Project description:The proto-oncogenes ETV1, ETV4, and ETV5 encode members of the E26 transformation-specific (ETS) transcription factor family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COnstitutive Photomorphogenic-1 (COP1, also called RFWD2) as a tumor suppressor that negatively regulates ETV1, ETV4, and ETV5. ETV1, which is the member mutated more frequently in prostate cancer, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs (degrons) and was 50-fold more stable than wild-type ETV1. Almost all patient translocations eliminate these ETV1 degrons, implying that translocations rendering ETV1 insensitive to COP1 confer a significant selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 levels and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. The combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, relatively rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein expression, and abnormally elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a bona fide tumor suppressor whose down-regulation promotes prostatic epithelial cell proliferation and tumorigenesis. LNCap prostate cancer cell line were treated with 5 different sets of siRNAs: (1) control siRNA; (2) COP1 (RFWD2) siRNA; (3) COP1 siRNA + ETV1 siRNA; (4) COP1 siRNA + c-JUN siRNA; (5) COP1 siRNA + ETV1 siRNA + c-JUN siRNA. The experiments were conducted in two batches; each batch has its own control siRNA group, so that the batch effect can be properly modelled. Each group has 4-6 replicates; there are 31 samples in total.
Project description:In order to assess the physiological role of Cop1 in vivo we generated mice that do no longer express the protein. Cop1KO mice die at around E10.5 of embryonic development. In order to gain insights into the molecular mechanisms that cause the embryonic death we compared the genome-wide gene expression profile of E9.5 wild-tytpe and Cop1-null embryos. The data do not support a role for Cop1 in the regulation of the p53 pathway in vivo and highlight a role for Cop1 in cardiovascular development and/or angiogenesis. The abstract of the associated publication is as follows:Biochemical data have suggested conflicting roles for the E3 ubiquitin ligase Cop1 in tumourigenesis. Here we present the first in vivo investigation of the role of Cop1 in cancer aetiology. We used an innovative genetic approach to generate an allelic series of Cop1 and show that Cop1 hypomorphic mice spontaneously develop malignancy at a high frequency in their first year of life and are highly susceptible to radiation-induced lymphomagenesis. Biochemically, we show that Cop1 regulates c-Jun oncoprotein stability and modulates c-Jun/AP1 transcriptional activity in vivo. Cop1-deficiency stimulates cell proliferation in a c-Jun-dependent manner. We conclude that Cop1 is a tumour suppressor that antagonizes c-Jun oncogenic activity in vivo. RNA from 6 different control embryos (+/+ or +/-) were mixed and subdivided into control pool 1 and pool 2. RNA from 6 different Cop-null embryos were mixed and subdivided into KO pool 1 and pool 2.
Project description:Although many regulatory components of light signaling have been functionally characterized, only a few of them have been reported to cross talk with other signaling cascades. In this study, we have analyzed the expression profiles of Arabidopsis genes in wild-type, atmyc2 mutant, cop1-6 mutant, and atmyc2 cop1-6 double mutant seedlings grown under constant dark, constant blue-light, and constant blue-light along with abscisic acid (ABA) to illustrate the interplay of negative regulators, AtMYC2 and COP1, in light and ABA signaling Keywords: Light and abscisic acid treatment Expression data for wild-type, atmyc2, cop1-6, and atmyc2 cop1-6 seedlings under constant dark, blue-light and blue-light along with abscisic acid Ten-day-old Arabidopsis (wild-type, atmyc2 mutant, cop1-6 mutant, and atmyc2 cop1-6 double mutant) seedlings grown under constant dark, constant blue-light (35 µmol/sec/m2), and constant blue-light along with ABA (0.5 µm) were used for RNA extraction and hybridization on Affymetrix microarrays. Two biological replicates of each sample were used for microarray analysis.
Project description:Cop1 regulates stability of transcription factors. We found that Cop1 can regulate the proteasomal degradation of Cebpd. We generated 4T1 breast cancer cells with CRISPR/Cas9 mediated Cop1 (gene symbol RFWD2) knockout. We used Cebpd antibody to pulldown the DNA fragments binding with Cebpd and did ChIP-seq.
Project description:Cop1 regulates stability of transcription factors. We determined the role of Cop1 in the regulation of Cebpd transciptional output. We generated 4T1breast cancer cells with CRISPR/Cas9 mediated Cop1 (gene symbol RFWD2) knockout. We treated control sgRosa26 and sgCop1 cells with vehicle or IFNgamma (20ng/ml) for 24 hours and did ATAC-seq.
Project description:The proper accumulation of BBX22 mediated by COP1 is crucial for plants to maintain better growth fitness when growing in the dark environment as well as responding to seasonal changes in day-length. The constitutive-photomorphogenic development of the cop1 mutant is enhanced in cop1BBX22-GFPox plants, which show a short hypocotyl, high anthocyanin accumulation and expression of light-responsive genes. Target genes responsible for the exaggerated light phenotype in cop1BBX22-GFPox plants were revealed by comparing transcriptomes among dark-grown wild-type, cop1 and cop1BBX22-GFPox plants. Expression of genes regulated by light and multiple hormones are altered in plants over-accumulating BBX22, implying a coordination role of BBX22 in light- and hormone-mediated seedling development. Three biological replicates for 4-day-old etiolated seedlings of wild type (Col-0) and two mutants (cop1-4 and cop1-4BBX22-GFPox).
