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Identifying transcriptional targets of ONECUT3 by RNAseq

ABSTRACT: We report the direct target genes of the aberrant expression of ONECUT3 through next generation sequencing. We combine RNA-seq and ChIP-seq to identify target genes of ONECUT3 overexpression. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) assays were performed on TP53-KO mouse embryonic fibroblast MEF with or without Doxycycline (100 ng/ml) to identify target genes both bound and regulated by ONECUT3. We found that genes related with sister chromosome exchange separation, mitotic nuclear division, and chromosome segregation were significantly upregulated by high level of ONECUT3. The top 10 directly activated target genes predicted by Binding and Expression Target Analysis (BETA) include Incenp and Cdca8, which are the critical components of Chromosomal Passenger Complex (CPC). Through this observation, we conclude that main role of ONECUT3 as transcription factors is to upregulate mRNA transcription of mitotic process. This sutdy provide new insight of genetic network of how dysfunctional ONECUT3 underlies mitotic defects and Chromosomal instability.

ORGANISM(S): Mus musculus

PROVIDER: GSE202693 | GEO | 2024/03/01

REPOSITORIES: GEO

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Identifying transcriptional targets of ONECUT3 by ChIP-seq

Project description:We report the direct target genes of the aberrant expression of ONECUT3 through next generation sequencing. We combine RNA-seq and ChIP-seq to identify target genes of ONECUT3 overexpression. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) assays were performed on TP53-KO mouse embryonic fibroblast MEF with or without Doxycycline (100 ng/ml) to identify target genes both bound and regulated by ONECUT3. We found that genes related with sister chromosome exchange separation, mitotic nuclear division, and chromosome segregation were significantly upregulated by high level of ONECUT3. The top 10 directly activated target genes predicted by Binding and Expression Target Analysis (BETA) include Incenp and Cdca8, which are the critical components of Chromosomal Passenger Complex (CPC). Through this observation, we conclude that main role of ONECUT3 as transcription factors is to upregulate mRNA transcription of mitotic process. This sutdy provide new insight of genetic network of how dysfunctional ONECUT3 underlies mitotic defects and Chromosomal instability.

2024-03-01 | GSE202680 | GEO

Methylation-dependent and -independent roles of EZH2 coordinately contribute to CDCA8 activation in prostate cancer

Project description:Cell division cycle associated 8 (CDCA8) is a component of chromosomal passenger complex (CPC) that participates in mitotic regulation. Although cancer-related CDCA8 hyperactivation has been widely observed, its molecular mechanism remains elusive. Here, we report that CDCA8 overexpression maintains tumorigenicity and is associated with poor clinical outcome in patients with prostate cancer (PCa). Notably, enhancer of zeste homologue 2 (EZH2) is identified to be responsible for CDCA8 activation in PCa. Genome-wide assays revealed that EZH2-induced H3K27 trimethylation represses let-7b expression and thus protects the let-7b-targeting CDCA8 transcripts. More importantly, EZH2 facilitates the self-activation of E2F1 by recruiting E2F1 to its own promoter region in a methylation-independent manner. The high level of E2F1 further promotes transcription of CDCA8 along with the other CPC subunits. Taken together, our study suggests that EZH2-mediated cell cycle regulation in PCa relies on both its methyltransferase and non-methyltransferase activities.

2021-09-30 | GSE184740 | GEO

Utilizing HXMS to Uncover Structural Basis of CPC Phase Separation

Project description:The centromere is the locus that directs accurate chromosome segregation, and a mater regulatory complex ,the chromosome passenger comlex (CPC) is enriched in a region of the mitotic centromre termed the inner centromre. Recent findings support a role of CPC phase separation in its localization and function in mitotic error correction. Currently there are no teqhniques to globaly map the motifs on proteins that drive the multivalent interactions. In this context, we now employ hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) to study dynamics of CPC phase separation.

2024-03-13 | PXD034374 | Pride

Next Generation Sequencing Facilitates Quantitative Analysis of HCC cells transfected with NCsiRNA or CDCA8siRNA

Project description:We hypothesized if targeting of CDCA8 with small interfering (si) RNA could inhibit HCC progression. We also investigated molecular mechanism to mediate HCC cell death caused by CDCA8 silencing. To accomplish this, Huh1 and Huh7 human HCC cells were transfected with CDCA8 siRNA and tested for growth inhibition and apoptotic induction using MTS, FACS and microscopic analysis. To obtain insights into the molecular changes in response to CDCA8 knockdown, global changes in gene expression were examined using RNA sequencing. As results, siRNA silencing of CDCA8 inhibited HCC cell growth by blocking cell-cycle progression and inducing apoptosis. RNA sequencing showed that, representatively, the anti-proliferative effects were driven by a subset of molecular alterations including the upregulation of tumor suppressive ATF3 and GADD34 genes, whereas a key regulator of cell growth and invasiveness BGLAP was repressed. Subsequent Western blotting also revealed that CDCA8 silencing decreases the levels of pro-caspase 3 and PARP-1, accelerating apoptotic signaling in HCC cells. In addition, targeting CDCA8 effectively suppressed HCC tumor growth growth in a murine xenograft model. Taken together, these findings suggest that CDCA8 could be a promising molecular target for systemic therapy of HCC.

