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Two modes of transcriptional activation at native promoters by NF-kappaB p65.

ABSTRACT: The NF-kappaB 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-kappaB 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-kappaB-driven transcription at native promoters, we disrupted the interaction between NF-kappaB p65 and the Mediator complex. We found that expression of many endogenous NF-kappaB 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-kappaB 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-kappaB-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-kappaB.

SUBMITTER: van Essen D

PROVIDER: S-EPMC2661965 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Two modes of transcriptional activation at native promoters by NF-kappaB p65.

van Essen Dominic D Engist Bettina B Natoli Gioacchino G Saccani Simona S

PLoS biology 20090301 3

The NF-kappaB 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-kappaB 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-kappaB-driven transcription at native promoters, we disrupted the interaction betwe ...[more]

PMID: 19338389

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Project description:Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that threatens over one billion people. Nuclear translocation of the transcription factor, NF-?B, is the central initiating cellular event in the antimicrobial response. Here, we report that NF-?B p65 nuclear accumulation and NF-?B-dependent transcription are inhibited in O. tsutsugamushi infected HeLa cells and/or primary macrophages, even in the presence of TNF?. The bacterium modulates p65 subcellular localization by neither degrading it nor inhibiting I?B? degradation. Rather, it exploits host exportin 1 to mediate p65 nuclear export, as this phenomenon is leptomycin B-sensitive. O. tsutsugamushi antagonizes NF-?B-activated transcription even when exportin 1 is inhibited and NF-?B consequently remains in the nucleus. Two ankyrin repeat-containing effectors (Anks), Ank1 and Ank6, each of which possess a C-terminal F-box and exhibit 58.5% amino acid identity, are linked to the pathogen's ability to modulate NF-?B. When ectopically expressed, both translocate to the nucleus, abrogate NF-?B-activated transcription in an exportin 1-independent manner, and pronouncedly reduce TNF?-induced p65 nuclear levels by exportin 1-dependent means. Flag-tagged Ank 1 and Ank6 co-immunoprecipitate p65 and exportin 1. Both also bind importin ?1, a host protein that is essential for the classical nuclear import pathway. Importazole, which blocks importin ?1 activity, abrogates Ank1 and Ank6 nuclear translocation. The Ank1 and Ank6 regions that bind importin ?1 also mediate their transport into the nucleus. Yet, these regions are distinct from those that bind p65/exportin 1. The Ank1 and Ank6 F-box and the region that lies between it and the ankyrin repeat domain are essential for blocking p65 nuclear accumulation. These data reveal a novel mechanism by which O. tsutsugamushi modulates the activity and nuclear transport of NF-?B p65 and identify the first microbial proteins that co-opt both importin ?1 and exportin 1 to antagonize a critical arm of the antimicrobial response.

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Two modes of transcriptional activation at native promoters by NF-kappaB p65.

Publications

Two modes of transcriptional activation at native promoters by NF-kappaB p65.

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