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A role for the Cockayne Syndrome B (CSB)-Elongin ubiquitin ligase complex in signal-dependent RNA polymerase II transcription.

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.

SUBMITTER: Weems JC

PROVIDER: S-EPMC8294581 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cockayne syndrome B protein regulates recruitment of the Elongin A ubiquitin ligase to sites of DNA damage.

Project description:Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and ?-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and ?-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage.

| S-EPMC5399097 | biostudies-literature

Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase.

Project description:Growth hormone (GH) signaling is tightly controlled by ubiquitination of GH receptors, phosphorylation levels, and accessibility of binding sites for downstream signaling partners. Members of the suppressors of cytokine signaling (SOCS) family function as key regulators at all levels of this pathway, and mouse knockout studies implicate SOCS2 as the primary suppressor. To elucidate the structural basis for SOCS2 function, we determined the 1.9-A crystal structure of the ternary complex of SOCS2 with elongin C and elongin B. The structure defines a prototypical SOCS box ubiquitin ligase with a Src homology 2 (SH2) domain as a substrate recognition motif. Overall, the SOCS box and SH2 domain show a conserved spatial domain arrangement with the BC box and substrate recognition domain of the von Hippel-Lindau (VHL) tumor suppressor protein, suggesting a common mechanism of ubiquitination in these cullin-dependent E3 ligases. The SOCS box binds elongin BC in a similar fashion to the VHL BC box and shows extended structural conservation with the F box of the Skp2 ubiquitin ligase. A previously unrecognized feature of the SOCS box is revealed with the burial of the C terminus, which packs together with the N-terminal extended SH2 subdomain to create a stable interface between the SOCS box and SH2 domain. This domain organization is conserved in SOCS1-3 and CIS1, which share a strictly conserved length of their C termini, but not in SOCS4, 5, and 7, which have extended C termini defining two distinct classes of inter- and intramolecular SOCS box interactions.

| S-EPMC1472497 | biostudies-literature

Structure of transcribing RNA polymerase II-Elongin complex

Project description:Elongin is a hetero-trimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. We solved three structures of human Elongin bound to transcribing Pol II using cryo-EM assisted by crosslinking mass spectrometry. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB-8 ELOC subunit heterodimer. ELOA contains an N-terminal ‘latch’ that binds between the end of the RPB1 bridge helix and the funnel helices, thereby inducing a conformational change near the Pol II active center. The latch is strictly required for the elongation-stimulatory activity of Elongin, but not for its binding to Pol II, indicating that Elongin functions by allosterically influencing the conformational mobility of the active center. Structural comparisons show that Elongin binding to Pol II is incompatible with association of super elongation complex, the PAF1 complex, and RTF1, which also contains a latch element that stimulates Pol II.

2023-10-11 | PXD045446 | Pride

Cockayne syndrome (CSB) fibroblasts

Project description:Cockayne syndrome (CS) is an inherited neurodevelopmental disorder with progeroid features. Although the genes responsible for CS have been implicated in a variety of DNA repair- and transcription-related pathways, the nature of the molecular defect in CS remains mysterious. We sought to define this defect by expression analysis of cells lacking functional CSB, a SWI/SNF-like ATPase that is responsible for most CS cases. Keywords: primary disease rescue

2006-07-14 | GSE3407 | GEO

Signal-induced disassembly of the SCF ubiquitin ligase complex by Cdc48/p97.

Project description:A large group of E3 ubiquitin ligases is formed by the multisubunit SCF complex, whose core complex (Rbx1/Cul1-Cdc53/Skp1) binds one of many substrate recruiting F-box proteins to form an array of SCF ligases with diverse substrate specificities. It has long been thought that ubiquitylation by SCF ligases is regulated at the level of substrate binding. Here we describe an alternative mechanism of SCF regulation by active dissociation of the F-box subunit. We show that cadmium stress induces selective recruitment of the AAA(+) ATPase Cdc48/p97 to catalyze dissociation of the F-box subunit from the yeast SCF(Met30) ligase to block substrate ubiquitylation and trigger downstream events. Our results not only provide an additional layer of ubiquitin ligase regulation but also suggest that targeted, signal-dependent dissociation of multisubunit enzyme complexes is an important mechanism in control of enzyme function.

| S-EPMC3483439 | biostudies-literature

Assembly of the Elongin A Ubiquitin Ligase Is Regulated by Genotoxic and Other Stresses.

Project description:Elongin A performs dual functions in cells as a component of RNA polymerase II (Pol II) transcription elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that has been shown to target Pol II stalled at sites of DNA damage. Here we investigate the mechanism(s) governing conversion of the Elongin complex from its elongation factor to its ubiquitin ligase form. We report the discovery that assembly of the Elongin A ubiquitin ligase is a tightly regulated process. In unstressed cells, Elongin A is predominately present as part of Pol II elongation factor Elongin. Assembly of Elongin A into the ubiquitin ligase is strongly induced by genotoxic stress; by transcriptional stresses that lead to accumulation of stalled Pol II; and by other stimuli, including endoplasmic reticulum and nutrient stress and retinoic acid signaling, that activate Elongin A-dependent transcription. Taken together, our findings shed new light on mechanisms that control the Elongin A ubiquitin ligase and suggest that it may play a role in Elongin A-dependent transcription.

| S-EPMC4463447 | biostudies-literature

Elongin A regulates transcription in vivo through enhanced RNA polymerase processivity.

