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Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes

Abstract

Ligand-induced proteolysis of Notch produces an intracellular effector domain that transduces essential signals by regulating the transcription of target genes. This function relies on the formation of transcriptional activation complexes that include intracellular Notch, a Mastermind co-activator and the transcription factor CSL bound to cognate DNA. These complexes form higher-order assemblies on paired, head-to-head CSL recognition sites. Here we report the X-ray structure of a dimeric human Notch1 transcription complex loaded on the paired site from the human HES1 promoter. The small interface between the Notch ankyrin domains could accommodate DNA bending and untwisting to allow a range of spacer lengths between the two sites. Cooperative dimerization occurred on the human and mouse Hes5 promoters at a sequence that diverged from the CSL-binding consensus at one of the sites. These studies reveal how promoter organizational features control cooperativity and, thus, the responsiveness of different promoters to Notch signaling.

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Figure 1: Structure of a dimer of Notch transcription complexes on paired-site DNA.
Figure 2: NTC dimer formation on putative Notch targets hHES4 and hFJX1 and on SPS elements with 15–17-bp spacers.
Figure 3: Dimer-dependent and dimer-independent DNA elements in the promoters of the Notch-responsive genes HEYL, Hey2 and Hes5.
Figure 4: The mouse and human HES5 proximal promoters each contain two conserved high-affinity CSL binding sites, one of which is essential for the activation of expression.
Figure 5: Identification of a non-consensus SPS element in the HES5 promoter.

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Acknowledgements

We thank W. Pear for discussions and review of the manuscript. Supported in part by US National Institutes of Health grants CA-092433 (to S.C.B.) and CA-119070 (to S.C.B. and J.C.A.). K.L.A. is a Postdoctoral Fellow of the American Cancer Society.

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Contributions

K.L.A. solved the structure. K.L.A. and D.G.M. performed EMSAs. M.H., M.X.G.I. and D.G.M. performed reporter assays. K.L.A., M.H., D.G.M., M.X.G.I., J.C.A., R.K. and S.C.B. analyzed data. K.L.A. and S.C.B. wrote the manuscript with input from all authors.

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Correspondence to Stephen C Blacklow.

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The authors declare no competing financial interests.

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Arnett, K., Hass, M., McArthur, D. et al. Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nat Struct Mol Biol 17, 1312–1317 (2010). https://doi.org/10.1038/nsmb.1938

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