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

Kizilirmak et al., iScience, 2023 asked why genetically identical cells respond so differently to the same inflammatory signal. Working with immortalised mouse embryonic fibroblasts carrying a GFP-tagged p65, all derived from a single embryo, the authors grew single cell clones and filmed their NF-κB responses to tumour necrosis factor alpha and interleukin 1 beta. The clones behaved distinctly and reproducibly: some oscillated sharply, others held a persistent nuclear signal. Transcriptomic profiling showed the clones differ only modestly, in the amount of NF-κB itself, in the number of activating complexes formed at the receptor, and in the expression of Nfkbia, which encodes the inhibitor IκBα and forms the pathway's main negative feedback. A mathematical model constrained by those measured differences reproduced the observed dynamics.

The prediction the model made was testable: lower IκBα in the oscillating clone and its dynamics should shift toward the persistent one. To test it the authors used AUMsilence sdASOs against Nfkbia, four custom oligonucleotides at 5 μM against a scrambled control at the same concentration, on the fibroblasts for 24 hours. The paper describes the oligonucleotides as designed to be self-delivered and fully complementary to the target messenger RNA, cutting it through RNase H.

The prediction held. A moderate reduction in the transcript was enough to change the response qualitatively rather than only quantitatively. The peak height was almost unaltered in the oscillating clone, but the area under the curve rose in both clones, and the oscillating clone's dynamics became similar to those of the persistent one. The authors report a caveat with it: the scrambled control was not completely inert, producing a smaller but still significant effect of its own, and they lean on the agreement between the measured knockdown and the model to argue the change is specific.