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

Cell Rep. 2019 Jan 22;26(4):845-854.e6. doi: 10.1016/j.celrep.2018.12.097.

Abstract

Notch is a conserved signaling pathway that is essential for metazoan development and homeostasis; dysregulated signaling underlies the pathophysiology of numerous human diseases. Receptor-ligand interactions result in gene expression changes, which are regulated by the transcription factor RBPJ. RBPJ forms a complex with the intracellular domain of the Notch receptor and the coactivator Mastermind to activate transcription, but it can also function as a repressor by interacting with corepressor proteins. Here, we determine the structure of RBPJ bound to the corepressor SHARP and DNA, revealing its mode of binding to RBPJ. We tested structure-based mutants in biophysical and biochemical-cellular assays to characterize the role of RBPJ as a repressor, clearly demonstrating that RBPJ mutants deficient for SHARP binding are incapable of repressing transcription of genes responsive to Notch signaling in cells. Altogether, our structure-function studies provide significant insights into the repressor function of RBPJ.

Keywords: CSL; MINT; Notch signaling; RBPJ; SHARP; SPEN; X-ray crystallography; isothermal titration calorimetry; signal transduction; transcriptional regulation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism
  • DNA-Binding Proteins* / chemistry
  • DNA-Binding Proteins* / genetics
  • DNA-Binding Proteins* / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein* / chemistry
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein* / genetics
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein* / metabolism
  • Mice
  • Multiprotein Complexes* / chemistry
  • Multiprotein Complexes* / genetics
  • Multiprotein Complexes* / metabolism
  • Protein Structure, Quaternary
  • RNA-Binding Proteins* / chemistry
  • RNA-Binding Proteins* / genetics
  • RNA-Binding Proteins* / metabolism
  • Receptors, Notch / chemistry
  • Receptors, Notch / genetics
  • Receptors, Notch / metabolism
  • Signal Transduction*
  • Transcription, Genetic*

Substances

  • DNA-Binding Proteins
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein
  • Multiprotein Complexes
  • RNA-Binding Proteins
  • Rbpj protein, mouse
  • Receptors, Notch
  • Spen protein, mouse
  • DNA