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NEMO lysine mutant mice reveal ROS-RelA signaling required for optimal CD40-induced humoral immunity

ABSTRACT: Oxidative and genotoxic stresses activate NF-κB signaling through intracellular pathways distinct from those initiated by membrane receptors, but their physiological roles remain incompletely defined. We created the NEMODK mouse model with germline arginine substitutions at NEMO (NF-κB essential modulator) lysines 270 and 302, key sites for post-translational modifications selectively induced by these intracellular stresses to enable NF-κB signaling in vitro. NEMODK mice exhibited B cell-intrinsic defects in the generation of germinal center B cells and humoral responses. Mechanistically, NEMODK B cells displayed a CD40-specific NF-κB signaling defect, unrelated to acute canonical and delayed noncanonical NF-κB activity, which was accompanied by the lack of sustained NEMO monoubiquitination downstream of mitochondrial reactive oxygen species generation without relying on nuclear DNA damage. This specifically reduced persistent RelA activity and a failure to mount transcriptomic and epigenetic changes critical for homotypic B cell aggregation, cell proliferation, class switch recombination and antibody secreting cell generation. Thus, CD40 induces a uniquely sustained NEMO modification and persistent RelA transcriptional activity to efficiently generate class-switched antibodies.

ORGANISM(S): Mus musculus

PROVIDER: GSE325910 | GEO | 2026/06/09

REPOSITORIES: GEO

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NEMO lysine mutant mice reveal ROS-RelA signaling required for optimal CD40-induced humoral immunity [NEMODK_CUTTAG]

Project description:Oxidative and genotoxic stresses activate NF-κB signaling through intracellular pathways distinct from those initiated by membrane receptors, but their physiological roles remain incompletely defined. We created the NEMODK mouse model with germline arginine substitutions at NEMO (NF-κB essential modulator) lysines 270 and 302, key sites for post-translational modifications selectively induced by these intracellular stresses to enable NF-κB signaling in vitro. NEMODK mice exhibited B cell-intrinsic defects in the generation of germinal center B cells and humoral responses. Mechanistically, NEMODK B cells displayed a CD40-specific NF-κB signaling defect, unrelated to acute canonical and delayed noncanonical NF-κB activity, which was accompanied by the lack of sustained NEMO monoubiquitination downstream of mitochondrial reactive oxygen species generation without relying on nuclear DNA damage. This specifically reduced persistent RelA activity and a failure to mount transcriptomic and epigenetic changes critical for homotypic B cell aggregation, cell proliferation, class switch recombination and antibody secreting cell generation. Thus, CD40 induces a uniquely sustained NEMO modification and persistent RelA transcriptional activity to efficiently generate class-switched antibodies.

2026-06-09 | GSE325580 | GEO

NEMO lysine mutant mice reveal ROS-RelA signaling required for optimal CD40-induced humoral immunity [NEMODK_ATACSeq]

2026-06-09 | GSE325579 | GEO

CD40-induced humoral immunity

Project description:CD40CD40 induces a uniquely sustained NEMO modification and persistent RelA transcriptional activity to efficiently generate class-switched antibodies.

2026-06-09 | GSE325803 | GEO

ATM-Dependent Phosphorylation of Nemo SQ Motifs is Dispensable for Nemo-Mediated Gene Expression Changes in Response to DNA Double Strand Breaks

Project description:In response to DNA double strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm where it promotes monoubiquitylation of Nemo and resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as lipopolysaccharides (LPS). To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy, fertile, and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.

