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

Abstract

SUMMARY 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 as-says 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.

Decoding the Notch signal

“…The two-foot binding conferred by dimerization could be one factor that favors binding of NICD containing complexes at target enhancers. SHARP interacts with two distinct surfaces on RBPJ, contacting both the CTD and BTD in regions that overlap with those interacting with NICD (Yuan et al, 2019). bind the BTD of RBPJ (Collins, Yuan, & Kovall, 2014;Tabaja, Yuan, Oswald, & Kovall, 2017), similar to the RAM domain of NICD.…”

“…However, the extent that dimerization is important for the recruitment of CSL-NICD-Mam complexes remains to be established. Interestingly, although there is no sequence similarity between SHARP and Hairless, these two corepressors have evolved to bind the same CTD interface on RBPJ and Su(H) (Yuan et al, 2019). SHARP interacts with two distinct surfaces on RBPJ, contacting both the CTD and BTD in regions that overlap with those interacting with NICD (Yuan et al, 2019).…”

“…CSL also participates in other complexes. For example, it interacts directly with several co-repressors, such as Hairless (H), SHARP/MINT or SMRT (silencing mediator for retinoid and thyroid receptor), which have the potential to recruit histone deacetylases (HDAC) and other chromatin remodeling proteins to repress target enhancers (Tanigaki & Honjo, 2010;Yuan et al, 2016Yuan et al, , 2019. Studies on the structure of repressor complexes indicate repressors of different species bind different CSL domains.…”

“…Hairless binds the CTD of fly Su(H) (Figure 2a; Yuan et al, 2016), whereas FHL1 and RITA1 bind the BTD of RBPJ (Collins, Yuan, & Kovall, 2014;Tabaja, Yuan, Oswald, & Kovall, 2017), similar to the RAM domain of NICD. SHARP interacts with two distinct surfaces on RBPJ, contacting both the CTD and BTD in regions that overlap with those interacting with NICD (Yuan et al, 2019). Interestingly, although there is no sequence similarity between SHARP and Hairless, these two corepressors have evolved to bind the same CTD interface on RBPJ and Su(H) (Yuan et al, 2019).…”

“…For example, it interacts directly with several co-repressors, such as Hairless (H), SHARP/MINT or SMRT (silencing mediator for retinoid and thyroid receptor), which have the potential to recruit histone deacetylases (HDAC) and other chromatin remodeling proteins to repress target enhancers(Tanigaki & Honjo, 2010;Yuan et al, 2016Yuan et al, , 2019. For example, it interacts directly with several co-repressors, such as Hairless (H), SHARP/MINT or SMRT (silencing mediator for retinoid and thyroid receptor), which have the potential to recruit histone deacetylases (HDAC) and other chromatin remodeling proteins to repress target enhancers(Tanigaki & Honjo, 2010;Yuan et al, 2016Yuan et al, , 2019.…”