EWSR1 maintains centromere identity.

The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance.

Single-cell RNA profiling identifies diverse cellular responses to EWSR1/FLI1 downregulation in Ewing sarcoma cells.

Background: The EWSR1/FLI1 gene fusion is the most common rearrangement leading to cell transformation in Ewing sarcoma (ES). Previous studies have indicated that expression at the cellular level is heterogeneous, and that levels of expression may oscillate, conferring different cellular characteristics. In ES the role of EWSR1/FLI1 in regulating subpopulation dynamics is currently unknown.

The Role of Human Centromeric RNA in Chromosome Stability.

Chromosome instability is a hallmark of cancer and is caused by inaccurate segregation of chromosomes. One cellular structure used to avoid this fate is the kinetochore, which binds to the centromere on the chromosome. Human centromeres are poorly understood, since sequencing and analyzing repeated alpha-satellite DNA regions, which can span a few megabases at the centromere, are particularly difficult.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A).

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e.

CENP-A Ubiquitylation Is Indispensable to Cell Viability.

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity, but how CENP-A is deposited at the centromere remains obscure. We previously reported that CENP-A K124 ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere. However, a recent report stated that CENP-A K124R mutants show no defects in centromere localization and cell viability.

CENP-A Ubiquitylation Contributes to Maintaining the Chromosomal Location of the Centromere.

The centromere plays an essential role in accurate chromosome segregation, and the chromosomal location of the centromere is determined by the presence of a histone H3 variant, centromere protein A (CENP-A), in centromeric nucleosomes. However, the precise mechanisms of deposition, maintenance, and inheritance of CENP-A at centromeres are unclear. We have reported that CENP-A deposition requires ubiquitylation of CENP-A lysine 124 mediated by the E3 ligase activity of Cullin 4A (CUL4A)-RING-box protein 1 (RBX1)-COP9 signalsome complex subunit 8 (COPS8).

SGT1-HSP90 complex is required for CENP-A deposition at centromeres.

The centromere plays an essential role in accurate chromosome segregation, and defects in its function lead to aneuploidy and thus cancer. The centromere-specific histone H3 variant CENP-A is proposed to be the epigenetic mark of the centromere, as active centromeres require CENP-A-containing nucleosomes to direct the recruitment of multiple kinetochore proteins. CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere.

The inheritance of centromere identity.

CENP-A (Centromere protein A) is a histone H3 variant that epigenetically determines the centromere position, but the mechanism of its centromere inheritance is obscure. We propose that CENP-A ubiquitylation, which is inherited through dimerization between rounds of cell division, is a candidate for the epigenetic mark of centromere identity.

CENP-A Ubiquitylation Is Inherited through Dimerization between Cell Divisions.

The presence of chromatin containing the histone H3 variant CENP-A dictates the location of the centromere in a DNA sequence-independent manner. But the mechanism by which centromere inheritance occurs is largely unknown. We previously reported that CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere.

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins.

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores.

CENP-A K124 Ubiquitylation Is Required for CENP-A Deposition at the Centromere.

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity to ensure kinetochore assembly and proper chromosome segregation, but the precise mechanism of its specific localization within centromeric heterochromatin remains obscure. We have discovered that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A ubiquitylation on lysine 124 (K124) and CENP-A centromere localization. A mutation of CENP-A, K124R, reduces interaction with HJURP (a CENP-A-specific histone chaperone) and abrogates localization of CENP-A to the centromere.

Degradation of centromeric histone H3 variant Cse4 requires the Fpr3 peptidyl-prolyl Cis-Trans isomerase.

The centromeric histone H3 variant Cse4 in Saccharomyces cerevisiae is polyubiquitylated and degraded in a proteasome-dependent manner. We report here that the proline isomerase Fpr3 regulates Cse4 proteolysis. Structural change in Cse4 by Fpr3 might be important for the interaction between Cse4 and the E3 ubiquitin ligase Psh1.