2017-02-14 | GSE77992 | GEO

Haspin-dependent and independent effects of the kinase inhibitor 5-Iodotubercidin on self-renewal and differentiation.

Project description:The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting "off-target" effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the "off-target" effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.

2020-01-16 | GSE119737 | GEO

Interaction between TBP and condensing drives the organization and faithful segregation of mitotic chromosomes

Project description:Genome/chromosome organization is highly ordered and controls nuclear events. Here, we show that the TATA box-binding protein (TBP) interacts with the Cnd2 kleisin subunit of condensin to mediate interphase and mitotic chromosome organization in fission yeast. TBP recruits condensin onto RNA polymerase III-transcribed (Pol III) genes and highly transcribed Pol II genes; condensin in turn associates these genes with centromeres. Inhibition of the Cnd2-TBP interaction disrupts condensin localization across the genome and the proper assembly of mitotic chromosomes, leading to severe defects in chromosome segregation and eventually causing cellular lethality. We propose that the Cnd2-TBP interaction coordinates transcription with chromosomal architecture by linking dispersed gene loci with centromeres. This chromosome arrangement can contribute to the efficient transmission of physical force at the kinetochore to chromosomal arms, thereby supporting the fidelity of chromosome segregation.

2015-08-12 | GSE60273 | GEO

Interaction between TBP and condensing drives the organization and faithful segregation of mitotic chromosomes

2015-08-12 | E-GEOD-60273 | biostudies-arrayexpress

Slx5/8 alleviates replication stress by promoting mitotic progression

Project description:MCM-10 vs. wild type yeast differential analysis using label-free relative quantification and directed mass spectometry using RIPPER. Loss of Mcm10 causes replication stress. We uncovered that S. cerevisiae mcm10-1 mutants rely on the SUMO-targeted E3 ubiquitin ligase complex Slx5/8 and SUMO network for survival. How Slx5/8 suppresses replication stress has remained elusive. We developed a novel intensity-based label-free quantitative mass spectrometry approach and identified SUMO conjugates that were either enriched or depleted in mcm10-1 cells. We reasoned that sumoylated proteins that were decreased in mcm10-1 cells were potential Slx5/8 targets. Such candidates included subunits of the chromosome passenger complex (CPC), Bir1 and Sli15, which we verified to be substrates of the Slx5/8 pathway. CPC is known to positively regulate the spindle assembly checkpoint (SAC), and we show here that the ablation of CPC components and other SAC proteins supports growth of mcm10-1 cells by relieving mitotic arrest. Together, our data provide mechanistic insight into how SUMO and Slx5/8 orchestrate the replication stress response to promote cell survival.

2016-04-05 | PXD002607 | Pride

Mass spectrometry analysis of chromosomal passenger complex proteins and kinetochore proteins in Trypanosoma brucei

Project description:We performed chemical crosslinking mass spectrometry of native protein complexes purified from Trypanosoma brucei procyclic cells to obtain protein-protein interaction information for the chromosomal passenger complex (CPC) and kinetochore complex. Crosslinks among CPC subunits were detected using YFP-AUK1, while those among KKT8 complex subunits were detected in the YFP-KKIP1 sample. We also performed immunoprecipitation and mass spectrometry of CPC subunits (full-length proteins and fragments) to identify co-purifying proteins.

2023-10-13 | PXD045987 | Pride

MKLP2 walks along microtubules and takes the CPC along for a ride

Project description:The mitotic kinesin-like Protein 2 (MKLP2, KIF20A) is essential for the proper execution of cytokinesis and required to ‘passage’ the chromosomal passenger complex (CPC) from the chromosomes in (pro)metaphase to the spindle midzone and equatorial cortex in anaphase. Together with MKLP1 (KIF23) and MPP1 (KIF20B), MKLP2 forms the kinesin-6 subfamily of motor proteins. Whilst MKLP1 and MPP1 are both plus-end directed processive motors, the typical structure of the MKLP2 motor domain along with the extended neck-linker region, suggested that MKLP2 might not be able to function as a transport motor but is more likely to act as a microtubule bundler. This notion contradicts the prevailing hypothesis that MKLP2 can transport the CPC in anaphase, but appears to be in line with in vitro and in cellulo studies showing that recombinant MKLP2 efficiently bundles microtubules, and that knock-down of MKLP2, disturbs the organization of the anaphase spindle midzone. Using TIRF microscopy and purified fluorescent proteins, we here demonstrate that MKLP2 is a plus-end directed processive motor that can transport the CPC along microtubules in vitro. Live imaging of MKLP2::GFP and INCENP::GFP in early anaphase cells revealed that a fraction of both MKLP2 and INCENP displays directed movements towards the (equatorial) cortex. In line, inhibition of MKLP2 ATPase activity at the start of anaphase disturbed the localization of MKLP2 and CPC at the equatorial cortex. Our data suggest that MKLP2 is a processive microtubule-based motor that transports itself and the CPC to the equatorial cortex.

2020-06-10 | PXD014665 | Pride

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