Cockayne syndrome A and B proteins regulate the transcription arrest upon genotoxic stress through a ubiquitin/proteasome degradation process
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE87564 | GEO | 2017/12/01
SECONDARY ACCESSION(S): PRJNA345198
REPOSITORIES: GEO
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Project description:The rare genetic disease Cockayne syndrome (CS) results in mutations in CSA and CSB. Upon UV irradiation, RNA synthesis was arrested: RNA-seq showed 70% of down-regulated genes in common between CSA and CSB deficient cells. ATF3, the product of an immediate early gene was overexpressed and bound to its CRE/ATF site to inhibit its responsive genes. ChIP experiments showed that CSA/CUL4A/DDB1 together with CSB and MDM2, target ATF3. In vivo and in vitro experiments showed that ATF3 was ubiquitilated by a concerted action of CSA and MDM2 ubiquitin-ligases and was further eliminated by the proteasome concomitantly with the recruitment of RNA polymerase II to restart transcription. In CS cells, dysfunctional CSA or CSB were unable to assemble the ubiquitin/proteasome complex, thereby maintaining the ATF3-dependent transcription arrested. Though, in addition to their function in DNA repair, CSA and CSB might thus regulate the timing of DNA binding factors on its specific target site via the ubiquitin/proteasome machinery.
2017-12-01 | GSE87562 | GEO
Project description:The rare genetic disease Cockayne syndrome (CS) results in mutations in CSA and CSB. Upon UV irradiation, RNA synthesis was arrested: RNA-seq showed 70% of down-regulated genes in common between CSA and CSB deficient cells. ATF3, the product of an immediate early gene was overexpressed and bound to its CRE/ATF site to inhibit its responsive genes. ChIP experiments showed that CSA/CUL4A/DDB1 together with CSB and MDM2, target ATF3. In vivo and in vitro experiments showed that ATF3 was ubiquitilated by a concerted action of CSA and MDM2 ubiquitin-ligases and was further eliminated by the proteasome concomitantly with the recruitment of RNA polymerase II to restart transcription. In CS cells, dysfunctional CSA or CSB were unable to assemble the ubiquitin/proteasome complex, thereby maintaining the ATF3-dependent transcription arrested. Though, in addition to their function in DNA repair, CSA and CSB might thus regulate the timing of DNA binding factors on its specific target site via the ubiquitin/proteasome machinery.
2017-12-01 | GSE87540 | GEO
Project description:Cockayne syndrome A and B proteins regulate the transcription arrest upon genotoxic stress through a ubiquitin/proteasome degradation process
| PRJNA345198 | ENA
Project description:Cytokinesis is monitored by a molecular machinery that promotes the degradation of the intercellular bridge, a transient protein structure connecting the two daughter cells. Here, we found that CSA and CSB, primarily defined as DNA repair factors, are located at the midbody, a transient structure in the middle of the intercellular bridge, where they recruit CUL4 and MDM2 ubiquitin ligases and the proteasome. As a part of this molecular machinery, CSA and CSB contribute to the ubiquitination and the degradation of proteins such as PRC1, the Protein Regulator of Cytokinesis, to ensure the correct separation of the two daughter cells. Defects in CSA or CSB result in perturbation of the abscission leading to the formation of long intercellular bridges and multinucleated cells, which might explain part of the Cockayne syndrome phenotypes. Our results enlighten the role played by CSA and CSB as part of a ubiquitin/proteasome degradation process involved in transcription, DNA repair, and cell division.
| S-EPMC7720219 | biostudies-literature
Project description:Oocyte meiosis and early mitotic divisions in developing embryos rely on the timely production of cell cycle regulators and their clearance via proteasomal degradation. Ret Finger Protein-Like 4 (Rfpl4), encoding a RING finger-like protein with a B30.2 domain, was discovered during an in silico search for germ cell-specific genes. To study the expression and functions of RFPL4 protein, we performed immunolocalizations and used yeast two-hybrid and other protein-protein interaction assays. Immunohistochemistry and immunofluorescence showed that RFPL4 accumulates in all growing oocytes and quickly disappears during early embryonic cleavage. We used a yeast two-hybrid model to demonstrate that RFPL4 interacts with the E2 ubiquitin-conjugating enzyme HR6A, proteasome subunit beta type 1, ubiquitin B, as well as a degradation target protein, cyclin B1. Coimmunoprecipitation analyses of in vitro translated proteins and extracts of transiently cotransfected Chinese hamster ovary (CHO)-K1 cells confirmed these findings. We conclude that, like many RING-finger containing proteins, RFPL4 is an E3 ubiquitin ligase. The specificity of its expression and these interactions suggest that RFPL4 targets cyclin B1 for proteasomal degradation, a key aspect of oocyte cell cycle control during meiosis and the crucial oocyte-to-embryo transition to mitosis.