Synthetic genetic array screen identifies PP2A as a therapeutic target in Mad2-overexpressing tumors.

The spindle checkpoint is essential to ensure proper chromosome segregation and thereby maintain genomic stability. Mitotic arrest deficiency 2 (Mad2), a critical component of the spindle checkpoint, is overexpressed in many cancer cells. Thus, we hypothesized that Mad2 overexpression could specifically make cancer cells susceptible to death by inducing a synthetic dosage lethality defect.

Structure-specific endonucleases xpf and mus81 play overlapping but essential roles in DNA repair by homologous recombination.

DNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intact sister chromatid. HR, therefore, plays pivotal roles in cellular proliferation and cellular tolerance to camptothecin.

Role of transcription at centromeres in budding yeast.

Centromeres are specialized chromosomal loci that are essential for proper chromosome segregation. Recent data show that a certain level of active transcription, regulated by transcription factors Cbf1 and Ste12, makes a direct contribution to centromere function in Saccharomyces cerevisiae. Here, we discuss the requirement and function of transcription at centromeres.

Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint.

The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage.

Endogenous transcription at the centromere facilitates centromere activity in budding yeast.

Background: The centromere (CEN) DNA-kinetochore complex is the specialized chromatin structure that mediates chromosome attachment to the spindle and is required for high-fidelity chromosome segregation. Although kinetochore function is conserved from budding yeast to humans, it was thought that transcription had no role in centromere function in budding yeast, in contrast to other eukaryotes including fission yeast.

Results: We report here that transcription at the centromere facilitates centromere activity in the budding yeast Saccharomyces cerevisiae.

Bub1-mediated adaptation of the spindle checkpoint.

During cell division, the spindle checkpoint ensures accurate chromosome segregation by monitoring the kinetochore-microtubule interaction and delaying the onset of anaphase until each pair of sister chromosomes is properly attached to microtubules. The spindle checkpoint is deactivated as chromosomes start moving toward the spindles in anaphase, but the mechanisms by which this deactivation and adaptation to prolonged mitotic arrest occur remain obscure. Our results strongly suggest that Cdc28-mediated phosphorylation of Bub1 at T566 plays an important role for the degradation of Bub1 in anaphase, and the phosphorylation is required for adaptation of the spindle checkpoint to prolonged mitotic arrest.

Dishevelled, a Wnt signalling component, is involved in mitotic progression in cooperation with Plk1.

Wnt signalling is known to promote G1/S progression through the stimulation of gene expression, but whether this signalling regulates mitotic progression is not clear. Here, the function of dishevelled 2 (Dvl2), which transmits the Wnt signal, in mitosis was examined. Dvl2 localized to the spindles and spindle poles during mitosis.

Sgt1 dimerization is negatively regulated by protein kinase CK2-mediated phosphorylation at Ser361.

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism.

Sgt1 dimerization is required for yeast kinetochore assembly.

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses.

Caspase-independent mitotic death (CIMD).

The spindle checkpoint, which monitors kinetochore-microtubule attachment, is required for high fidelity of chromosome transmission. A failure in this mechanism causes aneuploidy, thereby promoting progression to tumorigenesis. However, the cell death mechanism that prevents the aneuploidy caused by failure of the spindle checkpoint is yet unknown.

Ybp2 associates with the central kinetochore of Saccharomyces cerevisiae and mediates proper mitotic progression.

The spindle checkpoint ensures the accurate segregation of chromosomes by monitoring the status of kinetochore attachment to microtubules. Simultaneous mutations in one of several kinetochore and cohesion genes and a spindle checkpoint gene cause a synthetic-lethal or synthetic-sick phenotype. A synthetic genetic array (SGA) analysis using a mad2Delta query mutant strain of yeast identified YBP2, a gene whose product shares sequence similarity with the product of YBP1, which is required for H(2)O(2)-induced oxidation of the transcription factor Yap1.