Heat Shock Promoter Research Articles

Mosaic Analysis with a Repressible Cell Marker (MARCM) Clone Generation in Drosophila Dendritic Arborization Neurons.

Mosaic analysis with a repressible cell marker (MARCM) is used in Drosophila research to create labeled homozygous mutant clones of cells in an otherwise heterozygous fly. It allows the study of the effect of embryonically lethal genes and the determination of cell autonomy for a mutant phenotype. When used in dendritic arborization (da) neurons with a fluorescent protein targeted to the plasma membrane, MARCM allows the identification of homozygous mutant neurons and clear imaging of the dendrite arbor in both live and fixed preparations. Previous protocols that outlined experimental procedures to create MARCM clones in da neurons used a heat shock promoter to drive Flippase (FLP) expression; such an approach requires laborious embryo collection and heat shock steps, and it creates clones in other tissues besides the da neurons. The updated protocol described here outlines the use of FLP expression driven by a sensory organ precursor promoter (SOP-FLP); it requires no embryo collection or manipulation steps and creates clones exclusively in the peripheral sensory neuron lineage.

Sonogenetic control of multiplexed genome regulation and base editing

Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.

An Engineered Heat-Inducible Expression System for the Production of Casbene in Nicotiana benthamiana.

Plants respond to heat stress by producing heat-shock proteins. These are regulated by heat-shock promoters containing regulatory elements, which can be harnessed to control protein expression both temporally and spatially. In this study, we designed heat-inducible promoters to produce the diterpene casbene in Nicotiana benthamiana, through a multi-step metabolic pathway. To potentially increase gene transcription, we coupled heat-shock elements from Arabidopsis thaliana Hsp101 or Glycine max GmHsp17.3-B promoters, CAAT and TATA boxes from CaMV 35S, and the 5'UTR from the tobacco mosaic virus. The resulting four chimeric promoters fused to a green fluorescent protein (GFP) reporter showed that the variant Ara2 had the strongest fluorescent signal after heat shock. We next created a 4-gene cassette driven by the Ara2 promoter to allow for exogenous synthesis of casbene and transformed this multigene construct along with a selectable marker gene into Nicotiana benthamiana. Metabolic analysis on the transgenic lines revealed that continuous heat outperforms heat shock, with up to 1 μg/mg DW of casbene detected after 32 h of uninterrupted 40 °C heat. These results demonstrate the potential of heat-inducible promoters as synthetic biology tools for metabolite production in plants.

Targeted single-cell gene induction by optimizing the dually regulated CRE/loxP system by a newly defined heat-shock promoter and the steroid hormone in Arabidopsis thaliana.

Multicellular organisms rely on intercellular communication systems to organize their cellular functions. In studies focusing on intercellular communication, the key experimental techniques include the generation of chimeric tissue using transgenic DNA recombination systems represented by the CRE/loxP system. If an experimental system enables the induction of chimeras at highly targeted cell(s), it will facilitate the reproducibility and precision of experiments. However, multiple technical limitations have made this challenging. The stochastic nature of DNA recombination events, especially, hampers reproducible generation of intended chimeric patterns. Infrared laser-evoked gene operator (IR-LEGO), a microscopic system that irradiates targeted cells using an IR laser, can induce heat shock-mediated expression of transgenes, for example, CRE recombinase gene, in the cells. In this study, we developed a method that induces CRE/loxP recombination in the target cell(s) of plant roots and leaves in a highly specific manner. We combined IR-LEGO, an improved heat-shock-specific promoter, and dexamethasone-dependent regulation of CRE. The optimal IR-laser power and irradiation duration were estimated via exhaustive irradiation trials and subsequent statistical modeling. Under optimized conditions, CRE/loxP recombination was efficiently induced without cellular damage. We also found that the induction efficiency varied among tissue types and cellular sizes. The developed method offers an experimental system to generate a precisely designed chimeric tissue, and thus, will be useful for analyzing intercellular communication at high resolution in roots and leaves.

Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free PDH promoter.

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (P AOX1 ) and/or the constitutive GAP promoter (P GAP ). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (PDH) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical P GAP was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under PDH outperformed P GAP controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that PDH is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for PDH under different feeding and osmolarity conditions. In all of them the PDH controlled process outperformed the P GAP one in regard to CalB titer. The best PDH approach reached 3.6-fold more specific productivity than P GAP fed-batch at low μ. Compared to the optimum approach for P GAP -based process, the best PDH fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, PDH is an inducible promoter that exhibited a non-coupled growth regulation showing high performance, which provides a methanol-free additional solution to the usual growth-coupled systems for RPP. Thus, this novel system emerges as a potential alternative for K. phaffii RPP bioprocess and for revaluing crude glycerol, promoting the transition towards a circular economy.

Domain analysis of Drosophila Blimp-1 reveals the importance of its repression function and instability in determining pupation timing.

The PRDM family transcription repressor Blimp-1 is present in almost all multicellular organisms and plays important roles in various developmental processes. This factor has several conserved motifs among different species, but the function of each motif is unclear. Drosophila Blimp-1 plays an important role in determining pupation timing by acting as an unstable transcriptional repressor of the βftz-f1 gene. Thus, Drosophila provides a good system for analyzing the molecular and biological functions of each region in Blimp-1. Various Blimp-1 mutants carrying deletions at the conserved motifs were induced under the control of the heat shock promoter in prepupae, and the expression patterns of βFTZ-F1 and Blimp-1 and pupation timing were observed. The results showed that the regions with strong and weak repressor functions exist within the proline-rich middle section of the factor and near the N-terminal conserved motif, respectively. Rapid degradation was supported by multiple regions that were mainly located in a large proline-rich region. Results revealed that pupation timing was affected by the repression ability and stability of Blimp-1. This suggests that both the repression function and instability of Blimp-1 are indispensable for the precise determination of pupation timing.

Hoxa9/meis1-transgenic zebrafish develops acute myeloid leukaemia-like disease with rapid onset and high penetrance.

HOXA9 and MEIS1 are co-expressed in over 50% of acute myeloid leukaemia (AML) and play essential roles in leukaemogenesis, but the mechanisms involved are poorly understood. Diverse animal models offer valuable tools to recapitulate different aspects of AML and link in vitro studies to clinical trials. We generated a double transgenic zebrafish that enables hoxa9 overexpression in blood cells under the draculin (drl) regulatory element and an inducible expression of meis1 through a heat shock promoter. After induction, Tg(drl:hoxa9;hsp70:meis1) embryos developed a preleukaemic state with reduced myeloid and erythroid differentiation coupled with the poor production of haematopoietic stem cells and myeloid progenitors. Importantly, most adult Tg(drl:hoxa9;hsp70:meis1) fish at 3 months old showed abundant accumulations of immature myeloid precursors, interrupted differentiation and anaemia in the kidney marrow, and infiltration of myeloid precursors in peripheral blood, resembling human AML. Genome-wide transcriptional analysis also confirmed AML transformation by the transgene. Moreover, the dihydroorotate dehydrogenase (DHODH) inhibitor that reduces leukaemogenesis in mammals effectively restored haematopoiesis in Tg(drl:hoxa9;hsp70:meis1) embryos and improved their late survival. Thus, Tg(drl:hoxa9;hsp70:meis1) zebrafish is a rapid-onset high-penetrance AML-like disease model, which provides a novel tool to harness the unique advantages of zebrafish for mechanistic studies and drug screening against HOXA9/MEIS1 overexpressed high-risk AML.

Mosaic gene expression analysis of semaphorin-plexin interactions in Caenorhabditis elegans using the IR-LEGO single-cell gene induction system.

