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1. Engineering combinatorial and dynamic decoders using synthetic immediate-early genes

   https://doi.org/10.1038/s42003-020-01171-1  

   Ravindran, Pavithran T.; Wilson, Maxwell Z.; Jena, Siddhartha G.; Toettcher, Jared E. (December 2020, Communications Biology)

   null (Ed.)

   Abstract Many cell- and tissue-level functions are coordinated by intracellular signaling pathways that trigger the expression of context-specific target genes. Yet the input–output relationships that link pathways to the genes they activate are incompletely understood. Mapping the pathway-decoding logic of natural target genes could also provide a basis for engineering novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (SynIEGs), target genes of Erk signaling that implement complex, user-defined regulation and can be monitored by using live-cell biosensors to track their transcription and translation. We demonstrate the power of this approach by confirming Erk duration-sensing by FOS , elucidating how the BTG2 gene is differentially regulated by external stimuli, and designing a synthetic immediate-early gene that selectively responds to the combination of growth factor and DNA damage stimuli. SynIEGs pave the way toward engineering molecular circuits that decode signaling dynamics and combinations across a broad range of cellular contexts. 

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2. FosGFP expression does not capture a sensory learning-related engram in superficial layers of mouse barrel cortex

   https://doi.org/10.1073/pnas.2112212118  

   Lee, Jiseok; Urban-Ciecko, Joanna; Park, Eunsol; Zhu, Mo; Myal, Stephanie E.; Margolis, David J.; Barth, Alison L. (December 2021, Proceedings of the National Academy of Sciences)

   Immediate-early gene (IEG) expression has been used to identify small neural ensembles linked to a particular experience, based on the principle that a selective subset of activated neurons will encode specific memories or behavioral responses. The majority of these studies have focused on “engrams” in higher-order brain areas where more abstract or convergent sensory information is represented, such as the hippocampus, prefrontal cortex, or amygdala. In primary sensory cortex, IEG expression can label neurons that are responsive to specific sensory stimuli, but experience-dependent shaping of neural ensembles marked by IEG expression has not been demonstrated. Here, we use a fosGFP transgenic mouse to longitudinally monitor in vivo expression of the activity-dependent gene c-fos in superficial layers (L2/3) of primary somatosensory cortex (S1) during a whisker-dependent learning task. We find that sensory association training does not detectably alter fosGFP expression in L2/3 neurons. Although training broadly enhances thalamocortical synaptic strength in pyramidal neurons, we find that synapses onto fosGFP+ neurons are not selectively increased by training; rather, synaptic strengthening is concentrated in fosGFP− neurons. Taken together, these data indicate that expression of the IEG reporter fosGFP does not facilitate identification of a learning-specific engram in L2/3 in barrel cortex during whisker-dependent sensory association learning. 

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3. Analysis of localized cAMP perturbations within a tissue reveal the effects of a local, dynamic gap junction state on ERK signaling

   https://doi.org/10.1371/journal.pcbi.1009873  

   Fonseca, João Pedro; Aslankoohi, Elham; Ng, Andrew H.; Chevalier, Michael (March 2022, PLOS Computational Biology)

   Meier-Schellersheim, Martin (Ed.)

   Beyond natural stimuli such as growth factors and stresses, the ability to experimentally modulate at will the levels or activity of specific intracellular signaling molecule(s) in specified cells within a tissue can be a powerful tool for uncovering new regulation and tissue behaviors. Here we perturb the levels of cAMP within specific cells of an epithelial monolayer to probe the time-dynamic behavior of cell-cell communication protocols implemented by the cAMP/PKA pathway and its coupling to the ERK pathway. The time-dependent ERK responses we observe in the perturbed cells for spatially uniform cAMP perturbations (all cells) can be very different from those due to spatially localized perturbations (a few cells). Through a combination of pharmacological and genetic perturbations, signal analysis, and computational modeling, we infer how intracellular regulation and regulated cell-cell coupling each impact the intracellular ERK response in single cells. Our approach reveals how a dynamic gap junction state helps sculpt the intracellular ERK response over time in locally perturbed cells. 

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4. Heterogeneous NF-κB activation and enhancer features shape transcription in Drosophila immunity

   https://doi.org/10.1101/2025.05.19.654881  

   Nawar, Noshin; Rits, Emma; Cohen, Lianne; Wunderlich, Zeba (May 2025, bioRxiv)

   Conserved NF-κB signaling pathways shape immune responses in animals. In mammals, NF-κB activation patterns and downstream transcription vary with stimulus, cell type, and stochastic differences among identically treated cells. Whether animals without adaptive immunity exhibit similar heterogeneity or rely on distinct immune strategies remains unknown. We engineered Drosophila melanogaster S2* reporter cells as an immune-responsive model to monitor the dynamics of an NF-κB transcription factor, Relish, and downstream transcription in single, living cells. Following immune stimulation, Relish exhibits diverse nuclear localization dynamics that fall into distinct categories, with both the fraction of responsive cells and their activation speed rising with stimulus dose. Pre-stimulus features, including Relish nuclear fraction, predict a cell's responsiveness to stimulation. Simultaneous measurement of Relish and downstream transcription revealed that the probability of transcriptional bursts from immune-responsive enhancers correlates with Relish nuclear fraction. The number of NF-κB binding sites tunes transcriptional activity among immune enhancers. Our study uncovers heterogeneity in NF-κB activation and target gene expression within Drosophila, illustrating how dynamic NF-κB behavior and enhancer architecture tune gene regulation. 

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5. Crosstalk between ERK and MRTF‐A signaling regulates TGFβ1‐induced epithelial‐mesenchymal transition

   https://doi.org/10.1002/jcp.30705  

   Nalluri, Sandeep M.; Sankhe, Chinmay S.; O'Connor, Joseph W.; Blanchard, Paul L.; Khouri, Joelle N.; Phan, Steven H.; Virgi, Gage; Gomez, Esther W. (February 2022, Journal of Cellular Physiology)

   Abstract Epithelial‐mesenchymal transition (EMT) is a physiological process that is essential during embryogenesis and wound healing and also contributes to pathologies including fibrosis and cancer. EMT is characterized by marked gene expression changes, loss of cell–cell contacts, remodeling of the cytoskeleton, and acquisition of enhanced motility. In the late stages of EMT, cells can exhibit myofibroblast‐like properties with enhanced expression of the mesenchymal protein marker α‐smooth muscle actin and contractile activity. Transforming growth factor (TGF)‐β1 is a well‐known inducer of EMT and it activates a plethora of signaling cascades including extracellular signal‐regulated kinase (ERK). Previous reports have demonstrated a role for ERK signaling in the early stages of EMT, but the molecular impacts of ERK signaling on the late stages of EMT are still unknown. Here, we found that inhibition of the phosphorylation of ERK enhances focal adhesions, stress fiber formation, cell contractility, and gene expression changes associated with TGFβ1‐induced EMT in mammary epithelial cells. These effects are mediated in part by the phosphorylation state and subcellular localization of myocardin‐related transcription factor‐A. These findings indicate that the intricate crosstalk between signaling cascades plays an important role in regulating the progression of EMT and suggests new approaches to control EMT processes. 

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