Two Modes of Transcriptional Activation at Native Promoters by NF-κB p65

Abstract

The NF-κB family of transcription factors is crucial for the expression of multiple genes involved in cell survival, proliferation, differentiation, and inflammation. The molecular basis by which NF-κB activates endogenous promoters is largely unknown, but it seems likely that it should include the means to tailor transcriptional output to match the wide functional range of its target genes. To dissect NF-κB–driven transcription at native promoters, we disrupted the interaction between NF-κB p65 and the Mediator complex. We found that expression of many endogenous NF-κB target genes depends on direct contact between p65 and Mediator, and that this occurs through the Trap-80 subunit and the TA1 and TA2 regions of p65. Unexpectedly, however, a subset of p65-dependent genes are transcribed normally even when the interaction of p65 with Mediator is abolished. Moreover, a mutant form of p65 lacking all transcription activation domains previously identified in vitro can still activate such promoters in vivo. We found that without p65, native NF-κB target promoters cannot be bound by secondary transcription factors. Artificial recruitment of a secondary transcription factor was able to restore transcription of an otherwise NF-κB–dependent target gene in the absence of p65, showing that the control of promoter occupancy constitutes a second, independent mode of transcriptional activation by p65. This mode enables a subset of promoters to utilize a wide choice of transcription factors, with the potential to regulate their expression accordingly, whilst remaining dependent for their activation on NF-κB.

Author Summary

Transcriptional activation by the NF-κB family of transcription factors is crucial for the expression of multiple genes involved in cell survival, proliferation, differentiation, and inflammation. The activation domain of the p65 subunit of NF-κB has been extensively studied in vitro and on artificial reporter plasmids, but the molecular basis by which it drives expression of natural target genes in vivo is still not well understood. Moreover, it is unclear how any single activation mechanism could allow different target genes to fine tune their timing and expression according to their biological requirements. To address this, we experimentally blocked the interaction of p65 with the Mediator complex—a key factor for transcription by most, if not all, activators. While this prevented expression of many NF-κB–dependent genes, others were unaffected, revealing that p65 is able to drive their expression by an independent mode, which does not depend on direct contact with Mediator. Further experiments indicated that p65 accomplishes this by controlling the recruitment of other, secondary transcription factors to its target promoters. This may enable NF-κB to retain overall control over activation of its target genes, but at the same time allow secondary transcription factors to specify appropriate expression levels according to the cell-type and stimulus.

Abstract

The p65 subunit of NF-κB drives expression of target genes not only as a classical activator, via direct interactions with the basic transcriptional machinery, but also independently by controlling the recruitment of secondary transcription factors to target promoters.