Sustained Oscillations of NF-κB Produce Distinct Genome Scanning and Gene Expression Profiles

Abstract

NF-κB is a prototypic stress-responsive transcription factor that acts within a complex regulatory network. The signaling dynamics of endogenous NF-κB in single cells remain poorly understood. To examine real time dynamics in living cells, we monitored NF-κB activities at multiple timescales using GFP-p65 knock-in mouse embryonic fibroblasts. Oscillations in NF-κB were sustained in most cells, with several cycles of transient nuclear translocation after TNF-α stimulation. Mathematical modeling suggests that NF-κB oscillations are selected over other non-oscillatory dynamics by fine-tuning the relative strengths of feedback loops like IκBα. The ability of NF-κB to scan and interact with the genome in vivo remained remarkably constant from early to late cycles, as observed by fluorescence recovery after photobleaching (FRAP). Perturbation of long-term NF-κB oscillations interfered with its short-term interaction with chromatin and balanced transcriptional output, as predicted by the mathematical model. We propose that negative feedback loops do not simply terminate signaling, but rather promote oscillations of NF-κB in the nucleus, and these oscillations are functionally advantageous.

Interrogation of Inhibitor of Nuclear Factor κB α/Nuclear Factor κB (IκBα/NF-κB) Negative Feedback Loop Dynamics

“…3, B and E). This correlates with the NF-B nuclear/cytoplasmic oscillations observed by others with a period of ϳ100 min (15)(16)(17)(21)(22)(23)(24). The slightly longer duty cycle in this study may relate to cell type-specific differences in the pathway or to the time required to transcribe and translate the larger ⌱B␣-FLuc chimeric reporter protein.…”

“…Characterizing TNF␣-induced Regulation of the ⌱B␣/NF-B Negative Feedback Loop in Cell Populations-The ⌱B␣/NF-B negative feedback loop represents a major regulatory node within the NF-B pathway and is a critical determinant of NF-B oscillatory behaviors that can encode stimulus-specific gene expression programs (15,(21)(22)(23)(24)30). Bioluminescent reporters are ideally suited for medium to high throughput studies of stimulus-induced cellular signaling in real time in populations of cells.…”

“…negative feedback loops) (20). The frequency of these NF-B protein oscillations can encode distinct gene expression profiles, as determined with cell population studies (15,(21)(22)(23)(24). Furthermore, single cell studies have revealed heterogeneous and asynchronous NF-B responses in single cells (18,21,25), especially in response to low concentrations of TNF␣ (17).…”

β-Transducin Repeat-containing Protein 1 (β-TrCP1)-mediated Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) Protein Degradation Promotes Tumor Necrosis Factor α (TNFα)-induced Inflammatory Gene Expression

“…A body of studies have elucidated the role of TNF␣ in inflammatory responses (38), but how it precisely controls inflammatory gene expression is still not completely understood. Activation of NF-B by TNF␣ is a major pathway that controls inflammatory gene expression, although it has been shown to be biphasic and depend on the status of its associated coregulators (11)(12)(13)(14)(15)39). On the basis of our findings, the reduction of SMRT protein levels by TNF␣ downstream ␤-TrCP1 E3 ligase may partly account for the activation of NF-B activity on the expression of its target genes in HUVECs.…”

“…Upon TNF␣ stimulation, an early response is the selective induction of inflammatory genes driven by NF-B (11)(12)(13)(14)(15). Although several mechanisms underlying the activation of NF-B have been proposed (15), a complete picture includes less well studied nuclear events.…”