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Transcriptome profiling of HepG2 cells in Cdc20 overexpressed and downregulated condition

ABSTRACT: Cdc20 acts as a regulatory protein interacting with many other proteins at multiple points in the cell cycle. Here we tried to explore the novel transcription regulatory function of Cdc20 by means of altering Cdc20 level in HepG2 cells. We transiently over expressed Flag tagged Cdc20 (F) and siRNA mediated knocked down Cdc20(S) in HepG2 cell line and performed whole genome microarray analysis using illumina BeadChip Array. Significantly altered genes were subjected to the analysed by Ingenuity pathway analysis software (Build version: 302937) and crucial cellcycle and apoptotic pathways were found to be altered. These pathways could further be explored as anticancer therapy in liver carcinoma.

ORGANISM(S): Homo sapiens

PROVIDER: GSE99756 | GEO | 2021/12/06

REPOSITORIES: GEO

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Effect of indel in promoter of CDC20 on gene expression in A375 human melanoma cell line.

Project description:CDC20 promoter variants are present in ~26% of cutaneous melanomas. We generated an indel in the promoter of CDC20 and performed RNA-sequencing to determine the effect on CDC20 expression and other secondary transcriptional changes.

2023-11-01 | GSE206639 | GEO

PRMT6-CDC20 facilitates glioblastoma progression via the degradation of CDKN1B

Project description:PRMT6, a type I arginine methyltransferase, di-methylates the arginine residues of both histones and non-histones asymmetrically. Increasing evidence indicates that PRMT6 plays a tumor mediator involved in human malignancies. Here, we aim to uncover the essential role and underlying mechanisms of PRMT6 in promoting glioblastoma (GBM) proliferation. Investigation of PRMT6 expression in glioma tissues demonstrated that PRMT6 is overexpressed, and elevated expression of PRMT6 is negatively correlated with poor prognosis in glioma/GBM patients. Silencing PRMT6 inhibited GBM cell proliferation and induced cell cycle arrest at the G0/G1 phase, while overexpressing PRMT6 had opposite results. Further, we found that PRMT6 attenuates the protein stability of CDKN1B by promoting its degradation. Subsequent mechanistic investigations showed that PRMT6 maintains the transcription of CDC20 by activating histone methylation mark (H3R2me2a), and CDC20 interacts with and destabilizes CDKN1B. Rescue experimental results confirmed that PRMT6 promotes the ubiquitinated degradation of CDKN1B and cell proliferation via CDC20. We also verified that the PRMT6 inhibitor (EPZ020411) could attenuate the proliferative effect of GBM cells. Our findings illustrate that PRMT6, an epigenetic mediator, promotes CDC20 transcription via H3R2me2a to mediate the degradation of CDKN1B to facilitate GBM progression. Targeting PRMT6-CDC20-CDKN1B axis might be a promising therapeutic strategy for GBM.

2023-03-31 | GSE221971 | GEO

Coupling of Cdc20 inhibition and activation by BubR1

Project description:Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.

2021-02-18 | MSV000086910 | MassIVE

Bulk RNA sequencing from sorted thymic populations and tumors from control Cdc20(+/D) and aneuploid Cdc20(D/AAA)mice

Project description:Purpose: The goals of this study are to compare the transcriptome profile of specific thymic populations in control Cdc20(+/D) and Cdc20(D/AAA)mice after the induction of chromosome missegregation as well the profile of tumors generated in those mice. Methods: Total RNA was isolated from primary cells or tumors, using the RNeasy Plus Mini Kit (Qiagen) and sequenced on an Illumina HiSeq2000. At least 5000 cells were sorted directly in lysis buffer for specifc T cell progenitor populations in the thymus while at least 5 million cells have used for RNA isolation form thymic lymphomas. Unless otherwise indicated samples from tumors were isolated without sorting specific polulations.

