify a novel mechanism for preservation of the satellite cell pool by E2 via promotion of satellite cell cycling.
Subject(s)
Estradiol , Muscle, Skeletal , Animals , Cell Division , Estradiol/pharmacology , Female , Humans , Mice , Muscle, Skeletal/metabolism , Myoblasts/metabolism , OvariectomyABSTRACT
The dynamics of p53 expression provide a mechanism to increase differentiation between cellular stresses and specificity in appropriate responses. Here, we review recent advances in our understanding of the molecular mechanisms regulating p53 dynamics and the functions of the dynamics in the regulation of p53-dependent cell stress responses. We also compare dynamic encoding in the p53 system with that found in other important cell signaling systems, many of which can interact with the p53 network. Finally, we highlight some of the current challenges in understanding dynamic cell signaling within a larger cellular network context.
Subject(s)
Signal Transduction/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Animals , Cell Cycle Checkpoints/genetics , Cell Proliferation/genetics , DNA Damage/genetics , DNA Repair/genetics , Gene Expression , Gene Expression Regulation , Humans , Kinetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
DNA double strand breaks induce oscillatory expression of the transcription factor p53 that is dependent on ataxia telangiectasia mutated (ATM) activity and the rate of double strand break resolution. Although p53 dynamics are known to play a role in the regulation of cell fate determination, the consequences of the variability in dynamics associated with differences in repair rates and utilized repair pathways are unknown. Using single-cell time-lapse microscopy, we found that disruption of specific repair pathways has distinct impacts on p53 dynamics. The small-molecule rucaparib, an inhibitor of the alternative end-joining-associated protein poly (ADP-ribose) polymerase (PARP), increased p53 pulse duration, altering the temporal expression of multiple p53 target genes. As a result, combination treatments of the radiomimetic drug neocarzinostatin with rucaparib drove prolonged growth arrest beyond that of DNA damage alone. This study highlights how pharmacological manipulation of DNA repair pathways may be used to alter p53 dynamics to enhance therapeutic regimens.
Subject(s)
Cell Cycle Checkpoints , DNA Breaks, Double-Stranded , Indoles/pharmacology , Single-Cell Analysis , Tumor Suppressor Protein p53/metabolism , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , DNA Breaks, Double-Stranded/drug effects , DNA Repair/drug effects , DNA-Activated Protein Kinase , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , MCF-7 Cells , Zinostatin/pharmacologyABSTRACT
Physiologically, MYC levels must be precisely set to faithfully amplify the transcriptome, but in cancer MYC is quantitatively misregulated. Here, we study the variation of MYC amongst single primary cells (B-cells and murine embryonic fibroblasts, MEFs) for the repercussions of variable cellular MYC-levels and setpoints. Because FUBPs have been proposed to be molecular "cruise controls" that constrain MYC expression, their role in determining basal or activated MYC-levels was also examined. Growing cells remember low and high-MYC setpoints through multiple cell divisions and are limited by the same expression ceiling even after modest MYC-activation. High MYC MEFs are enriched for mRNAs regulating inflammation and immunity. After strong stimulation, many cells break through the ceiling and intensify MYC expression. Lacking FUBPs, unstimulated MEFs express levels otherwise attained only with stimulation and sponsor MYC chromatin changes, revealed by chromatin marks. Thus, the FUBPs enforce epigenetic setpoints that restrict MYC expression.
Subject(s)
DNA-Binding Proteins/physiology , Epigenesis, Genetic , Proto-Oncogene Proteins c-myc/metabolism , RNA-Binding Proteins/physiology , Animals , B-Lymphocytes/metabolism , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Fibroblasts/metabolism , Gene Expression Regulation , Mice , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolismABSTRACT
Supraphysiological MYC levels are oncogenic. Originally considered a typical transcription factor recruited to E-boxes (CACGTG), another theory posits MYC a global amplifier increasing output at all active promoters. Both models rest on large-scale genome-wide "-omics'. Because the assumptions, statistical parameter and model choice dictates the '-omic' results, whether MYC is a general or specific transcription factor remains controversial. Therefore, an orthogonal series of experiments interrogated MYC's effect on the expression of synthetic reporters. Dose-dependently, MYC increased output at minimal promoters with or without an E-box. Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors made expression more MYC-responsive, effectively increasing MYC-amplifier gain. Mutations of conserved MYC-Box regions I and II impaired amplification, whereas MYC-box III mutations delivered higher reporter output indicating that MBIII limits over-amplification. Kinetic theory and experiments indicate that MYC activates at least two steps in the transcription-cycle to explain the non-linear amplification of transcription that is essential for global, supraphysiological transcription in cancer.
