Centromere Protein A (CENPA) Regulates Metabolic Reprogramming in the Colon Cancer Cells by Transcriptionally Activating Karyopherin Subunit Alpha 2 (KPNA2).

The karyopherin α2 subunit gene (KPNA2), an oncogene, is involved in metabolic reprogramming in cancer. This study aimed to explore the function of KPNα2 in the growth and glycolysis in colon cancer (CC) cells. Genes from the Oncomine database that were differentially expressed in multiple CC types were screened. Bioinformatics analysis suggested that KPNA2 was highly expressed in CC, and consequently, high expression of KPNA2 was detected in the CC cell lines. Down-regulation of KPNA2 reduced viability and DNA-replication ability, and increased apoptosis of HCT116 and LoVo cells. It also reduced glucose consumption, extracellular acidification rate, and the ATP production in cells. Centromere protein A (CENPA) was confirmed as an upstream transcription activator of KPNA2. There was significant H3K27ac modification in the promoter region of KPNA2. CENPA primarily recruited histone acetyltransferase general control of amino acid synthesis (GCN)-5 to the promoter region of KPNA2 to induce transcription activation. Overexpression of either CENPA or GCN-5 blocked the role of short hairpin KPNα2 and restored growth and glycolysis in CC cells. To conclude, the findings from this study suggest that CENPA recruits GCN-5 to the promoter region of KPNA2 to induce KPNα2 activation, which strengthens growth and glycolysis in, and augments the development of, CC.

A natural histone H2A variant lacking the Bub1 phosphorylation site and regulated depletion of centromeric histone CENP-A foster evolvability in Candida albicans.

Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis, and yet specific aneuploidies can be adaptive during environmental stress. Here, we identify a chromatin-based system required for inducible aneuploidy in a human pathogen. Candida albicans utilizes chromosome missegregation to acquire tolerance to antifungal drugs and for nonmeiotic ploidy reduction after mating. We discovered that the ancestor of C. albicans and 2 related pathogens evolved a variant of histone 2A (H2A) that lacks the conserved phosphorylation site for kinetochore-associated Bub1 kinase, a key regulator of chromosome segregation. Using engineered strains, we show that the relative gene dosage of this variant versus canonical H2A controls the fidelity of chromosome segregation and the rate of acquisition of tolerance to antifungal drugs via aneuploidy. Furthermore, whole-genome chromatin precipitation analysis reveals that Centromere Protein A/ Centromeric Histone H3-like Protein (CENP-A/Cse4), a centromeric histone H3 variant that forms the platform of the eukaryotic kinetochore, is depleted from tetraploid-mating products relative to diploid parents and is virtually eliminated from cells exposed to aneuploidy-promoting cues. We conclude that genetically programmed and environmentally induced changes in chromatin can confer the capacity for enhanced evolvability via chromosome missegregation.

Adaptations for centromere function in meiosis.

The aim of mitosis is to segregate duplicated chromosomes equally into daughter cells during cell division. Meiosis serves a similar purpose, but additionally separates homologous chromosomes to produce haploid gametes for sexual reproduction. Both mitosis and meiosis rely on centromeres for the segregation of chromosomes. Centromeres are the specialized regions of the chromosomes that are attached to microtubules during their segregation. In this review, we describe the adaptations and layers of regulation that are required for centromere function during meiosis, and their role in meiosis-specific processes such as homolog-pairing and recombination. Since female meiotic divisions are asymmetric, meiotic centromeres are hypothesized to evolve quickly in order to favor their own transmission to the offspring, resulting in the rapid evolution of many centromeric proteins. We discuss this observation using the example of the histone variant CENP-A, which marks the centromere and is essential for centromere function. Changes in both the size and the sequence of the CENP-A N-terminal tail have led to additional functions of the protein, which are likely related to its roles during meiosis. We highlight the importance of CENP-A in the inheritance of centromere identity, which is dependent on the stabilization, recycling, or re-establishment of CENP-A-containing chromatin during meiosis.

CENP-A nucleosome-a chromatin-embedded pedestal for the centromere: lessons learned from structural biology.