2023-06-06 | GSE174012 | GEO

Paradoxical mitotic exit induced by a small molecule inhibitor of APC/C Cdc20

Project description:The Anaphase Promoting Complex/Cyclosome (APC/C) is a mega-dalton ubiquitin ligase that initiates mitotic exit by targeting substrates for degradation. The APC/C is activated by Cdc20, which acts as a substrate receptor, and is inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated. We previously identified apcin, a small molecule ligand of Cdc20, as an inhibitor of APC/CCdc20. Surprisingly, we found that apcin paradoxically accelerates substrate degradation and promotes mitotic exit in cells with high SAC activity. Biochemical studies indicate that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin’s behavior as an antagonist of Cdc20 can thus result in either net inhibition of APC/C, delaying mitotic exit when SAC activity is slow, or net activation of APC/C, promoting mitotic exit when SAC activity is high. Genetic experiments suggest that the dual behaviors of apcin arise from targeting a common binding site in Cdc20 that is required for both substrate ubiquitination as well as efficient APC/C inhibition by MCC. We therefore establish a new mechanism through which a small molecule, by targeting a single site on a dynamic protein interface, can lead to opposing biological effects depending on the regulatory context.

2020-01-07 | PXD013786 | Pride

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling

Project description:Accurate chromosome segregation is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), to inhibit the anaphase-promoting complex or cyclosome (APC/C). Cdc20 is an essential mitotic regulator since it promotes mitotic exit through activating the APC/C and monitors kinetochore-microtubule attachment through activating the SAC. The proper functioning of Cdc20 requires multiple interactions with APC/C and MCC subunits. To functionally assess each of these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNAi. Here we generated Cdc20 RNAi sensitive cell lines by CRISPR/Cas9 which display a penetrant metaphase arrest phenotype by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron was found to be critical for the SAC by promoting theMCC formation and stabilizing the interaction between the MCC and APC/C. These data reveal additionalregulatory components within the SAC and establish a novel method to interrogate Cdc20 function.

2023-12-06 | PXD045411 | Pride

Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly (IGX-MS data)

Project description:During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC) to inhibit the anaphase-promoting complex/cyclosome (APC/C). The Mad1-Mad2 complex provides a catalytic platform for MCC assembly. Mad1-bound Mad2 recruits open-Mad2 through asymmetric dimerization and Mad1 phosphorylation by Mps1 promotes conversion of Mad2 from an open (O-Mad2) to closed (C-Mad2) state, which binds Cdc20 to form the MCC. How Mad1 phosphorylation catalytically activates MCC formation is poorly understood. This study characterises Mad1 phosphorylation by Mps1 and provides structural and biochemical insights into a phosphorylation-specific Mad1-Cdc20 interaction, which allows for a tripartite assembly of Bub1-Mad1-Cdc20 on the C-terminal domain of Mad1. We also identify a folded state of Mad1-Mad2 complex, suggesting a model by which the Cdc20-Mad1 interaction brings the Cdc20 MIM motif near Mad2. The Cdc20 MIM motif is then entrapped by the Mad2 safety belt to form a stable complex, allowing spontaneous MCC assembly.

2023-03-11 | PXD031872 | Pride

ChIP-seq in SUNE1 cells that overexpressed CBX1

Project description:ChIP experiment against anti-FLAG in SUNE1 cells trasfected with FLAG-CBX1 plasmid.

2023-01-04 | GSE201232 | GEO

Immunoprecipitates of KDM1A in HepG2 cells with stably overexpressed KDM1A

Project description:KDM1A requires an adaptor protein with specific DNA-binding activity to recognize chromatin sequence. We constructed HepG2 cells stably overexpressing KDM1A-Flag tag using lentivirus. We performed Co-IP with Flag-gel and we used LC-MS to determine the immunoprecipitates of KDM1A.

2023-05-19 | PXD043284 | iProX

Identification of CDC20/CDC20B partners in HEK cells demonstrates that CDC20, but not CDC20B, interacts with multiple APC/C components

Project description:Multiciliated cells (MCCs) harbour dozens to hundreds of motile cilia, which beat in a synchronized and directional manner, thus generating hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation critically depends on the synthesis and release of numerous centrioles by specialized structures called deuterosomes. Little is known about the composition, organization and regulation of deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome-stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate specific cell division cycle 20B (CDC20B) gene, the host gene of microRNA-449abc. We show that the CDC20B protein associates to deuterosomes and is required for the release of centrioles and the subsequent production of cilia in mouse and Xenopus MCCs. CDC20B interacts with PLK1, which has been shown to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescued centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of a new biological function, deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules. A specific aim of this mass spectrometry experiment was to verify on immunoprecipitated protein complexes from CDC20 or CDC20B transfected HEK cells that CDC20, but not CDC20B, interacts with multiple APC/C components.

2019-04-03 | PXD010629 | Pride

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