Genetic mosaic analysis is a powerful means of addressing the sites of gene action in multicellular organisms. In conventional genetic analysis, the generation of desired mosaic patterns is difficult to control due to the randomness of generating the genetic mosaic which often renders the analysis laborious and time consuming. The infrared laser-evoked gene operator (IR-LEGO) microscope system facilitates genetic mosaic analysis by enabling gene induction in targeted single cells in a living organism. However, the level of gene induction is not controllable due to the usage of a heat-shock promoter. Here, we applied IR-LEGO to examine the cell-cell interactions mediated by semaphoring-plexin signaling in Caenorhabditis elegans by inducing wild-type semaphorin/plexin in single cells within the population of mutant cells lacking the relevant proteins. We found that the cell contact-dependent termination of the extension of vulval precursor cells is elicited by the forward signaling mediated by the semaphorin receptor, PLX-1, but not by the reverse signaling via the transmembrane semaphorin, SMP-1. By utilizing Cre/loxP recombination coupled with the IR-LEGO system to induce SMP-1 at a physiological level, we found that SMP-1 interacts with PLX-1 only in trans upon contact between vulval precursor cells. In contrast, when overexpressed, SMP-1 exhibits the ability to cis-interact with PLX-1 on a single cell. These results indicate that mosaic analysis with IR-LEGO, especially when combined with an in vivo recombination system, efficiently complements conventional methods.

Using De Novo Designed Protein Switches in C. elegans

The ‘Latching Orthogonal Cage/Key pRoteins’ (LOCKR) system is a de novo designed protein Switch and Key. The Switch functions to cage a functional protein sequence, such as a degron, in the off or “locked” state. Upon Key addition, the Switch is turned on or “unlocked,” which exposes the degron, simultaneously degrading the Switch and any proteins fused to the Switch via the proteasome. Key expression can be tailored for tissue‐specific expression, which gives LOCKR exquisite spatial protein control. LOCKR provides novel post‐translational control over a protein of interest but has not yet been applied to a multicellular organism. We are developing the LOCKR technology for conditional protein depletion in the model organism C. elegans. Although genetic RNAi and similar gene editing techniques benefit from ease of delivery and can target essentially any gene, they have various limitations, including 1) slow protein turnover can prohibit use of RNAi; 2) genetic perturbation of essential genes via genetic deletion prevents characterization of later functions. The degronLOCKR system requires two parts: 1) a gene fused to the ‘degronSwitch’ and 2) an inducer peptide (the Key). The Switch can be fused to any gene of interest using CRISPR‐Cas9 gene editing. We fused degronSwitch to dhc‐1 (dynein heavy chain), an essential cytoplasmic motor protein along with mScarlet and a 3xFLAG tag to aid in protein quantitation via microscopic and Western analysis. We also cloned a negative control, which lacks degronSwitch, to analyze the effect of inserting the Switch in vivo. To complement the degronSwitch, we designed six tissue‐specific Keys for Mos‐Single Copy Insertion (MosSCI), which allows for spatial control of degronSwitch protein degradation. We predict that ubiquitous expression of Key with the degronSwitch fused to dhc‐1 will result in embryonic lethality, whereas germline‐specific Key will permit viability but result in meiotic defects. These phenotypes are easily identifiable and serve as effective proof of concept. Future work includes expanding the LOCKR system to incorporate temporal control of the degronSwitch fusion protein by toggling the Key on and off using heat‐shock promoters and RNAi against the Key. This work establishes a novel and tissue‐specific method for regulating protein function in an animal model.

