Contribution of CENP-F to FOXM1-Mediated Discordant Centromere and Kinetochore Transcriptional Regulation.

Proper chromosome segregation is required to ensure chromosomal stability. The centromere (CEN) is a unique chromatin domain defined by CENP-A and is responsible for recruiting the kinetochore (KT) during mitosis, ultimately regulating microtubule spindle attachment and mitotic checkpoint function. Upregulation of many CEN/KT genes is commonly observed in cancer. Here, we show that although FOXM1 occupies promoters of many CEN/KT genes with MYBL2, FOXM1 overexpression alone is insufficient to drive the FOXM1-correlated transcriptional program. CENP-F is canonically an outer kinetochore component; however, it functions with FOXM1 to coregulate G2/M transcription and proper chromosome segregation. Loss of CENP-F results in altered chromatin accessibility at G2/M genes and reduced FOXM1-MBB complex formation. We show that coordinated CENP-FFOXM1 transcriptional regulation is a cancer-specific function. We observe a small subset of CEN/KT genes including CENP-C, that are not regulated by FOXM1. Upregulation of CENP-C in the context of CENP-A overexpression leads to increased chromosome missegregation and cell death suggesting that escape of CENP-C from FOXM1 regulation is a cancer survival mechanism. Together, we show that FOXM1 and CENP-F coordinately regulate G2/M genes, and this coordination is specific to a subset of genes to allow for maintenance of chromosome instability levels and subsequent cell survival.

DNAJC9 prevents CENP-A mislocalization and chromosomal instability by maintaining the fidelity of histone supply chains.

The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3-H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3-H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.

Cdc48 and its co-factor Ufd1 extract CENP-A from centromeric chromatin and can induce chromosome elimination in the fission yeast Schizosaccharomyces pombe.

CENP-A determines the identity of the centromere. Because the position and size of the centromere and its number per chromosome must be maintained, the distribution of CENP-A is strictly regulated. In this study, we have aimed to understand mechanisms to regulate the distribution of CENP-A (Cnp1SP) in fission yeast. A mutant of the ufd1+ gene (ufd1-73) encoding a cofactor of Cdc48 ATPase is sensitive to Cnp1 expressed at a high level and allows mislocalization of Cnp1. The level of Cnp1 in centromeric chromatin is increased in the ufd1-73 mutant even when Cnp1 is expressed at a normal level. A preexisting mutant of the cdc48+ gene (cdc48-353) phenocopies the ufd1-73 mutant. We have also shown that Cdc48 and Ufd1 proteins interact physically with centromeric chromatin. Finally, Cdc48 ATPase with Ufd1 artificially recruited to the centromere of a mini-chromosome (Ch16) induce a loss of Cnp1 from Ch16, leading to an increased rate of chromosome loss. It appears that Cdc48 ATPase, together with its cofactor Ufd1 remove excess Cnp1 from chromatin, likely in a direct manner. This mechanism may play a role in centromere disassembly, a process to eliminate Cnp1 to inactivate the kinetochore function during development, differentiation, and stress response.

HJURP is recruited to double-strand break sites and facilitates DNA repair by promoting chromatin reorganization.

HJURP is overexpressed in several cancer types and strongly correlates with patient survival. However, the mechanistic basis underlying the association of HJURP with cancer aggressiveness is not well understood. HJURP promotes the loading of the histone H3 variant, CENP-A, at the centromeric chromatin, epigenetically defining the centromeres and supporting proper chromosome segregation. In addition, HJURP is associated with DNA repair but its function in this process is still scarcely explored. Here, we demonstrate that HJURP is recruited to DSBs through a mechanism requiring chromatin PARylation and promotes epigenetic alterations that favor the execution of DNA repair. Incorporation of HJURP at DSBs promotes turnover of H3K9me3 and HP1, facilitating DNA damage signaling and DSB repair. Moreover, HJURP overexpression in glioma cell lines also affected global structure of heterochromatin independently of DNA damage induction, promoting genome-wide reorganization and assisting DNA damage response. HJURP overexpression therefore extensively alters DNA damage signaling and DSB repair, and also increases radioresistance of glioma cells. Importantly, HJURP expression levels in tumors are also associated with poor response of patients to radiation. Thus, our results enlarge the understanding of HJURP involvement in DNA repair and highlight it as a promising target for the development of adjuvant therapies that sensitize tumor cells to irradiation.

The cysteine-rich domain in CENP-A chaperone Scm3HJURP ensures centromere targeting and kinetochore integrity.