| S-EPMC141033 | biostudies-literature
Project description:Cockayne syndrome (CS) is a premature aging disorder characterized by developmental defects, multisystem progressive degeneration and sensitivity to ultraviolet light. CS is divided into two primary complementation groups, A and B, with the CSA and CSB proteins presumably functioning in DNA repair and transcription. Using laser microirradiation and confocal microscopy, we characterized the nature and regulation of the CS protein response to oxidative DNA damage, double-strand breaks (DSBs), angelicin monoadducts and trioxsalen interstrand crosslinks (ICLs). Our data indicate that CSB recruitment is influenced by the type of DNA damage and is most rapid and robust as follows: ICLs>DSBs>monoadducts>oxidative lesions. Transcription inhibition reduced accumulation of CSB at sites of monoadducts and ICLs, but it did not affect recruitment to (although slightly affected retention at) oxidative damage. Inhibition of histone deacetylation altered the dynamics of CSB assembly, suggesting a role for chromatin status in the response to DNA damage, whereas the proteasome inhibitor MG132 had no effect. The C-terminus of CSB and, in particular, its ubiquitin-binding domain were critical to recruitment, while the N-terminus and a functional ATPase domain played a minor role at best in facilitating protein accumulation. Although the absence of CSA had no effect on CSB recruitment, CSA itself localized at sites of ICLs, DSBs and monoadducts but not at oxidative lesions. Our results reveal molecular components of the CS protein response and point to a major involvement of complex lesions in the pathology of CS.
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Project description:Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine ?-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.
| S-EPMC7312034 | biostudies-literature
Project description:ObjectivesUbiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown.MethodsWe examined the expression levels of E3 ligases and NF-κB members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in fracture callus from Itch knockout (KO) mice and their WT littermates were examined by qPCR. The effect of NF-κB on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay.ResultsThe expression levels of WW Domain Containing E3 Ubiquitin Protein Ligase 1 (Wwp1), SMAD Specific E3 Ubiquitin Protein Ligase 1 (Smurf1), SMAD Specific E3 Ubiquitin Protein Ligase 2 (Smurf2) and Itch were all significantly increased in the fracture callus of WT mice, which was associated with elevated expression of NF-κB members and total ubiquitinated proteins. Callus tissue isolated from Itch KO mice expressed higher levels of osteoblast-associated genes, including Runx2, a positive regulator of osteoblast differentiation, but osteoclast-associated genes were not increased. Both NF-κB RelA and RelB proteins were found to bind to the NF-κB binding site in the mouse Itch promoter.ConclusionsOur findings indicate that Itch depletion may have a strong positive effect on osteoblast differentiation in fracture callus. Thus, ubiquitin E3 ligase Itch could be a potential target for enhancing bone fracture healing.Cite this article: J. Liu, X. Li, H. Zhang, R. Gu, Z. Wang, Z. Gao, L. Xing. Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins. Bone Joint Res 2017;6:154-161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1.
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Project description:Optic fissure fusion is a critical event during retinal development. Failure of fusion leads to coloboma, a potentially blinding congenital disorder. Pax2a is an essential regulator of optic fissure fusion and the target of numerous morphogenetic pathways. In our current study, we examined the negative regulator of pax2a expression, Nz2, and the mechanism modulating Nlz2 activity during optic fissure fusion. Upregulation of Nlz2 in zebrafish embryos resulted in downregulation of pax2a expression and fissure fusion failure. Conversely, upregulation of pax2a expression also led to fissure fusion failure suggesting Pax2 levels require modulation to ensure proper fusion. Interestingly, we discovered Nlz2 is a target of the E3 ubiquitin ligase Siah. We show that zebrafish siah1 expression is regulated by Hedgehog signaling and that Siah1 can directly target Nlz2 for proteasomal degradation, in turn regulating the levels of pax2a mRNA. Finally, we show that both activation and inhibition of Siah activity leads to failure of optic fissure fusion dependent on ubiquitin-mediated proteasomal degradation of Nlz2. In conclusion, we outline a novel, proteasome-mediated degradation regulatory pathway involved in optic fissure fusion.
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