Combination of baicalein and trans‐chalcone synergistically reduces amyloid beta and its toxicity in yeast

AbstractBackgroundThere has been a long quest to uncover effective therapeutic strategy to prevent or cure AD as current strategies only support as palliative care. This study utilizes unmodified and genetically modified Saccharomyces cerevisiae as model organisms to screen and find potential natural bioactive compounds that can reduce the levels of amyloid beta and associated toxicity.MethodsEleven selected naturally occurring bioactive compounds namely mangiferin, quercetin, rutin, resveratrol, epigallocatechin gallate (EGCG), urolithin A, oleuropein, rosmarinic acid, salvianolic acid B, baicalein and trans‐chalcone at specific concentration (50µM) were screened for their ability to change intracellular green fluorescent protein (GFP) tagged Aβ42 levels, in vivo anti‐amyloid properties using thioflavin‐S fluorescent dye and induce heat stress response using the yeast heat stress reporter that expresses mCherry red fluorescent protein under control of a heat shock promoter containing heat shock elements (HSEs). Furthermore, the two compounds with highest activity in all three screens were combined together in varying concentrations of each to study the underlying combination ability to reduce GFP‐Aβ42, their effect on growth, turnover of native Aβ42 and reactive oxygen species.ResultsThe bioactive compounds reduced intracellular GFP‐Aβ42 levels (except mangiferin) and intracellular amyloid fibrils (except resveratrol and EGCG) in yeast models significantly with highest effect shown by baicalein and trans‐chalcone. Among the bioactive compounds, quercetin, rutin, rosmarinic acid, baicalein and trans‐chalcone significantly induced heat stress reporter with highest effect demonstrated by baicalein and trans‐chalcone. Furthermore, the combinations of baicalein and trans‐chalcone showed synergistic ability to reduce GFP‐Aβ42. Although some of the combinations were found growth inhibitory, a combination of 50μM trans‐chalcone and 30μM baicalein was found to be cytoprotective as well as an effective reducer of GFP‐Aβ42. In addition, the combination of the two compounds showed synergy in reducing ROS in yeast cells expressing native Aβ42 and reduced the Aβ42 protein levels by nearly 80%.ConclusionThe findings of the study suggest that the combination of baicalein and trans‐chalcone can be a therapeutic strategy to cure or at least prevent AD. However, further studies are recommended to ascertain that similar cytoprotective activity is observed in humans and optimization of dosage to observe best effect.

Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering

The lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. However, no knock-in technique for this animal has been established yet, preventing application of advanced genetic techniques. Here, we report efficient generation of F0 knock-in lampreys by CRISPR-Cas9-mediated genome editing. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) for genome digestion, a sgRNA for donor plasmid digestion, and Cas9 mRNA. Targeting different genetic loci, we succeeded in generating knock-in lampreys expressing photoconvertible protein Dendra2 as well as those expressing EGFP. With its simplicity, design flexibility, and high efficiency, we propose that the present method has great versatility for various experimental uses in lamprey research and that it can also be applied to other “non-model” organisms.

Near-Infrared-Light Remote-Controlled Activation of Cancer Immunotherapy Using Photothermal Conjugated Polymer Nanoparticles.

Remote control of the therapeutic process is an ideal strategy for maximizing efficacy and avoiding side effects, especially for cancer immunotherapy. Herein, a conjugated polymer nanoparticles (CPNs)-mediated optogenetic system for in situ activation of immunotherapy under near-infrared laser irradiation is reported. This system is composed of photothermal CPNs and interferon-gamma (IFN-γ) plasmid driven by heat shock promoter HSP70. The photothermally responsive CPNs serve as a photo-heat nanotransducer to trigger the gene transcription of IFN-γ cytokine. The secreted IFN-γ from cancer cells can sufficiently elicit surrounding tumor-associated macrophages activation through IFN-γ-JAK-STAT1 transcription-factor signaling pathway and finally induce cancer cell killing by immunotherapy. Therefore, this synergetic optogenetic system provides a promising approach to remotely control the process of cancer immunotherapy.

Development of a genetically modified hepatoma cell line with heat-inducible high liver function.

The online version of this article (10.1007/s10616-021-00457-4) contains supplementary material, which is available to authorized users.