Subject(s)
Gene Amplification , Proto-Oncogene Proteins c-myc/genetics , Transcription Factors/genetics , Transcriptional Activation , Animals , Cell Line , Humans , Proto-Oncogene Proteins c-myc/metabolism , Rats , Transcription Factors/metabolismABSTRACT
The p53 tumor suppressor regulates distinct responses to cellular stresses. Although different stresses generate different p53 dynamics, the mechanisms by which cells decode p53 dynamics to differentially regulate target genes are not well understood. Here, we determined in individual cells how canonical p53 target gene promoters vary in responsiveness to features of p53 dynamics. Employing a chemical perturbation approach, we independently modulated p53 pulse amplitude, duration, or frequency, and we then monitored p53 levels and target promoter activation in individual cells. We identified distinct signal processing features-thresholding in response to amplitude modulation, a refractory period in response to duration modulation, and dynamic filtering in response to frequency modulation. We then showed that the signal processing features not only affect p53 target promoter activation, they also affect p53 regulation and downstream cellular functions. Our study shows how different promoters can differentially decode features of p53 dynamics to generate distinct responses, providing insight into how perturbing p53 dynamics can be used to generate distinct cell fates.
Subject(s)
Cell Cycle Checkpoints/drug effects , Gene Expression Regulation/drug effects , Promoter Regions, Genetic/drug effects , Tumor Suppressor Protein p53 , Computational Biology , HEK293 Cells , Humans , MCF-7 Cells , Microfluidic Analytical Techniques , Signal Transduction/drug effects , Transcription Factors/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/pharmacologyABSTRACT
Synthetic biological circuits that can generate outputs with distinct expression dynamics are useful for a variety of biomedical and industrial applications. We present a method to control output dynamics by altering output mRNA decay rates. Using oscillatory expression of the transcription factor p53 as the circuit regulator, we use two approaches for controlling target gene transcript degradation rates based on the output gene's 3'-untranslated region (3'-UTR): introduction of copies of destabilizing AU-rich elements into the 3'-UTR or swapping in naturally occurring 3'-UTRs conferring different transcript stabilities. As a proof of principle, we apply both methods to control the expression dynamics of a fluorescent protein and visualize the circuit output dynamics in single living cells. We then use the naturally occurring 3'-UTR approach to restore apoptosis in a tunable manner in a cancer cell line deficient for caspase-3 expression. Our method can be readily adapted to regulate multiple outputs each with different expression dynamics under the control of a single naturally occurring or synthetically constructed biological oscillator.
Subject(s)
3' Untranslated Regions/genetics , Gene Expression Regulation , RNA Stability/genetics , RNA, Messenger/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis/genetics , Apoptosis/physiology , Caspase 3/deficiency , Caspase 3/genetics , Cell Line, Tumor , Genetic Engineering/methods , Humans , Luminescent Proteins/metabolism , Periodicity , Proof of Concept Study , RNA Stability/physiology , RNA, Messenger/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
In response to DNA damage, the transcription factor p53 accumulates in a series of pulses. While p53 dynamics play a critical role in regulating stress responses, how p53 pulsing affects target protein expression is not well understood. Recently, we showed that p53 pulses generate diversity in target mRNA expression dynamics; however, given that mRNA and protein expression are not necessarily well correlated, it remains to be determined how p53 pulses impact target protein expression. Using computational and experimental approaches, we show that target protein decay rates filter p53 pulses: Distinct target protein expression dynamics are generated depending on the relationship between p53 pulse frequency and target mRNA and protein stability. Furthermore, by mutating the targets MDM2 and PUMA to alter their stabilities, we show that downstream pathways are sensitive to target protein decay rates. This study delineates the mechanisms by which p53 dynamics play a crucial role in orchestrating the timing of events in the DNA damage response network.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Breast Neoplasms/metabolism , DNA Breaks, Double-Stranded , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis Regulatory Proteins/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Kinetics , MCF-7 Cells , Models, Biological , Mutation , Protein Stability , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-mdm2/genetics , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Tumor Suppressor Protein p53/genetics , Zinostatin/pharmacologyABSTRACT
Understanding the mechanisms supporting tumor-initiating cells (TIC) is vital to combat advanced-stage recurrent cancers. Here, we show that in advanced ovarian cancers NFκB signaling via the RelB transcription factor supports TIC populations by directly regulating the cancer stem-like associated enzyme aldehyde dehydrogenase (ALDH). Loss of RelB significantly inhibited spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in subcutaneous and intrabursal mouse xenograft models of human ovarian cancer. RelB also affected expression of the ALDH gene ALDH1A2 Interestingly, classical NFκB signaling through the RelA transcription factor was equally important for tumorigenesis in the intrabursal model, but had no effect on ALDH. In this case, classical signaling via RelA was essential for proliferating cells, whereas the alternative signaling pathway was not. Our results show how NFκB sustains diverse cancer phenotypes via distinct classical and alternative signaling pathways, with implications for improved understanding of disease recurrence and therapeutic response. Cancer Res; 77(24); 6927-40. ©2017 AACR.
