Biology subjects

Olson, E. J.

Publications and source records attributed to Olson, E. J..

6 recordsLinked to original sources

bioRxiv2022

Perfect adaptation achieved by transport limitations governs the inorganic phosphate response in S. cerevisiae.

Cells cope with and adapt to ever-changing environmental conditions. Sophisticated regulatory networks allow cells to adjust to these fluctuating environments. One such archetypal system is the S. cerevisiae Pho regulon. When external inorganic phosphate (Pi) concentration is low, the Pho regulon activates, expressing genes that scavenge external and internal Pi. However, the precise mechanism controlling this regulon remains elusive. We conducted a systems analysis of the Pho regulon on the single cell level under well-controlled environmental conditions. This analysis identified a robust, perfectly adapted Pho regulon state in intermediate Pi conditions, and we discovered a hitherto unknown intermediate nuclear localization state of the transcriptional master regulator Pho4p. The existence of an intermediate nuclear Pho4p state unifies and resolves outstanding incongruities associated with the Pho regulon, explains the observed programmatic states of the Pho regulon, and improves our general understanding of how nature evolves and controls sophisticated gene regulatory networks. We further propose that robustness and perfect adaptation are not achieved through complex network-centric control, but by simple transport biophysics. The ubiquity of multi-transporter systems suggests that similar mechanisms could govern the function of other regulatory networks as well.

systems biology↗

bioRxiv2021

Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance

Transcription factor binding to a single binding site and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain. We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.

biophysics↗

bioRxiv2021

High content screening and computational prediction reveal viral genes that suppress innate immune response

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-{kappa}B and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins. IMPORTANCEInfectious diseases caused by viral pathogens exacerbate healthcare and economic burdens. Numerous viral biomolecules suppress the human innate immune system, enabling viruses to evade an immune response from the host. Despite our current understanding of viral replications and immune evasion, new viral proteins, including those encoded by uncultivated viruses or emerging viruses, are being unearthed at a rapid pace from large scale sequencing and surveillance projects. The use of medium- and high-throughput functional assays to characterize immunosuppressive functions of viral proteins can advance our understanding of viral replication and possibly treatment of infections. In this study we assembled a large viral gene library from diverse viral families and developed a high content assay to test for inhibition of innate immunity pathways. Our work expands the tools that can rapidly link sequence and protein function, representing a practical step towards early-stage evaluation of emerging and understudied viruses.

systems biology↗

bioRxiv2021

High-content screening of coronavirus genes for innate immune suppression revealsenhanced potency of SARS-CoV-2 proteins

Suppression of the host intracellular innate immune system is an essential aspect of viral replication. Here, we developed a suite of medium-throughput high-content cell-based assays to reveal the effect of individual coronavirus proteins on antiviral innate immune pathways. Using these assays, we screened the 196 protein products of seven coronaviruses (SARS-CoV-2, SARS-CoV-1, 229E, NL63, OC43, HKU1 and MERS). This includes a previously unidentified gene in SARS-CoV-2 encoded within the Spike gene. We observe immune-suppressing activity in both known host-suppressing genes (e.g., NSP1, Orf6, NSP3, and NSP5) as well as other coronavirus genes, including the newly identified SARS-CoV-2 protein. Moreover, the genes encoded by SARS-CoV-2 are generally more potent immune suppressors than their homologues from the other coronaviruses. This suite of pathway-based and mechanism-agnostic assays could serve as the basis for rapid in vitro prediction of the pathogenicity of novel viruses based on provision of sequence information alone.

systems biology↗

bioRxiv2020

Hydrazines as versatile chemical biology probes and drug-discovery tools for cofactor-dependent enzymes

Known chemoproteomic probes generally use warheads that tag a single type of amino acid or modified form thereof to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electron-poor enzyme cofactors, transient intermediates and chemically-labile regulatory modifications, but probes for such species are underdeveloped. Here, we have innovated a versatile class of chemoproteomic probes for this less charted hemisphere of the proteome by using hydrazine as the common chemical warhead. Its electron-rich nature allows it to react by both polar and radicaloid mechanisms and to target multiple, pharmacologically important functional classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment can be blocked by active-site-directed inhibitors, and elaboration of the warhead supports connection of a target to a lead compound. The capacity of substituted hydrazines to profile, discover and inhibit diverse cofactor-dependent enzymes enables cell and tissue imaging and makes this platform useful for enzyme and drug discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/154864v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1e1a54forg.highwire.dtl.DTLVardef@12be680org.highwire.dtl.DTLVardef@129e027org.highwire.dtl.DTLVardef@3b2317_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2016

A two-state photoconversion model predicts the spectral response dynamics of optogenetic systems

In optogenetics, light signals are used to control genetically engineered photoreceptors, and in turn manipulate biological pathways with unmatched precision. Recently, evolved photoreceptors with diverse in vitro-measured wavelength and intensity-dependent photoswitching properties have been repurposed for synthetic control of gene expression, proteolysis, and numerous other cellular processes. However, the relationship between the input light spectrum and in vivo photoreceptor response dynamics is poorly understood, restricting the utility of these optogenetic tools. Here, we advance a classic in vitro two-state photoreceptor model to reflect the in vivo environment, and combine it with simplified mathematical descriptions of signal transduction and output gene expression through our previously engineered green/red and red/far red photoreversible bacterial two-component systems (TCSs). Additionally, we leverage our recent open-source optical instrument to develop a workflow of spectral and dynamical characterization experiments to parameterize the model for both TCSs. To validate our approach, we challenge the model to predict experimental responses to a series of complex light signals very different from those used during parameterization. We find that the model generalizes remarkably well, predicting the results of all categories of experiments with high quantitative accuracy for both systems. Finally, we exploit this predictive power to program two simultaneous and independent dynamical gene expression signals in bacteria expressing both TCSs. This multiplexed gene expression programming approach will enable entirely new studies of how metabolic, signaling, and decision-making pathways integrate multiple gene expression signals. Additionally, our approach should be compatible with a wide range of optogenetic tools and model organisms.\n\nSignificance statementLight-switchable signaling pathways (optogenetic tools) enable precision studies of how biochemical networks underlie cellular behaviors. We have developed a versatile mathematical model based on a two-state photoconversion mechanism that we have applied to the E. coli CcaSR and Cph8-OmpR optogenetic tools. This model enables accurate prediction of the gene expression response to virtually any light source or mixture of light sources. We express both optogenetic tools in the same cell and apply our model to program two simultaneous and independent gene expression signals in the same cell. This method can be used to study how biological pathways integrate multiple inputs and should be extensible to other optogenetic tools and host organisms.

synthetic biology↗