A Toolbox for Efficient Proximity-Dependent Biotinylation in Zebrafish Embryos

Understanding how proteins are organized in compartments is essential to elucidating their function. While proximity-dependent approaches such as BioID have enabled a massive increase in information about organelles, protein complexes, and other structures in cell culture, to date there have been only a few studies on living vertebrates. Here, we adapted proximity labeling for protein discovery in vivo in the vertebrate model organism, zebrafish. Using lamin A (LMNA) as bait and green fluorescent protein (GFP) as a negative control, we developed, optimized, and benchmarked in vivo TurboID and miniTurbo labeling in early zebrafish embryos. We developed both an mRNA injection protocol and a transgenic system in which transgene expression is controlled by a heat shock promoter. In both cases, biotin is provided directly in the egg water, and we demonstrate that 12 h of labeling are sufficient for biotinylation of prey proteins, which should permit time-resolved analysis of development. After statistical scoring, we found that the proximal partners of LMNA detected in each system were enriched for nuclear envelope and nuclear membrane proteins and included many orthologs of human proteins identified as proximity partners of lamin A in mammalian cell culture. The tools and protocols developed here will allow zebrafish researchers to complement genetic tools with powerful proteomics approaches.

Generation of Gene-Engineered Human Hepatoma Cells with Heat-Inducible Liver Functions

Hepatoma cells derived from liver carcinoma are a candidate cell source for bioartificial liver (BAL) systems due to their high proliferative capacity, although liver function of hepatoma cells is considerably low compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells with inducible high liver function were established by transducing liver-enriched transcription factor (LETF) genes. In this study, we aimed to develop new gene-engineered human hepatoma cells, in which high liver functions are inducible by heat treatment. For this purpose, we constructed a gene expression system for eight LETF genes under control of tetracycline-dependent transactivator (tTA), and the system was introduced into the genome of HepG2-HSP cells, in which a tTA expression system induced by a heat-shock protein promoter with transcriptional amplification was introduced into HepG2 cells. Thus, the heat-inducible tTA promotes LETF genes to induce liver function. Upon the heat treatment of the cells (HepG2-HSP/8F) at 43°C for 30 min, liver functions such as albumin secretion and cytochrome P450 were significantly enhanced. The cells with heat-inducible liver function can be used as a new cell source for various hepatic studies including construction of BAL systems.

Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish.

Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3–5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1ΔRING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1ΔRING, resulted in a decrease in the number of rods and cones at 72 h post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar cells was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did, however, detect increased cell death, confirmed by an increase in the number of TUNEL + cells in the ONL, which was proteasome-dependent, as proteasome inhibition rescued the cell death phenotype. Furthermore, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1a mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Lastly, CRISPR induced loss of Cdhr1a function phenocopied Siah1 overexpression resulting in a significant reduction of rods and cones. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS.

Inhibition of mTOR decreases insoluble proteins burden by reducing translation in C. elegans.

Aging animals accumulate insoluble proteins as a consequence of a decline of proteostatic maintenance with age. In Caenorhabditis elegans, for instance, levels of detergent-insoluble proteins increase with age. In longer-livedstrains of C. elegans, this accumulation occurs more slowly, implying a link to lifespan determination. We further explored this link and found that detergent-insoluble proteins accumulate more rapidly at higher temperatures, a condition where lifespan is short. We employed aC. elegansstrain carrying a GFP transcriptional reporter under the control of a heat shock (hsp-16.2) promoter to investigate the dynamics of proteostatic failure in individual nematodes. We found that early, sporadic activation of hsp-16.2was predictive of shorter remaining lifespan in individual nematodes. Exposure to rapamycin, resulting in reduced mTOR signaling, delayed spurious expression, extended lifespan, and delayed accumulation of insoluble proteins, suggesting that targets downstream of the mTOR pathway regulate the accumulation of insoluble proteins. We specifically explored ribosomal S6 kinase (rsks-1) as one such candidate and found that RNAi against rsks-1 also resulted in less age-dependent accumulation of insoluble proteins and extended lifespan. Our results demonstrate that inhibition of protein translation via reduced mTOR signaling resulted in slower accumulation of insoluble proteins, delayed proteostatic crisis, and extended lifespan inC. elegans.