2024-08-08 | GSE264315 | GEO

Atypical NF-κB activation by endogenous DNA damage drives senescence and aging

Project description:Chronic activation of NF-κB contributes to senescence and aging, but how endogenous DNA damage engages this pathway in vivo remains unclear. NF-κB can be activated by an atypical pathway in which genotoxic stress triggers ATM-dependent sumoylation of the regulatory subunit NEMO, yet the role of this axis in senescence and aging has not been tested. Here we introduced a knock-in NEMO double-lysine mutant (NEMO-DK) that prevents sumoylation at the conserved sites required for DNA damage-induced NF-κB activation, and examined its impact in Ercc1-/∆ mice, a model of accelerated, DNA damage-driven aging. We demonstrated that NEMO-DK markedly reduced senescence and SASP in primary fibroblasts and across multiple tissues, improved liver and skeletal muscle pathology, attenuated intervertebral disc degeneration, significantly delayed the onset and progression of aging symptoms and extended healthspan. These findings identify DNA damage-induced NEMO sumoylation as a key driver of NF-κB-mediated senescence in vivo and suggest that the atypical NF-κB pathway is a potential therapeutic target to mitigate aging-associated diseases.

2026-01-01 | GSE314144 | GEO

A single Thr505 phospho-site mutation reveals an important role for the RelA NF-κB subunit in liver regeneration and cancer

Project description:Post-translational modification of NF-κB subunits provides a mechanism to differentially regulate their activity in response to the many stimuli that can induce this pathway. However, the physiological significance of these modifications is largely unknown and it remains unclear if these have a critical role in the normal and pathological functions of NF-κB in vivo. Among these, phosphorylation of the RelA(p65) Thr505 residue has been described as an important regulator of NF-κB activity in cell lines but its physiological significance was not known. Therefore, to learn more about the role of this pathway in vivo, we generated a knockin mouse with a RelA T505A mutation. Unlike RelA knockout mice, the RelA T505A mice develop normally but exhibit aberrant hepatocyte proliferation following liver partial hepatectomy or damage resulting from carbon tetrachloride treatment. Consistent with these effects, RelA T505A mice exhibit earlier onset of cancer in the N-nitrosodiethylamine (DEN) model of hepatocellular carcinoma. This data reveals a critical pathway controlling NF-κB function in the liver that acts to suppress tumour-promoting activities of RelA.

2015-11-30 | E-MTAB-3596 | biostudies-arrayexpress

RelA and RelB-dependent transcriptome analysis in lymphotoxin-ß receptor stimulated mouse embryonic fibroblasts

Project description:Background: Lymphotoxin signaling via the lymphotoxin-β receptor (LTβR) has been implicated in several biological processes, ranging from development of secondary lymphoid organs, maintenance of splenic tissue, host defense against pathogens, autoimmunity, and lipid homeostasis. The major transcription factor that is activated by LTβR crosslinking is NF-κB. Two signaling pathways have been described that result in the activation of classical p50-RelA and alternative p52-RelB NF-κB heterodimers. Results: Using microarray analysis, we investigated the transcriptional response downstream of the LTβR in mouse embryoni fibroblasts (MEF) and its regulation by the RelA and RelB subunits of NF-κB. We describe novel LTβR-responsive genes that are regulated by RelA and/or RelB. Interestingly, we found that the majority of LTβR-regulated genes require the presence of both RelA and RelB, suggesting significant crosstalk between the two NF-κB activation pathways. Gene Ontology (GO) analysis confirmed that LTβR-NF-κB target genes are predominantly involved in the regulation of immune responses. However, other biological processes, such as apoptosis/cell death, cell cycle, angiogenesis, and taxis were also regulated by LTβR signaling. Moreover, we show that activation of the LTβR inhibits the expression of a key adipogenic transcription factor, peroxisome proliferator activated receptor-γ (pparg), suggesting that LTβR signaling may interfere with adipogenic differentiation. Conclusions: Thus, microarray analysis of LTβR-stimulated fibroblasts revealed further insight into the transcriptional response of LTβR signaling and its regulation by the NF-κB family members RelA and RelB. Keywords: cell type comparison (wt vs relA-/- vs relB-/-) after genetic modification using a time course for each cell type (wt, relA-/-, relB-/-) two time points were analysed (0h as control and 10h) using 3 technical replicates resulting in 18 samples in total

