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p53 activates transcription by directing structural shifts in Mediator

Abstract

It is not well understood how the human Mediator complex, transcription factor IIH and RNA polymerase II (Pol II) work together with activators to initiate transcription. Activator binding alters Mediator structure, yet the functional consequences of such structural shifts remain unknown. The p53 C terminus and its activation domain interact with different Mediator subunits, and we find that each interaction differentially affects Mediator structure; strikingly, distinct p53–Mediator structures differentially affect Pol II activity. Only the p53 activation domain induces the formation of a large pocket domain at the Mediator–Pol II interaction site, and this correlates with activation of stalled Pol II to a productively elongating state. Moreover, we define a Mediator requirement for TFIIH-dependent Pol II C-terminal domain phosphorylation and identify substantial differences in Pol II C-terminal domain processing that correspond to distinct p53–Mediator structural states. Our results define a fundamental mechanism by which p53 activates transcription and suggest that Mediator structural shifts trigger activation of stalled Pol II complexes.

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Figure 1: The p53AD is not required for PEC assembly in vitro or in cells.
Figure 2: p53AD is required to activate stalled Pol II in vitro and in cells; PECs assembled in the absence of p53 or with p53CTD remain inactive.
Figure 3: p53AD activates stalled Pol II in the context of the native tetramer in vitro and in cells.
Figure 4: Mediator is required for TFIIH-dependent Pol II CTD phosphorylation in the PEC; oncogenic mutations in p53AD (p53QS) prevent activation of stalled Pol II.
Figure 5: p53AD triggers a structural shift in Mediator distinct from p53CTD.
Figure 6: Model outlining how p53AD appears to trigger Pol II promoter escape and transcription elongation.
Figure 7: The pocket domain represents a common structural feature in activator-bound Mediator.

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Acknowledgements

We thank A. Donner and J. Espinosa for help with the ChIP assays, RT-qPCR experiments and other useful advice, C. Schwartz and M. Morphew for assistance with electron microscopy data collection, J. Blaydes (University of Southampton) for the gift of the HDM2 promoter fragment and J. Goodrich and J. Espinosa for helpful comments on the manuscript. Protein expression in insect cells was completed at the Tissue Culture Core Facility at the University of Colorado Cancer Center (Support Grant #P30 CA046934). This work was supported by the US National Cancer Institute (R01 CA127364) and the Ellison Medical Foundation. K.D.M. and C.B. were supported in part by US National Institutes of Health grant T32 GM065103.

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Contributions

K.D.M. designed, analyzed and performed most experiments and helped write the paper; S.-c.L. completed immobilized template assays; C.B. assisted with cryo-EM, purified Pol II and provided Mediator–Pol II electron microscopy data; Y.G. mapped Med1 binding domain to p53CTD; D.J.T. designed and performed experiments and helped write the paper.

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Correspondence to Dylan J Taatjes.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Note, Supplementary Results and Supplementary Methods (PDF 2035 kb)

Supplementary Video 1

P53AD-Mediator (MP4 959 kb)

Supplementary Video 2

P53CTD-Mediator (MP4 1921 kb)

Supplementary Video 3

WT p53-Mediator (MP4 1384 kb)

Supplementary Video 4

P53ΔCTD-Mediator (MP4 1016 kb)

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Meyer, K., Lin, Sc., Bernecky, C. et al. p53 activates transcription by directing structural shifts in Mediator. Nat Struct Mol Biol 17, 753–760 (2010). https://doi.org/10.1038/nsmb.1816

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