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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.

2024-04-11 | PXD041717 | Pride

RNF4-knockdown and RNF4-TULIP identified proteins

Project description:In this project we aim to identify the spacific targets of the SUMO-Targeted Ubiquitin Ligase (STUBL) RNF4. For that, we combine the study of SUMOylation targets that are enriched after RNF4 knockdown with the use of Targeted Ubiquitin Ligases Identified by Proteomics (TULIP).

2017-11-23 | PXD005425 | Pride

Identification of heat induced, RNF4 regulated ubiquitylation sites

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.

2023-06-30 | PXD017887 | Pride

RNF4 interactome in heatshocked HeLa cells

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. To better understand the role of RNF4 in the proteotoxic stress response we aimed to identify RNF4-associated protein complexes in cells exposed to heat stress. To allow immuno-affinity purification of endogenous RNF4 on anti-Flag agarose beads we used CRISPR/Cas9-mediated gene editing to integrate a C-terminal 3xFlag epitope tag into the genomic locus of RNF4 in HeLa cells. For the IP-MS experiment HeLa-RNF4-3xFlag cells or parental untagged control cells were exposed to heat stress for 1 h and RNF4-3xFlag together with associated proteins was captured on anti-Flag affinity beads. Tryptic peptides were analyzed by LC-MS/MS and data from three replicates were quantified by the Max Quant label-free quantification (LFQ) algorithm.

2023-06-30 | PXD017888 | Pride

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

Project description:Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex-1 (PRC1)-associated protein, maintains human epidermal stem cells slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its anti-senescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors, such as Ezh2, Dnmt1, and Bmi1, to prevent the activate stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. Primary human keratinocytes were infected with control vector pBABE, Cbx4 wildtype and a Sumoylation mutant version of Cbx4 (the two Sumo Interaction Motifs were deleted), both fused to a Estrogen Receptor to render their activaty inducible by addition of 4OHT to the media. Cells were cultured for 5 days (after infection and selection) under 4OHT treatment, after which total RNA was collected.

2011-08-01 | E-GEOD-31094 | biostudies-arrayexpress

Polycomb Repressive Complex 1 subunit Cbx4 positively regulates effector responses in CD8 T cells

Project description:Cytotoxic T lymphocyte (CTL) differentiation is controlled by the crosstalk of various transcription factors and epigenetic modulators. Fully uncovering this process is fundamental to improving immunotherapy and designing novel therapeutic approaches. Here, we show that Polycomb Repressive Complex (PRC)1 subunit Chromobox (Cbx)4 favors effector CTL differentiation in murine model. Cbx4 deficiency in CTLs induced transcriptional signature and phenotype of memory cells, increasing the memory CTL population during acute viral infection. It has been previously shown that besides binding to H3K27me3 through its chromodomain, Cbx4 function as a SUMO E3 ligase in a SUMO interacting motifs (SIM)-dependent way. Overexpression of Cbx4 mutants in distinct domains showed that this protein regulates CTL differentiation primarily in a SIM-dependent way and partially through its chromodomain. Our data revealed a novel role of a Polycomb group protein Cbx4 controlling CTL differentiation and indicates the SUMOylation as a key molecular mechanism connected to chromatin modification in this process.

2023-07-10 | GSE236487 | GEO

SUMO-targeted ubiquitin ligases (STUbLs) reduce the toxicity and abnormal transcriptional activity associated with a mutant, aggregation-prone fragment of huntingtin

Project description:We report that the SUMO-targeted ubiquitin ligase (STUbL) Slx5 reduces the toxicity and abnormal transcriptional activity of a mutant, aggregation-prone fragment of huntingtin (htt), the causative agent of HD. An extra copy of SLX5 specifically reduces htt aggregates in the cytosol as well as chromatin-associated htt aggregates in the nucleus. Using RNA sequencing, we identified and confirmed specific targets of mHtt's transcriptional activity that are modulated by Slx5.

2018-06-20 | GSE115990 | GEO

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

Project description:Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex-1 (PRC1)-associated protein, maintains human epidermal stem cells slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its anti-senescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors, such as Ezh2, Dnmt1, and Bmi1, to prevent the activate stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. Primary human keratinocytes were infected with control vector pBABE, Cbx4 wildtype and a chromodomain mutant version of Cbx4 (F11A and W35L), both fused to a Estrogen Receptor to render their activaty inducible by addition of 4OHT to the media. Cells were cultured for 5 days (after infection and selection) under 4OHT treatment, after which total RNA was collected. For the knockdown, the pRetroSuperPuro was used as a control vector, in which a shRNA sequence for Cbx4 was introduced to interfere with Cbx4 expression. Cell were cultured in the same way as for the overexpression experiments.

2011-08-01 | E-GEOD-31093 | biostudies-arrayexpress

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

2011-08-02 | GSE31094 | GEO

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