2008-12-17 | E-GEOD-11963 | biostudies-arrayexpress

WSB1/2 target chromatin-bound lysine-methylated RelA for proteasomal degradation and NF-κB termination

Project description:Proteasome-mediated degradation of chromatin-bound NF-κB is critical in terminating the transcription of pro-inflammatory genes and can be triggered by Set9-mediated lysine methylation of RelA subunit. However, the E3 ligase targeting methylated RelA remains unknown. Here, we identified two structurally similar WD-40 repeat (WDR) proteins, WSB1 and WSB2, as the E3s that recognize chromatin-bound methylated RelA for polyubiquitination and proteasomal degradation. WSB1/2 specifically recognized methylated lysines (K) 314 and 315 of RelA via their WDR domains. Deletion of WRD in WSB1/2 or mutation of K314/315 of RelA to arginines abolished the interaction between WSB1/2 and RelA. RNA-sequencing of TNF--stimulated WSB1/2 knockdown cells revealed that WSB1/2 negatively regulated a subset of NF-B target genes with reduced polyubiquitination of chromatin-bound RelA. TNF- stimulated the methylation of RelA and the subsequent recruitment of WSB1/2 to the promoters of these NF-B target genes to terminate the transcription. Computational modeling demonstrated that a highly conserved Asp within repeat 3 of WDR domains of WSB1/2 coordinated its interaction with K314/K315 of RelA, with a higher pKa when either of the lysines is methylated. Together, these findings identify novel E3 ligases that target chromatin-bound methylated RelA for proteolysis to prevent sustained NF-κB activation, providing new targets for therapeutic intervention of NF-κB-mediated inflammatory diseases.

2024-05-29 | GSE233286 | GEO

Multi-species RELA ChIP-seq of mammalian aortic endothelial cells before and after TNFα stimulation

Project description:TNFα has an evolutionary conserved role in mediating inflammation via activation of the transcription factor NF-κB. The functions of individual NF-κB binding sites are not well understood. To identify conserved and functionally important NF-κB binding sites in mammals, we performed ChIP-seq to map the genome-wide binding of RELA and select histone modifications in primary vascular endothelial cells (ECs) isolated from the aortas of human (HAEC), mouse (MAEC) and cow (BAEC), before and after TNFα. The conserved RELA binding sites show strong epigenetic changes in response to TNFα and enrich near genes controlling vascular development and pro-inflammatory responses. Our method identifies novel modes of RELA-chromatin interactions that are conserved in mammals and shared between multiple cell-types. Particularly, genomic regions bound by RELA prior to stimulation are important responders during TNFα stimulation. We use CRISPR/Cas9 genome editing to validate the roles of the conserved RELA pre-bound sites near pro-inflammatory genes such as CCL2 and PLK2. Our evolutionary approach describes new aspects of mammalian NF-κB biology including its role within super-enhancers and relevance in inflammatory disorders.

2020-10-03 | E-MTAB-7889 | biostudies-arrayexpress

Transcriptional outcomes and kinetic patterning of gene expression in response to NF-κB activation [RNA-seq]

Project description:Transcription factor NF-κB regulates cellular responses to environmental cues. For many stimuli NF-κB resides only transiently in the nucleus. Consequently, time-dependent transcriptional outputs are a fundamental feature of NF-κB activation. Here we identify mechanisms that direct kinetic patterns of NF-κB-dependent gene expression and transcriptional outcomes in response to a transient NF-κB-inducing stimulus in B cells. By combining RELA binding, RNA polymerase II (Pol II) recruitment, and perturbation of NF-κB activation, we demonstrate that kinetic differences amongst early- and late-activated RELA target genes can be understood based on chromatin configuration prior to cell activation and RELA-dependent priming, respectively. Additionally, we identified genes that were repressed by RELA activation and others that responded to RELA-activated transcription factors. Cumulatively, our studies define an NF-κB-responsive inducible gene cascade in activated B cells.

2018-09-17 | GSE117254 | GEO

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