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Structural and Functional Studies of the RBPJ-SHARP Complex Reveal Conserved Corepressor Binding Site

51 Pages Posted: 26 Feb 2019 Sneak Peek Status: Published

See all articles by Zhenyu Yuan

Zhenyu Yuan

University of Cincinnati - Department of Molecular Genetics

Bradley D. VanderWielen

University of Cincinnati - Department of Molecular Genetics

Benedetto Daniele Giaimo

Giessen University - Institute of Biochemistry

Leiling Pan

University Medical Center Ulm - Department of Internal Medicine I

Courtney E. Collins

University of Cincinnati - Department of Molecular Genetics

Aleksandra Turkiewicz

Giessen University - Institute of Biochemistry

Kerstin Hein

Giessen University - Institute of Biochemistry

Franz Oswald

University Medical Center Ulm - Department of Internal Medicine I

Tilman Borggrefe

Giessen University - Institute of Biochemistry

Rhett Kovall

University of Cincinnati - Department of Molecular Genetics

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Abstract

The Notch pathway is a conserved signaling mechanism that is essential for cell fate decisions during pre and postnatal development. Dysregulated signaling underlies the pathophysiology of numerous human diseases, most notably T-cell acute lymphoblastic leukemia. Receptor-ligand interactions result in changes in gene expression, which are regulated by the transcription factor CSL. CSL forms a complex with the intracellular domain of the Notch receptor and the transcriptional coactivator Mastermind, which is required to activate transcription of all Notch target genes. CSL can also function as repressor by interacting with corepressor proteins, e.g. SHARP in mammals and Hairless in Drosophila melanogaster; however, its role as a transcriptional repressor is not well understood. Here we determine the high-resolution structure of RBPJ, the mouse CSL ortholog, bound to the corepressor SHARP and DNA, which reveals a new mode of corepressor binding to CSL and an interesting example for how ligand binding sites evolve in proteins. Based on the structure, we designed and tested a number of mutants in biophysical, biochemical, and cellular assays to characterize the role of RBPJ as a repressor of Notch target genes. Our cellular studies clearly demonstrate that RBPJ mutants that are deficient for binding SHARP are incapable of repressing transcription from genes responsive to Notch signaling. Altogether, our structure-function studies of the RBPJ-SHARP corepressor complex bound to DNA provide significant insights into the repressor function of RBPJ and identify a new binding pocket on RBPJ that could be targeted for therapeutic benefit.

Suggested Citation

Yuan, Zhenyu and VanderWielen, Bradley D. and Giaimo, Benedetto Daniele and Pan, Leiling and Collins, Courtney E. and Turkiewicz, Aleksandra and Hein, Kerstin and Oswald, Franz and Borggrefe, Tilman and Kovall, Rhett, Structural and Functional Studies of the RBPJ-SHARP Complex Reveal Conserved Corepressor Binding Site (2018). Available at SSRN: https://ssrn.com/abstract=3195335 or http://dx.doi.org/10.2139/ssrn.3195335
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Zhenyu Yuan

University of Cincinnati - Department of Molecular Genetics

260 Stetson Street, Suite 3200
P. O. Box 670559
Cincinnati, OH 45219
United States

Bradley D. VanderWielen

University of Cincinnati - Department of Molecular Genetics

260 Stetson Street, Suite 3200
P. O. Box 670559
Cincinnati, OH 45219
United States

Benedetto Daniele Giaimo

Giessen University - Institute of Biochemistry

Licher Str. 62
Giessen, 35394
Germany

Leiling Pan

University Medical Center Ulm - Department of Internal Medicine I

Ulm, 89081
Germany

Courtney E. Collins

University of Cincinnati - Department of Molecular Genetics

260 Stetson Street, Suite 3200
P. O. Box 670559
Cincinnati, OH 45219
United States

Aleksandra Turkiewicz

Giessen University - Institute of Biochemistry

Licher Str. 62
Giessen, 35394
Germany

Kerstin Hein

Giessen University - Institute of Biochemistry

Licher Str. 62
Giessen, 35394
Germany

Franz Oswald

University Medical Center Ulm - Department of Internal Medicine I

Ulm, 89081
Germany

Tilman Borggrefe

Giessen University - Institute of Biochemistry

Licher Str. 62
Giessen, 35394
Germany

Rhett Kovall (Contact Author)

University of Cincinnati - Department of Molecular Genetics ( email )

260 Stetson Street, Suite 3200
P. O. Box 670559
Cincinnati, OH 45219
United States

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