Project description:Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro. The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet, the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide. Our results suggest that Elongin A localizes to actively transcribed regions and potential enhancers, and the level of recruitment correlated with transcription levels. We also identified a large group of factors involved in transcription as Elongin A-associated factors. In addition, we found that loss of Elongin A leads to dramatically reduced levels of serine2-phosphorylated, but not total, RNAPII, and cells depleted of Elongin A show stronger promoter RNAPII pausing, suggesting that Elongin A may be involved in the release of paused RNAPII. Our RNA-seq studies suggest that loss of Elongin A did not alter global transcription, and unlike prior in vitro studies, we did not observe a dramatic impact on RNAPII elongation rates in our cell-based nascent RNA-seq experiments upon Elongin A depletion. Taken together, our studies provide the first comprehensive analysis of the role of Elongin A in regulating transcription in vivo. Our studies also revealed that unlike prior in vitro findings, depletion of Elongin A has little impact on global transcription profiles and transcription elongation in vivo.

| S-EPMC7948402 | biostudies-literature

Genome-wide regulations of the preinitiation complex formation and elongating RNA polymerase II by an E3 ubiquitin ligase, San1.

Project description:San1 is an E3 ubiquitin ligase involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and 26S proteasomal degradation. Since a number of transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in regulation of transcription. To address this, we carried out ChIP-seq (Chromatin immunoprecipitation sequencing) here to analyze the role of San1 in genome-wide association of TBP (TATA-box binding protein that nucleates the preinitiation complex formation at the promoter for transcription initiation) and RNA polymerase II (that is recruited to the promoter by TBP and subsequently engaged in transcription elongation at the coding sequence for mRNA biosynthesis). Our results reveal the genome-wide role of San1 in facilitating the recruitment of TBP to the promoter, thus indicating its involvement in promoting the preinitiation complex formation for transcription initiation. Further, we find the global role of San1 in regulating RNA polymerase II at the coding sequence.

2021-12-04 | GSE190065 | GEO

LEO1 is a partner for Cockayne syndrome protein B (CSB) in response to transcription-blocking DNA damage.

Project description:Cockayne syndrome (CS) is an autosomal recessive genetic disorder characterized by photosensitivity, developmental defects, neurological abnormalities, and premature aging. Mutations in CSA (ERCC8), CSB (ERCC6), XPB, XPD, XPG, XPF (ERCC4) and ERCC1 can give rise to clinical phenotypes resembling classic CS. Using a yeast two-hybrid (Y2H) screening approach, we identified LEO1 (Phe381-Ser568 region) as an interacting protein partner of full-length and C-terminal (Pro1010-Cys1493) CSB in two independent screens. LEO1 is a member of the RNA polymerase associated factor 1 complex (PAF1C) with roles in transcription elongation and chromatin modification. Supportive of the Y2H results, purified, recombinant LEO1 and CSB directly interact in vitro, and the two proteins exist in a common complex within human cells. In addition, fluorescently tagged LEO1 and CSB are both recruited to localized DNA damage sites in human cells. Cell fractionation experiments revealed a transcription-dependent, coordinated association of LEO1 and CSB to chromatin following either UVC irradiation or cisplatin treatment of HEK293T cells, whereas the response to menadione was distinct, suggesting that this collaboration occurs mainly in the context of bulky transcription-blocking lesions. Consistent with a coordinated interaction in DNA repair, LEO1 knockdown or knockout resulted in reduced CSB recruitment to chromatin, increased sensitivity to UVC light and cisplatin damage, and reduced RNA synthesis recovery and slower excision of cyclobutane pyrimidine dimers following UVC irradiation; the absence of CSB resulted in diminished LEO1 recruitment. Our data indicate a reciprocal communication between CSB and LEO1 in the context of transcription-associated DNA repair and RNA transcription recovery.

| S-EPMC8216283 | biostudies-literature

Identification of the endocytic sorting signal recognized by the Art1-Rsp5 ubiquitin ligase complex.

Project description:Targeted endocytosis of plasma membrane (PM) proteins allows cells to adjust their complement of membrane proteins to changing extracellular conditions. For a wide variety of PM proteins, initiation of endocytosis is triggered by ubiquitination. In yeast, arrestin-related trafficking adaptors (ARTs) enable a single ubiquitin ligase, Rsp5, to specifically and selectively target a wide range of PM proteins for ubiquitination and endocytosis. However, the mechanisms that allow ARTs to specifically recognize their appropriate substrates are unknown. We present the molecular features in the methionine permease Mup1 that are required for Art1-Rsp5-mediated ubiquitination and endocytosis. A combination of genetics, fluorescence microscopy, and biochemistry reveals three critical features that comprise an ART sorting signal in the Mup1 N-terminal cytosolic tail: 1) an extended acidic patch, 2) in close proximity to the first Mup1 transmembrane domain, and 3) close to the ubiquitinated lysines. We show that a functionally similar ART sorting signal is also required for the endocytosis of a second Art1-dependent cargo, Can1, suggesting a common mechanism for recognition of Art1 substrates. We isolate two separate suppressor mutations in the Art1 C-terminal domain that allele-specifically restore endocytosis of two Mup1 acidic patch mutants, consistent with an interaction between the Art1 C-terminus and the Mup1 acidic patch. We propose that this interaction is required for recruitment of the Art1-Rsp5 ubiquitination complex.

| S-EPMC5156545 | biostudies-literature

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