Subject(s)
Carcinogenesis/genetics , Cell Proliferation/genetics , Isoenzymes/metabolism , Neoplastic Stem Cells/physiology , Ovarian Neoplasms/genetics , Retinal Dehydrogenase/metabolism , Transcription Factor RelA/physiology , Aldehyde Dehydrogenase 1 Family , Animals , Carcinogenesis/metabolism , Carcinogenesis/pathology , Cell Line, Tumor , Female , Heterografts , Humans , Mice , Mice, Nude , NF-kappa B/genetics , NF-kappa B/physiology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Signal Transduction/genetics , Transcription Factor RelA/geneticsABSTRACT
In mammalian cells, the tumor suppressor p53 is activated upon a variety of cellular stresses and ensures an appropriate response ranging from arrest and repair to the induction of senescence and apoptosis. Quantitative measurements in individual living cells showed stimulus-dependent dynamics of p53 accumulation upon stress induction. Due to the complexity of the underlying biochemical interactions, mathematical models were indispensable for understanding the topology of the network regulating p53 dynamics. Recent work provides furhter insights into the causes of heterogeneous responses in individual cells, the rewiring of the network in response to different inputs and the role of the downstream processes in determining the cellular fate upon stress.
ABSTRACT
CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform.
Subject(s)
CRISPR-Cas Systems , Gene Dosage , Gene Knockout Techniques/methods , Green Fluorescent Proteins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , CRISPR-Associated Protein 9 , Cell Line, Tumor , Endonucleases/genetics , Endonucleases/metabolism , Flow Cytometry , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Kinetics , Mutation , Polymerase Chain Reaction , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Reproducibility of Results , Transgenes/geneticsABSTRACT
In response to stresses, cells often halt normal cellular processes, yet stress-specific pathways must bypass such inhibition to generate effective responses. We investigated how cells redistribute global transcriptional activity in response to DNA damage. We show that an oscillatory increase of p53 levels in response to double-strand breaks drives a counter-oscillatory decrease of MYC levels. Using RNA sequencing (RNA-seq) of newly synthesized transcripts, we found that p53-mediated reduction of MYC suppressed general transcription, with the most highly expressed transcripts reduced to a greater extent. In contrast, upregulation of p53 targets was relatively unaffected by MYC suppression. Reducing MYC during the DNA damage response was important for cell-fate regulation, as counteracting MYC repression reduced cell-cycle arrest and elevated apoptosis. Our study shows that global inhibition with specific activation of transcriptional pathways is important for the proper response to DNA damage; this mechanism may be a general principle used in many stress responses.
Subject(s)
Breast Neoplasms/genetics , DNA Breaks, Double-Stranded , Proto-Oncogene Proteins c-myc/genetics , Transcription, Genetic , Transcriptome , Tumor Suppressor Protein p53/genetics , Apoptosis , Binding Sites , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , CRISPR-Cas Systems , Cell Cycle Checkpoints , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , MCF-7 Cells , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Signal Transduction , Time Factors , Transfection , Tumor Suppressor Protein p53/metabolismABSTRACT
50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.
Subject(s)
B-Lymphocytes/metabolism , Cell Cycle , Cell Nucleus/metabolism , Chromatin Assembly and Disassembly , Chromatin/metabolism , Histones/metabolism , Lymphocyte Activation , Proto-Oncogene Proteins c-myc/metabolism , Acetyl Coenzyme A/metabolism , Acetylation , Adenosine Triphosphate/metabolism , Animals , B-Lymphocytes/immunology , Cell Line , Chromatin/chemistry , Chromatin/genetics , DNA Methylation , Epigenesis, Genetic , Genotype , Histones/chemistry , Immunity, Humoral , Methylation , Mice, Inbred C57BL , Mice, Knockout , Nucleic Acid Conformation , Phenotype , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/genetics , Single Molecule Imaging , Structure-Activity Relationship , Time Factors , Transcription, GeneticABSTRACT
Stochastic fluctuations, termed "noise," in the level of biological molecules can greatly impact cellular functions. While biological noise can sometimes be detrimental, recent studies have provided an increasing number of examples in which biological noise can be functionally beneficial. Rather than provide an exhaustive review of the growing literature in this field, in this review, we focus on single-cell studies based on quantitative microscopy that have generated a deeper understanding of the sources, characteristics, limitations, and benefits of biological noise. Specifically, we highlight studies showing how noise can help coordinate the expression of multiple downstream target genes, impact the channel capacity of signaling networks, and interact synergistically with oscillatory dynamics to enhance the sensitivity of signal processing. We conclude with a discussion of current challenges and future opportunities.
Subject(s)
Gene Expression Regulation , Microscopy/methods , Signal Transduction , Single-Cell Analysis/methods , Stochastic Processes , Signal Processing, Computer-Assisted , Signal Transduction/physiologyABSTRACT
Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.