Abstract
NF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as “NF-κB” in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant in specific cell types. Accurate characterization of stimulus-specific and tissue-specific dimer repertoires is fundamentally important for understanding the downstream gene regulation by NF-κB proteins. In vitro assays such as immunoprecipitation have been widely used to analyze subunit composition, but these methods do not provide information about dimerization status within the natural intracellular environment of intact live cells. Here we apply a live single cell microscopy technique termed Number and Brightness to examine dimers translocating to the nucleus in fibroblasts after pro-inflammatory stimulation. This quantitative assay suggests that RelA:RelA homodimers are more prevalent than might be expected. We also found that the relative proportion of RelA:RelA homodimers can be perturbed by small molecule inhibitors known to disrupt the NF-κB pathway. Our findings show that Number and Brightness is a useful method for investigating NF-κB dimer species in live cells. This approach may help identify the relevant targets in pathophysiological contexts where the dimer specificity of NF-κB intervention is desired.
Highlights
Nuclear Factor-kappaB (NF-κB) is arguably the most important signaling pathway involved in immune responses [1]
We discovered that the Number and Brightness (N&B) assay detects the presence of a mixed RelA-containing complexes (RelA) dimer status in the nuclei of stimulated immortalized and primary fibroblasts, with RelA homodimers seemingly comprising a substantial proportion of the overall RelA dimer species
Studies focusing on the dimerization states of NF-κB transcription factor (TF) have been relatively scarce due to the difficulties associated with obtaining and interpreting in vitro biochemical data
Summary
Nuclear Factor-kappaB (NF-κB) is arguably the most important signaling pathway involved in immune responses [1]. The specificity of NF-κB action as a transcription factor (TF) is partly mediated by the particular dimers that translocate into the nucleus in response to extracellular stimuli or stress (Figure 1A). NF-κB homo- and hetero-dimers (Figure 1B) reversibly interact with specific DNA sequence motifs to activate the transcription of hundreds of target genes [2, 3]. Depending on which of the 5 different NF-κB TF proteins comprise the dimers that translocate to the nucleus [4], different gene expression profiles can be induced [5, 6]. Because the NF-κB TF family is comprised of up to 15 different dimer species [1] (Figure 1B), developing a thorough understanding of how individual NF-κB dimers regulate transcription has proved to be an exceedingly difficult task
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