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NF-KBS SPECIFICITY Résumé de rapport

Project ID: 42509
Financé au titre de: FP6-MOBILITY
Pays: Germany

Final Activity Report Summary - NF-KBS SPECIFICITY (Control of specificity in the NF-kB system)

The aim of this project was to investigate the molecular mechanisms which underlie specific promoter activation by the NF-kB family of transcription factors, and which govern its cell-type specific functions. The NF-kB 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 was largely unknown, but it seemed likely that it should include the means to tailor transcriptional output to match the wide functional range of its target genes. To dissect NF-kB-driven transcription at native promoters, we disrupted the interaction between NF-kB 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 were transcribed normally even when the interaction of p65 with Mediator was abolished. Moreover, a mutant form of p65 lacking all transcription activation domains previously identified in vitro could still activate such promoters in vivo. We found that without p65, native NF-kB 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 utilise a wide choice of transcription factors, with the potential to regulate their expression accordingly, whilst remaining dependent for their activation on NF-kB. In many other biological contexts, activation of transcription from a silenced state is crucial to achieve specific gene expression. Methylation of lysine 9 on histone H3 (H3K9) is widely associated with transcriptional silencing, and its disappearance is linked to the activation of several inflammatory genes by NF-?B. We have shown that this event is controlled by a feed-forward circuit catalysed by the activity of the histone demethylase Aof1 (also known as Lsd2/Kdm1b).

We found that Aof1 is required for removal of dimethyl H3K9 at specific promoters, and thereby it controls stimulus-induced recruitment of NF-?B and gene expression. However, Aof1 is itself recruited by interaction with the c-Rel subunit of NF-?B, which is found at low levels associated with promoters in unstimulated cells. Thus, at these tightly regulated genes, NF-?B functions both as a transcriptional activator and as an upstream targeting signal that marks promoters to be derepressed by histone demethylation.


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