ABSTRACT
The Elongin complex was originally identified as an RNA polymerase II (RNAPII) elongation factor and subsequently as the substrate recognition component of a Cullin-RING E3 ubiquitin ligase. More recent evidence indicates that the Elongin ubiquitin ligase assembles with the Cockayne syndrome B helicase (CSB) in response to DNA damage and can target stalled polymerases for ubiquitylation and removal from the genome. In this report, we present evidence that the CSB-Elongin ubiquitin ligase pathway has roles beyond the DNA damage response in the activation of RNAPII-mediated transcription. We observed that assembly of the CSB-Elongin ubiquitin ligase is induced not just by DNA damage, but also by a variety of signals that activate RNAPII-mediated transcription, including endoplasmic reticulum (ER) stress, amino acid starvation, retinoic acid, glucocorticoids, and doxycycline treatment of cells carrying several copies of a doxycycline-inducible reporter. Using glucocorticoid receptor (GR)-regulated genes as a model, we showed that glucocorticoid-induced transcription is accompanied by rapid recruitment of CSB and the Elongin ubiquitin ligase to target genes in a step that depends upon the presence of transcribing RNAPII on those genes. Consistent with the idea that the CSB-Elongin pathway plays a direct role in GR-regulated transcription, mouse cells lacking the Elongin subunit Elongin A exhibit delays in both RNAPII accumulation on and dismissal from target genes following glucocorticoid addition and withdrawal, respectively. Taken together, our findings bring to light a new role for the CSB-Elongin pathway in RNAPII-mediated transcription.
Subject(s)
DNA Helicases/genetics , DNA Repair Enzymes/genetics , Elongin/genetics , Poly-ADP-Ribose Binding Proteins/genetics , RNA Polymerase II/genetics , Ubiquitin-Protein Ligases/genetics , Animals , Cockayne Syndrome/enzymology , Cockayne Syndrome/genetics , DNA Helicases/chemistry , DNA Helicases/ultrastructure , DNA Repair/genetics , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/ultrastructure , Elongin/chemistry , Elongin/ultrastructure , Humans , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/ultrastructure , Poly-ADP-Ribose Binding Proteins/chemistry , Poly-ADP-Ribose Binding Proteins/ultrastructure , RNA Polymerase II/chemistry , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/genetics , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/ultrastructure , Ubiquitination/geneticsABSTRACT
The Cullin 5 (CUL5) Ring E3 ligase uses adaptors Elongins B and C (ELOB/C) to bind different SOCS-box-containing substrate receptors, determining the substrate specificity of the ligase. The 18-member ankyrin and SOCS box (ASB) family is the largest substrate receptor family. Here we report cryo-EM data for the substrate, creatine kinase (CKB) bound to ASB9-ELOB/C, and for full-length CUL5 bound to the RING protein, RBX2, which binds various E2s. To date, no full structures are available either for a substrate-bound ASB nor for CUL5. Hydrogen-deuterium exchange (HDX-MS) mapped onto a full structural model of the ligase revealed long-range allostery extending from the substrate through CUL5. We propose a revised allosteric mechanism for how CUL-E3 ligases function. ASB9 and CUL5 behave as rigid rods, connected through a hinge provided by ELOB/C transmitting long-range allosteric crosstalk from the substrate through CUL5 to the RBX2 flexible linker.
