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1. Looking Down on NF-κB

   https://doi.org/10.1128/MCB.00104-20  

   Williams, Leah M.; Gilmore, Thomas D. (July 2020, Molecular and Cellular Biology)

   null (Ed.)

   ABSTRACT The diversified NF-κB transcription factor family has been extensively characterized in organisms ranging from flies to humans. However, homologs of NF-κB and many upstream signaling components have recently been characterized in basal phyla, including Cnidaria (sea anemones, corals, hydras, and jellyfish), Porifera (sponges), and single-celled protists, including Capsaspora owczarzaki and some choanoflagellates. Herein, we review what is known about basal NF-κBs and how that knowledge informs on the evolution and conservation of key sequences and domains in NF-κB, as well as the regulation of NF-κB activity. The structures and DNA-binding activities of basal NF-κB proteins resemble those of mammalian NF-κB p100 proteins, and their posttranslational activation appears to have aspects of both canonical and noncanonical pathways in mammals. Several studies suggest that the single NF-κB proteins found in some basal organisms have dual roles in development and immunity. Further research on NF-κB in invertebrates will reveal information about the evolutionary roots of this major signaling pathway, will shed light on the origins of regulated innate immunity, and may have relevance to our understanding of the responses of ecologically important organisms to changing environmental conditions and emerging pathogen-based diseases. 

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2. Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation

   https://doi.org/10.1093/nar/gkab874  

   Chen, Wei; Lu, Wei; Wolynes, Peter G; Komives, Elizabeth A (October 2021, Nucleic Acids Research)

   Abstract Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-κB from DNA is achieved through accelerated dissociation by the inhibitor protein IκBα. Using single-molecule FRET, we observed a continuum of conformations of NF-κB in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-κB is partially bound, allowing IκBα invasion to facilitate DNA dissociation. IκBα induces a locked conformation where the DNA-binding domains of NF-κB are too far apart to bind DNA, whereas a loss-of-function IκBα mutant retains the NF-κB conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors. 

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3. 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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4. NF-κB modifies the mammalian circadian clock through interaction with the core clock protein BMAL1

   https://doi.org/10.1371/JOURNAL.PGEN.1009933  

   Shen, Yang; Endale, Mehari; Wang, Wei; Morris, Andrew R.; Francey, Lauren J.; Harold, Rachel L.; Hammers, David W.; Huo, Zhiguang; Partch, Carrie L.; Hogenesch, John B.; et al (November 2021, PLOS Genetics)

   Kramer, Achim (Ed.)

   In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways. 

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5. The Interaction of NF-κB Transcription Factor with Centromeric Chromatin

   https://doi.org/10.1021/acs.jpcb.3c08388  

   Filliaux, Shaun; Bertelsen, Chloe; Baughman, Hannah; Komives, Elizabeth; Lyubchenko, Yuri (June 2024, The Journal of Physical Chemistry B)

   Centromeric chromatin is a subset of chromatin structure and governs chromosome segregation. The centromere is composed of both CENP-A nucleosomes (CENP-A(nuc)) and H3 nucleosomes (H3(nuc)) and is enriched with alpha-satellite (alpha-sat) DNA repeats. These CENP-A(nuc) have a different structure than H3(nuc), decreasing the base pairs (bp) of wrapped DNA from 147 bp for H3(nuc) to 121 bp for CENP-A(nuc). All these factors can contribute to centromere function. We investigated the interaction of H3(nuc) and CENP-A(nuc) with NF-kappaB, a crucial transcription factor in regulating immune response and inflammation. We utilized atomic force microscopy (AFM) to characterize complexes of both types of nucleosomes with NF-kappaB. We found that NF-kappaB unravels H3(nuc), removing more than 20 bp of DNA, and that NF-kappaB binds to the nucleosomal core. Similar results were obtained for the truncated variant of NF-kappaB comprised only of the Rel homology domain and missing the transcription activation domain (TAD), suggesting that RelA(TAD) is not critical in unraveling H3(nuc). By contrast, NF-kappaB did not bind to or unravel CENP-A(nuc). These findings with different affinities for two types of nucleosomes to NF-kappaB may have implications for understanding the mechanisms of gene expression in bulk and centromere chromatin. 

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