Double knockin mice show NF-κB trajectories in immune signaling and aging

Rahman, Shah Md Toufiqur; Aqdas, Mohammad; Martin, Erik W.; Tomassoni-Ardori, Francesco; Songkiatisak, Preeyaporn; Oh, Kyu-Seon; Uderhardt, Stefan; Yun, Sangwon; Claybourne, Quia C.; McDevitt, Ross A.; Greco, Valentina; Germain, Ronald N.; Tessarollo, Lino; Sung, Myong-Hee

doi:10.1016/j.celrep.2022.111682

Cited by 13 publications

(5 citation statements)

References 51 publications

(88 reference statements)

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“…This indicates that stimulus specificity in NF-κB dynamics is clone-dependent and, all the more so, it will vary among cell types within the same organism. This is in line with a recent study where a synthetic version of the NF-κB system ectopically expressed in yeast (Zhang et al, 2017) displays different types of dynamics and responses by manipulating the expression of key genes within the genetic circuit.Furthermore, since immortalized MEFs maintain certain characteristics of primary MEFs (Beg and Baltimore, 1996), our work suggests that a similar fine-tuning can take place naturally in primary mammalian cells; although a recently developed reporter mouse (Rahman et al, 2022) has allowed to show that primary cells and cell lines can have different NF-κB dynamical features. Only further analysis of primary cells will allow us to address this question.…”

Section: Discussionmentioning

confidence: 83%

Small transcriptional differences among cell clones lead to distinct NF-κB dynamics

Kizilirmak

Monteleone

García-Manteiga

et al. 2021

Preprint

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Transcription factor dynamics is fundamental to determine the activation of accurate transcriptional programs and yet is heterogeneous at single-cell level. The source of this dynamic variability is not completely understood. Here we focus on the nuclear factor κB (NF-κB), whose dynamics have been reported to cover a wide spectrum ranging from oscillatory to non-oscillatory. We show that clonal populations of immortalized fibroblasts derived from a single mouse embryo (that can hence be considered quasi-identical) display robustly distinct dynamics upon tumor necrosis α (TNF-α) stimulation. Combining transcriptomics, data-constrained mathematical modelling, and mRNA interference we show that small differences in the expression of genes belonging to the NF-κB regulatory circuit are predictive of the distinct responses to inflammatory stimuli observed among the clones. We propose that this transcriptional fine-tuning can be a general mechanism to produce cell subpopulations with distinct dynamic responses to stimuli within homogeneous cell populations.

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Section: Discussionmentioning

confidence: 83%

Small transcriptional differences among cell clones lead to distinct NF-κB dynamics

Kizilirmak

Monteleone

García-Manteiga

et al. 2021

Preprint

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“…Together, our results confirmed that the mTFP1-cRel reporter performs signaling and DNA binding functions with efficiencies comparable to its wild-type counterpart. This complements another fluorescent fusion protein cRel reporter that was recently made available, whose utility for a number of applications has also been documented ( 16 ).…”

Section: Resultsmentioning

confidence: 91%

“…Our choice of mTFP1-cRel in the blue wavelength spectrum distinguishes this mouse strain from a recently reported mScarlet-cRel mouse strain ( 16 ). We showed that this reporter is compatible for live cell imaging studies with other reporters, such as mVenus-RelA ( 19 ) or cMyc-EGFP ( 37 ), and leaves longer wavelengths open for multiplexing with other reporters for multidimensional trajectory measurements.…”

Section: Discussionmentioning

confidence: 93%

Direct observation correlates NFκB cRel in B cells with activating and terminating their proliferative program

Vaidehi Narayanan,

Xiang,

Chen

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Antibody responses require the proliferative expansion of B cells controlled by affinity-dependent signals. Yet, proliferative bursts are heterogeneous, varying between 0 and 8 divisions in response to the same stimulus. NFκB cRel is activated in response to immune stimulation in B cells and is genetically required for proliferation. Here, we asked whether proliferative heterogeneity is controlled by natural variations in cRel abundance. We developed a fluorescent reporter mTFP1-cRel for the direct observation of cRel in live proliferating B cells. We found that cRel is heterogeneously distributed among naïve B cells, which are enriched for high expressors in a heavy-tailed distribution. We found that high cRel expressors show faster activation of the proliferative program, but do not sustain it well, with population expansion decaying earlier. With a mathematical model of the molecular network, we showed that cRel heterogeneity arises from balancing positive feedback by autoregulation and negative feedback by its inhibitor IκBε, confirmed by mouse knockouts. Using live-cell fluorescence microscopy, we showed that increased cRel primes B cells for early proliferation via higher basal expression of the cell cycle driver cMyc. However, peak cMyc induction amplitude is constrained by incoherent feedforward regulation, decoding the fold change of cRel activity to terminate the proliferative burst. This results in a complex nonlinear, nonmonotonic relationship between cRel expression and the extent of proliferation. These findings emphasize the importance of direct observational studies to complement gene knockout results and to learn about quantitative relationships between biological processes and their key regulators in the context of natural variations.

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“…In this study we used cell line tools and an in vivo mouse model that overexpress a labelled IκB⍺ gene from a BAC that responds normally to an inflammatory stimulus. Recent studies have highlighted differences between cells derived from primary tissues with respect to NF-κB signalling dynamics ( Rahman et al, 2022 ), and our mouse would act as a useful physiological tool for investigation of this phenomenon. We used our models to demonstrate that IκB⍺ overexpression has a pronounced effect on inflammatory gene activation, mediated through elevated nuclear IκB⍺ that competes for translocating p65.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of IκB⍺ modulates NF-κB activation of inflammatory target gene expression

Downton

Bagnall

England

et al. 2023

Front. Mol. Biosci.

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Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-κB (NF-κB) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm in some cell types. We investigate the relationship between p65 and inhibitor-κB⍺ (IκBα) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of IκB⍺-eGFP protein overexpression in a pseudo-native genomic context. We find that cells with high levels of the negative regulator IκBα remain responsive to inflammatory stimuli and maintain dynamics for both p65 and IκBα. In contrast, canonical target gene expression is dramatically reduced by overexpression of IκBα, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of IκB⍺ also suppresses canonical target gene expression, suggesting a mechanism in which nuclear IκB⍺ accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immunoprecipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both IκB⍺ and p65. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response.

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Double knockin mice show NF-κB trajectories in immune signaling and aging

Cited by 13 publications

References 51 publications

Small transcriptional differences among cell clones lead to distinct NF-κB dynamics

Small transcriptional differences among cell clones lead to distinct NF-κB dynamics

Direct observation correlates NFκB cRel in B cells with activating and terminating their proliferative program

Overexpression of IκB⍺ modulates NF-κB activation of inflammatory target gene expression

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