Degringolade, a SUMO-targeted ubiquitin ligase, inhibits Hairy/Groucho-mediated repression

Abstract

Transcriptional cofactors are essential for proper embryonic development. One such cofactor in Drosophila, Degringolade (Dgrn), encodes a RING finger/E3 ubiquitin ligase. Dgrn and its mammalian ortholog RNF4 are SUMO‐targeted ubiquitin ligases (STUbLs). STUbLs bind to SUMOylated proteins via their SUMO interaction motif (SIM) domains and facilitate substrate ubiquitylation. In this study, we show that Dgrn is a negative regulator of the repressor Hairy and its corepressor Groucho (Gro/transducin‐like enhancer (TLE)) during embryonic segmentation and neurogenesis, as dgrn heterozygosity suppresses Hairy mutant phenotypes and embryonic lethality. Mechanistically Dgrn functions as a molecular selector: it targets Hairy for SUMO‐independent ubiquitylation that inhibits the recruitment of its corepressor Gro, without affecting the recruitment of its other cofactors or the stability of Hairy. Concomitantly, Dgrn specifically targets SUMOylated Gro for sequestration and antagonizes Gro functions in vivo. Our findings suggest that by targeting SUMOylated Gro, Dgrn serves as a molecular switch that regulates cofactor recruitment and function during development. As Gro/TLE proteins are conserved universal corepressors, this may be a general paradigm used to regulate the Gro/TLE corepressors in other developmental processes.

TheDrosophilaSTUbL protein Degringolade limits HES functions during embryogenesis

“…Luciferase and Renilla (control) activity were assayed using the Dual Reporter Assay (Promega) 48 hours after transfection of Drosophila Kc cells as described (Abed et al, 2011). In vitro ubiquitylation assays with ubiquitin were carried out as described previously (Ben-Saadon et al, 2006).…”

Regulation of Protein-Protein Interactions by the SUMO and Ubiquitin Pathways

“…Thus, Dgrn serves as a "molecular selector" that determines protein-protein interactions. We found that this "selector" activity of Dgrn/RNF4 is likely relevant also to HES independent processes and in other types of co-factor switches, and may directly impact chromatin structure (Abed et al, 2011a;Hu et al, 2010;Orian unpublished). We speculate that this activity of STUbLs will be highly relevant not only in transcription, but in the regulation of protein-protein interactions in other cellular process such as the selective recruitment of proteins to DNA repair foci.…”

“…For example RNF4 was shown to be essential for androgen and steroid receptor-mediated target gene activation (Moilanen et al, 1998;Poukka et al, 2000;). In addition, we found that in transcription the consequences of Dgrn activity are not strictly limited to targeting SUMOylated proteins for degradation (Abed et al, 2011a;Barry et al, 2011). Importantly, Dgrn/RNF4-mediated ubiquitylation impacts the affinity between proteins, inhibiting the interaction of a given protein with one protein but not affecting its ability to bind other protein.…”

“…Yet, no clear STUbL orthologs exist in the worm C. elegans. These members are structurally and functionally conserved, as RNF4 protein can substitute for the yeast and fly genes in functional assays (Abed et al, 2011aAbed et al, 2011bBarry et al, 2011;Praefcke et al, 2011). Recent structural work from the Hay lab uncovered that RNF4, and likely other STUbLs, function as dimers and that dimer formation is actively required to facilitate SUMO-dependent ubiquitin conjugation (Plechanovova' et al 2011).…”

“…For example, the recognition of the Mat2α repressor by Slx5: Slx8 does not require substrate SUMOylation, but does require intact SIM motifs (Xia et al, 2010) Furthermore, the binding of the Drosophila STUbL Dgrn to the Notch-related HES repressor proteins is independent of SUMOylation, and is mediated by Dgrn's RING domain and not the SIM motif. Yet, the SIM motifs are required for ubiquitylation in vitro and for Dgrn's impact in vivo (Abed et al, 2011a. Thus, it is highly likely that specific interactions are determined in vivo by a dual recognition machinery, where the SIM motif interacts with the poly-SUMO chain and the RING domain interacts with other non-SUMO determinants in the target protein or adjacent proteins.…”