The centromere is a chromosome locus that directs equal segregation of chromosomes during cell division. A nucleosome containing the histone H3 variant CENP-A epigenetically defines the centromere. Here, we summarize findings from recent structural biology studies, including several CryoEM structures, that contributed to elucidate specific features of the CENP-A nucleosome and molecular determinants of its interactions with CENP-C and CENP-N, the only two centromere proteins that directly bind to it. Based on those findings, we propose a role of the CENP-A nucleosome in the organization of centromeric chromatin beyond binding centromeric proteins.

Essentiality of CENP-A Depends on Its Binding Mode to HJURP.

CENP-A incorporation is critical for centromere specification and is mediated by the chaperone HJURP. The CENP-A-targeting domain (CATD) of CENP-A specifically binds to HJURP, and this binding is conserved. However, the binding interface of CENP-A-HJURP is yet to be understood. Here, we identify the critical residues for chicken CENP-A or HJURP. The A59Q mutation in the α1-helix of chicken CENP-A causes CENP-A mis-incorporation and subsequent cell death, whereas the corresponding mutation in human CENP-A does not. We also find that W53 of HJURP, which is a contact site of A59 in CENP-A, is also essential in chicken cells. Our comprehensive analyses reveal that the affinities of HJURP to CATD differ between chickens and humans. However, the introduction of two arginine residues to the chicken HJURP αA-helix suppresses CENP-A mis-incorporation in chicken cells expressing CENP-AA59Q. Our data explain the mechanisms and evolution of CENP-A essentiality by the CENP-A-HJURP interaction.

Guarding the Genome: CENP-A-Chromatin in Health and Cancer.

Faithful chromosome segregation is essential for the maintenance of genomic integrity and requires functional centromeres. Centromeres are epigenetically defined by the histone H3 variant, centromere protein A (CENP-A). Here we highlight current knowledge regarding CENP-A-containing chromatin structure, specification of centromere identity, regulation of CENP-A deposition and possible contribution to cancer formation and/or progression. CENP-A overexpression is common among many cancers and predicts poor prognosis. Overexpression of CENP-A increases rates of CENP-A deposition ectopically at sites of high histone turnover, occluding CCCTC-binding factor (CTCF) binding. Ectopic CENP-A deposition leads to mitotic defects, centromere dysfunction and chromosomal instability (CIN), a hallmark of cancer. CENP-A overexpression is often accompanied by overexpression of its chaperone Holliday Junction Recognition Protein (HJURP), leading to epigenetic addiction in which increased levels of HJURP and CENP-A become necessary to support rapidly dividing p53 deficient cancer cells. Alterations in CENP-A posttranslational modifications are also linked to chromosome segregation errors and CIN. Collectively, CENP-A is pivotal to genomic stability through centromere maintenance, perturbation of which can lead to tumorigenesis.

Centromere Dysfunction Compromises Mitotic Spindle Pole Integrity.

Centromeres and centrosomes are crucial mitotic players. Centromeres are unique chromosomal sites characterized by the presence of the histone H3-variant centromere protein A (CENP-A) [1]. CENP-A recruits the majority of centromere components, collectively named the constitutive centromere associated network (CCAN) [2]. The CCAN is necessary for kinetochore assembly, a multiprotein complex that attaches spindle microtubules (MTs) and is required for chromosome segregation [3]. In most animal cells, the dominant site for MT nucleation in mitosis are the centrosomes, which are composed of two centrioles, surrounded by a protein-rich matrix of electron-dense pericentriolar material (PCM) [4]. The PCM is the site of MT nucleation during mitosis [5]. Even if centromeres and centrosomes are connected via MTs in mitosis, it is not known whether defects in either one of the two structures have an impact on the function of the other. Here, using high-resolution microscopy combined with rapid removal of CENP-A in human cells, we found that perturbation of centromere function impacts mitotic spindle pole integrity. This includes release of MT minus-ends from the centrosome, leading to PCM dispersion and centriole mis-positioning at the spindle poles. Mechanistically, we show that these defects result from abnormal spindle MT dynamics due to defective kinetochore-MT attachments. Importantly, restoring mitotic spindle pole integrity following centromere inactivation lead to a decrease in the frequency of chromosome mis-segregation. Overall, our work identifies an unexpected relationship between centromeres and maintenance of the mitotic pole integrity necessary to ensure mitotic accuracy and thus to maintain genetic stability.

Cell cycle-dependent association of polo kinase Cdc5 with CENP-A contributes to faithful chromosome segregation in budding yeast.

Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle-dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle-regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle-regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.