An Efficient Gene Excision System in Maize.

Use of the morphogenic genes Baby Boom (Bbm) and Wuschel2 (Wus2), along with new ternary constructs, has increased the genotype range and the type of explants that can be used for maize transformation. Further optimizing the expression pattern for Bbm/Wus2 has resulted in rapid maize transformation methods that are faster and applicable to a broader range of inbreds. However, expression of Bbm/Wus2 can compromise the quality of regenerated plants, leading to sterility. We reasoned excising morphogenic genes after transformation but before regeneration would increase production of fertile T0 plants. We developed a method that uses an inducible site-specific recombinase (Cre) to excise morphogenic genes. The use of developmentally regulated promoters, such as Ole, Glb1, End2, and Ltp2, to drive Cre enabled excision of morphogenic genes in early embryo development and produced excised events at a rate of 25–100%. A different strategy utilizing an excision-activated selectable marker produced excised events at a rate of 53–68%; however, the transformation frequency was lower (13–50%). The use of inducible heat shock promoters (e.g. Hsp17.7, Hsp26) to express Cre, along with improvements in tissue culture conditions and construct design, resulted in high frequencies of T0 transformation (29–69%), excision (50–97%), usable quality events (4–15%), and few escapes (non-transgenic; 14–17%) in three elite maize inbreds. Transgenic events produced by this method are free of morphogenic and marker genes.

Cooperative Regulation of Campylobacter jejuni Heat-Shock Genes by HspR and HrcA.

The heat-shock response is defined by the transient gene-expression program that leads to the rapid accumulation of heat-shock proteins. This evolutionary conserved response aims at the preservation of the intracellular environment and represents a crucial pathway during the establishment of host–pathogen interaction. In the food-borne pathogen Campylobacter jejuni two transcriptional repressors, named HspR and HrcA, are involved in the regulation of the major heat-shock genes. However, the molecular mechanism underpinning HspR and HrcA regulatory function has not been defined yet. In the present work, we assayed and mapped the HspR and HrcA interactions on heat-shock promoters by high-resolution DNase I footprintings, defining their regulatory circuit, which governs C. jejuni heat-shock response. We found that, while DNA-binding of HrcA covers a compact region enclosing a single inverted repeat similar to the so-called Controlling Inverted Repeat of Chaperone Expression (CIRCE) sequence, HspR interacts with multiple high- and low-affinity binding sites, which contain HspR Associated Inverted Repeat (HAIR)-like sequences. We also explored the DNA-binding properties of the two repressors competitively on their common targets and observed, for the first time, that HrcA and HspR can directly interact and their binding on co-regulated promoters occurs in a cooperative manner. This mutual cooperative mechanism of DNA binding could explain the synergic repressive effect of HspR and HrcA observed in vivo on co-regulated promoters. Peculiarities of the molecular mechanisms exerted by HspR and HrcA in C. jejuni are compared to the closely related bacterium H. pylori that uses homologues of the two regulators.

Thermal Control of Engineered T-cells.

Genetically engineered T-cells are being developed to perform a variety of therapeutic functions. However, no robust mechanisms exist to externally control the activity of T-cells at specific locations within the body. Such spatiotemporal control could help mitigate potential off-target toxicity due to incomplete molecular specificity in applications such as T-cell immunotherapy against solid tumors. Temperature is a versatile external control signal that can be delivered to target tissues in vivo using techniques such as focused ultrasound and magnetic hyperthermia. Here, we test the ability of heat shock promoters to mediate thermal actuation of genetic circuits in primary human T-cells in the well-tolerated temperature range of 37-42 °C, and introduce genetic architectures enabling the tuning of the amplitude and duration of thermal activation. We demonstrate the use of these circuits to control the expression of chimeric antigen receptors and cytokines, and the killing of target tumor cells. This technology provides a critical tool to direct the activity of T-cells after they are deployed inside the body.