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1. Optimization of Transcription Factor Genetic Circuits

   https://doi.org/10.3390/biology11091294  

   Frank, Steven (September 2022, Biology)

   Transcription factors (TFs) affect the production of mRNAs. In essence, the TFs form a large computational network that controls many aspects of cellular function. This article introduces a computational method to optimize TF networks. The method extends recent advances in artificial neural network optimization. In a simple example, computational optimization discovers a four-dimensional TF network that maintains a circadian rhythm over many days, successfully buffering strong stochastic perturbations in molecular dynamics and entraining to an external day–night signal that randomly turns on and off at intervals of several days. This work highlights the similar challenges in understanding how computational TF and neural networks gain information and improve performance. 

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2. Clock-Controlled and Cold-Induced CYCLING DOF FACTOR6 Alters Growth and Development in Arabidopsis

   https://doi.org/10.3389/fpls.2022.919676  

   Blair, Emily J.; Goralogia, Greg S.; Lincoln, Matthew J.; Imaizumi, Takato; Nagel, Dawn H. (July 2022, Frontiers in Plant Science)

   The circadian clock represents a critical regulatory network, which allows plants to anticipate environmental changes as inputs and promote plant survival by regulating various physiological outputs. Here, we examine the function of the clock-regulated transcription factor, CYCLING DOF FACTOR 6 (CDF6), during cold stress in Arabidopsis thaliana . We found that the clock gates CDF6 transcript accumulation in the vasculature during cold stress. CDF6 mis-expression results in an altered flowering phenotype during both ambient and cold stress. A genome-wide transcriptome analysis links CDF6 to genes associated with flowering and seed germination during cold and ambient temperatures, respectively. Analysis of key floral regulators indicates that CDF6 alters flowering during cold stress by repressing photoperiodic flowering components, FLOWERING LOCUS T ( FT ), CONSTANS ( CO ), and BROTHER OF FT (BFT) . Gene ontology enrichment further suggests that CDF6 regulates circadian and developmental-associated genes. These results provide insights into how the clock-controlled CDF6 modulates plant development during moderate cold stress. 

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3. Regulatory links between the circadian clock and stress-induced biomolecular condensates

   https://doi.org/10.1038/s44323-025-00036-2  

   Brown, Gabriela; Nagel, Dawn H (December 2025, npj Biological Timing and Sleep)

   The circadian clock is a conserved timekeeping mechanism that is essential for integrating different environmental cues such as light and temperature to coordinate biological processes with the time of day. While much is known about transcriptional regulation by the clock, the role of post-transcriptional regulation, particularly through sequestration into biomolecular condensate such as stress granules, remains less understood. Stress granules are dynamic RNA-protein assemblies that play a critical role in the cellular response to stress by sequestering mRNAs to regulate translation during stressful conditions. In animals and fungi, the circadian clock regulates stress granule formation and mRNA translation by controlling key factors such as eIF2α, which orchestrates the rhythmic sequestration and translation of specific mRNAs. In plants, it has been shown that some transcripts, despite coming from arrhythmic expression, are rhythmically translated. In addition, some clock-controlled genes (CCGs) are induced in response to heat stress only at the transcriptional level and not at the translational level. Together this highlights a layer of clock regulation beyond transcription. This review discusses the intersection between the circadian clock and heat stress-related biomolecular condensates across eukaryotes, with a particular focus on plants. We discuss how the clock may regulate stress granule dynamics and preferential translation of mRNAs at specific times of the day or during stress responses, thereby enhancing cellular function and energy efficiency. By integrating evidence from animals, fungi, and plants, we highlight emerging questions regarding the role of biomolecular condensates as post-transcriptional mechanisms in controlling circadian rhythms and stress tolerance in plants. 

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4. Time of the day prioritizes the pool of translating mRNAs in response to heat stress

   https://doi.org/10.1093/plcell/koab113  

   Bonnot, Titouan; Nagel, Dawn H. (April 2021, The Plant Cell)

   Abstract The circadian clock helps organisms to anticipate and coordinate gene regulatory responses to changes in environmental stimuli. Under growth limiting temperatures, the time of the day modulates the accumulation of polyadenylated mRNAs. In response to heat stress, plants will conserve energy and selectively translate mRNAs. How the clock and/or the time of the day regulates polyadenylated mRNAs bound by ribosomes in response to heat stress is unknown. In-depth analysis of Arabidopsis thaliana translating mRNAs found that the time of the day gates the response of approximately one-third of the circadian-regulated heat-responsive translatome. Specifically, the time of the day and heat stress interact to prioritize the pool of mRNAs in cue to be translated. For a subset of mRNAs, we observed a stronger gated response during the day, and preferentially before the peak of expression. We propose previously overlooked transcription factors (TFs) as regulatory nodes and show that the clock plays a role in the temperature response for select TFs. When the stress was removed, the redefined priorities for translation recovered within 1 h, though slower recovery was observed for abiotic stress regulators. Through hierarchical network connections between clock genes and prioritized TFs, our work provides a framework to target key nodes underlying heat stress tolerance throughout the day. 

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5. Using Restriction Endonuclease, Protection, Selection, and Amplification to Identify Preferred DNA-Binding Sequences of Microbial Transcription Factors

   https://doi.org/10.1128/spectrum.04397-22  

   Barrows, John K.; Van Dyke, Michael W. (February 2023, Microbiology Spectrum)

   Polen, Tino (Ed.)

   ABSTRACT Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism’s genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro , iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms’ transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods. 

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