Project description:Bone marrow was extracted from mice that are COP1-wt Rosa26-CreERT2 or COP1-floxed Rosa26-CreERT2 BMDMs were obtained by culturing bone marrow precursors in media containing 20% of supernatant from L929 cells. At day 4 of differentiation 4-OHT was added at 1uM to induce deletion of COP1 in BMDMs derived from COP1-floxed mice. At day 7 of differentiation, BMDMs were treated with 100 ng/ml of LPS or not. BMDMs were directly harvested in lysis buffer (from Qiagen RNeasy mini kit) at different time points (0h, 2.5h, 2.5h, 4h, 6h, 9h and 13h) following LPS stimulation. Three BMDMs preparations per group: G1: BMDMs from COP1-wt mice (expressing the wt allele of COP1) CRE positive. G2: BMDMs from COP1-floxed mice (expressing the floxed allele of COP1) CRE positive
Project description:In order to assess the physiological role of Cop1 in vivo we generated mice that do no longer express the protein. Cop1KO mice die at around E10.5 of embryonic development. In order to gain insights into the molecular mechanisms that cause the embryonic death we compared the genome-wide gene expression profile of E9.5 wild-tytpe and Cop1-null embryos. The data do not support a role for Cop1 in the regulation of the p53 pathway in vivo and highlight a role for Cop1 in cardiovascular development and/or angiogenesis. The abstract of the associated publication is as follows:Biochemical data have suggested conflicting roles for the E3 ubiquitin ligase Cop1 in tumourigenesis. Here we present the first in vivo investigation of the role of Cop1 in cancer aetiology. We used an innovative genetic approach to generate an allelic series of Cop1 and show that Cop1 hypomorphic mice spontaneously develop malignancy at a high frequency in their first year of life and are highly susceptible to radiation-induced lymphomagenesis. Biochemically, we show that Cop1 regulates c-Jun oncoprotein stability and modulates c-Jun/AP1 transcriptional activity in vivo. Cop1-deficiency stimulates cell proliferation in a c-Jun-dependent manner. We conclude that Cop1 is a tumour suppressor that antagonizes c-Jun oncogenic activity in vivo.
Project description:MicroRNAs (miRNAs) play important roles in cell differentiation and self-renewal controlling post-transcriptional processing of mRNAs and attenuating production of the encoded proteins. Here, we unveil a novel oncogenic pathway leading to activation of STAT3 signaling through miRNA-mediated silencing of the E3 ubiquitin ligase COP1. miRNA profiling showed that miR-424 was upregulated in prostate cancer compared to normal prostate and specifically associated with reduced level of the ETS factor ESE3/EHF in an aggressive subgroup of tumors. MiR-424 was significantly elevated also in other epithelial cancers and amplified in 1-3% of various cancers. In normal prostate epithelial cells miR-424 was repressed by ESE3/EHF and when upregulated promoted oncogenic and cancer stem cell (CSC) properties. Conversely, ablation of miR-424 in metastatic prostate cancer cells reduced CSC self-renewal and prevented in vivo tumour initiation and metastatic spread. miR-424 targeted the 3' UTR of COP1 mRNA and reduced COP1 protein level. COP1 induced STAT3 ubiquitylation and degradation by the ubiquitin-proteasome system (UPS). Therefore, reduced levels of COP1 in prostate cancer cells, resulted in accumulation and increased STAT3 signaling. COP1 knockdown and over-expression phenocopied the effects of miR-424 deregulation on oncogenic phenotypes and STAT3 signalling, while STAT3 knockdown prevented the transforming effects of miR-424. Consistently, expression of EHF/ESE3 and RFWD2/COP1 were highly correlated in human prostate cancers and other epithelial tumors. Furthermore, miR-424 induced genes were enriched of STAT3 targets, converged with those induced by COP1 loss in mouse embryos and were associated with adverse prognosis in prostate and other epithelial cancers. In primary prostate tumours, low COP1 and high STAT3 protein level were also significantly associated and predictive of biochemical relapse. Collectively, this study reveals a novel miRNA-activated oncogenic axis in prostate cancer. Targeting miR-424 or miR-424 dependent pathways may represent a unique approach to attack a key node in tumorigenesis.
Project description:Light is an environmental factor that influences various aspects of plant development. Phytochromes (phys) as light sensors regulate myriads of downstream genes in response to changes in environmental factors. VIL1 (VIN3-LIKE 1)/VERNALIZATION 5 (VRN5), a Polycomb Repressive Complex 2 (PRC2) - associated protein, mediates vernalization pathway. VIL1 directly interacts with phyB and regulates photomorphogenesis through the formation of repressive chromatin loops at downstream growth-promoting genes in response to light. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is an E3 ligase for a number of substrates in light signaling, acting as a central repressor of photomorphogenesis. Here, we show that VIL1 is a substrate of COP1 and COP1 degrades VIL1 through the ubiquitin/26S proteosome system (UPS) in the dark. Our study illustrates that COP1 controls light-dependent formation of chromatin loop by regulating the stability of VIL1 and limits a repressive histone modification to fine-tune expressions of growth-promoting genes during photomorphogenesis. We performed Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications H3K27me3 in five Arabidopsis genotypes (Col-0, vil1-1, cop1-4, vil1-1cop1-4 and hy5-215).