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Multiple Arkadia/RNF111 structures coordinate its Polycomb body association and transcriptional control

ABSTRACT: The RING domain protein Arkadia/RNF111 is a ubiquitin ligase in the transforming growth factor beta (TGFβ) pathway. We previously identified Arkadia as a small ubiquitin-like modifier (SUMO)-binding protein with clustered SUMO-interacting motifs (SIMs) that together form a SUMO-binding domain (SBD). However, precisely how SUMO interaction contributes to the function of Arkadia was not resolved. Through analytical molecular and cell biology, we found that the SIMs share redundant function with Arkadia's M domain, a region distinguishing Arkadia from its paralogs ARKL1/ARKL2 and the prototypical SUMO-targeted ubiquitin ligase (STUbL) RNF4. The SIMs and M domain together promote both Arkadia's colocalization with CBX4/Pc2, a component of Polycomb bodies, and the activation of a TGFbeta pathway transcription reporter. Transcriptome profiling through RNA sequencing showed that Arkadia can both promote and inhibit gene expression, indicating that Arkadia's activity in transcriptional control may depend on the epigenetic context, defined by Polycomb repressive complexes and DNA methylation [Sun, Liu, and Hunter (2014) Mol Cell Biol 34(16):2981-2995].

ORGANISM(S): Mus musculus

PROVIDER: GSE65796 | GEO | 2015/02/11

SECONDARY ACCESSION(S): PRJNA275102

REPOSITORIES: GEO

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Multiple Arkadia/RNF111 structures coordinate its Polycomb body association and transcriptional control

Project description:The RING domain protein Arkadia/RNF111 is a ubiquitin ligase in the transforming growth factor beta (TGFβ) pathway. We previously identified Arkadia as a small ubiquitin-like modifier (SUMO)-binding protein with clustered SUMO-interacting motifs (SIMs) that together form a SUMO-binding domain (SBD). However, precisely how SUMO interaction contributes to the function of Arkadia was not resolved. Through analytical molecular and cell biology, we found that the SIMs share redundant function with Arkadia's M domain, a region distinguishing Arkadia from its paralogs ARKL1/ARKL2 and the prototypical SUMO-targeted ubiquitin ligase (STUbL) RNF4. The SIMs and M domain together promote both Arkadia's colocalization with CBX4/Pc2, a component of Polycomb bodies, and the activation of a TGFbeta pathway transcription reporter. Transcriptome profiling through RNA sequencing showed that Arkadia can both promote and inhibit gene expression, indicating that Arkadia's activity in transcriptional control may depend on the epigenetic context, defined by Polycomb repressive complexes and DNA methylation [Sun, Liu, and Hunter (2014) Mol Cell Biol 34(16):2981-2995]. To determine the role of Arkadia in TGFβ signaling at the transcriptome level, the profiles of TGFβ-stimulated gene expression were examined in Ark+/+ (Ark_WT), Ark-/- (Ark_null), and Arkadia (WT and sim mutant)-reconstituted Ark-/- MEFs. RNA sequencing was carried out using poly(A)-enriched RNA samples from unstimulated cells and cells treated with TGFβ for 1h, 4h, or 16h as indicated. Two batches of sequencing data for a total of 16 independent samples were submitted.

2015-02-11 | E-GEOD-65796 | biostudies-arrayexpress

Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation

Project description:Protein SUMOylation provides a principal driving force for cellular stress responses including DNA-protein crosslink (DPC) repair and arsenic-induced PML body degradation. In this study, using genome-scale screens, we identify the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL), combining ubiquitin ligase activity through its RING domain with poly-SUMO binding via SUMO-interacting motifs, analogous to the STUbL RNF4. Mechanistically, TOPORS is a SUMO1-selective STUbL that complements RNF4 in generating complex ubiquitin landscapes on SUMOylated targets including DPCs and PML, stimulating efficient p97/VCP unfoldase recruitment and proteasomal degradation. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, involving defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Our findings establish TOPORS as a STUbL whose parallel action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO-ubiquitin crosstalk.

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Project description:Exposure of cells to heat or oxidative stress causes misfolding of proteins. To avoid toxic protein aggregation, cells have evolved nuclear and cytosolic protein quality control (PQC) systems. In response to proteotoxic stress cells also limit protein synthesis by triggering transient storage of mRNAs and RNA binding proteins (RBPs) in cytosolic stress granules (SGs). We demonstrate that the SUMO-targeted ubiquitin ligase (StUbL) pathway, which is part of the nuclear proteostasis network, regulates SG dynamics. We provide evidence that inactivation of SUMO deconjugases under proteotoxic stress initiates SUMO-primed, RNF4-dependent ubiquitylation of RBPs that typically condense into SGs. Impairment of SUMO-primed ubiquitylation drastically delays SG resolution upon stress release. Importantly, the StUbL system regulates compartmentalization of an amyotrophic lateral sclerosis (ALS)-associated mutant of FUS in SGs. We propose that the StUbL system functions as surveillance pathway for aggregation-prone RBPs in the nucleus thereby linking the nuclear and cytosolic axis of proteotoxic stress response. Toidentify heat induced, RNF4 regulated ubiquitylation sites we performed an unbiased SILAC-based mass-spectrometry screen comparing heat-induced ubiquitylation in control and RNF4 depleted cells.

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Overexpression of Cbx4 wild type and of a Cbx4 sumoylation mutant in human epidermal stem cells

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