Centromeric chromatin plays a crucial role in kinetochore assembly and chromosome segregation. Centromeres are specified through the loading of the histone H3 variant CENP-A by the conserved chaperone Scm3/HJURP. The N-terminus of Scm3/HJURP interacts with CENP-A, while the C-terminus facilitates centromere localization by interacting with the Mis18 holocomplex via a small domain, called the Mis16-binding domain (Mis16-BD) in fission yeast. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) of unknown function. Here, we find that CYS binds zinc in vitro and is essential for the localization and function of fission yeast Scm3. Disrupting CYS by deletion or introduction of point mutations within its zinc-binding motif prevents Scm3 centromere localization and compromises kinetochore integrity. Interestingly, CYS alone can localize to the centromere, albeit weakly, but its targeting is greatly enhanced when combined with Mis16-BD. Expressing a truncated protein containing both Mis16-BD and CYS, but lacking the CENP-A binding domain, causes toxicity and is accompanied by considerable chromosome missegregation and kinetochore loss. These effects can be mitigated by mutating the CYS zinc-binding motif. Collectively, our findings establish the essential role of the cysteine-rich domain in fungal Scm3 proteins and provide valuable insights into the mechanism of Scm3 centromere targeting.

High expression of centromere protein A and its molecular mechanism and clinical significance in prostate cancer: A study based on data mining and immunohistochemistry.

The progression of prostate cancer (PCa) leads to poor prognosis. However, the molecular mechanism of PCa is still not completely clear. This study aimed to elucidate the important role of centromere protein A (CENPA) in PCa. Large numbers of bulk RNA sequencing (RNA-seq) data and in-house immunohistochemistry data were used in analysing the expression level of CENPA in PCa and metastatic PCa (MPCa). Single-cell RNA-seq data was used to explore the expression status of CENPA in different prostate subpopulations. Enrichment analysis was employed to detect the function of CENPA in PCa. Clinicopathological parameters analysis was utilised in analysing the clinical value of CENPA. The results showed that CENPA was upregulated in PCa (standardised mean difference [SMD] = 0.83, p = 0.001) and MPCa (SMD = 0.61, p = 0.029). CENPA was overexpressed in prostate cancer stem cells (CSCs) with androgen receptor (AR) negative compared to epithelial cells with AR positive. CENPA may influence the development of PCa through affecting cell cycle. Patients with nodal metastasis had higher expression level of CENPA. And patients with high CENPA expression had poor disease-free survival. Taken together, Overexpression of CENPA may influence the development of PCa by regulating cell cycle and promoting metastasis.

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. Despite its importance, the precise mechanism responsible for maintenance of centromere position remains obscure. Here, we report a mechanism to maintain centromere identity. We demonstrate that CENP-A interacts with EWSR1 (Ewing sarcoma breakpoint region 1) and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma). EWSR1 is required for maintaining CENP-A at the centromere in interphase cells. EWSR1 and EWSR1-FLI1 bind CENP-A through the SYGQ2 region within the prion-like domain, important for phase separation. EWSR1 binds to R-loops through its RNA-recognition motif in vitro. Both the domain and motif are required for maintaining CENP-A at the centromere. Therefore, we conclude that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA.

The histone H3/H4 chaperone CHAF1B prevents the mislocalization of CENP-A for chromosomal stability.

Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Among the top five candidates in the screen - the depletion of which showed increased nuclear YFP-CENP-A fluorescence - were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENP-A mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.

The ins and outs of CENP-A: Chromatin dynamics of the centromere-specific histone.

Centromeres are highly specialised chromosome domains defined by the presence of an epigenetic mark, the specific histone H3 variant called CENP-A (centromere protein A). They constitute the genomic regions on which kinetochores form and when defective cause segregation defects that can lead to aneuploidy and cancer. Here, we discuss how CENP-A is established and maintained to propagate centromere identity while subjected to dynamic chromatin remodelling during essential cellular processes like DNA repair, replication, and transcription. We highlight parallels and identify conserved mechanisms between different model organism with a particular focus on 1) the establishment of CENP-A at centromeres, 2) CENP-A maintenance during transcription and replication, and 3) the mechanisms that help preventing CENP-A localization at non-centromeric sites. We then give examples of how timely loading of new CENP-A to the centromere, maintenance of old CENP-A during S-phase and transcription, and removal of CENP-A at non-centromeric sites are coordinated and controlled by an intricate network of factors whose identity is slowly being unravelled.

CENP-A: A Histone H3 Variant with Key Roles in Centromere Architecture in Healthy and Diseased States.

Centromeres are key architectural components of chromosomes. Here, we examine their construction, maintenance, and functionality. Focusing on the mammalian centromere- specific histone H3 variant, CENP-A, we highlight its coevolution with both centromeric DNA and its chaperone, HJURP. We then consider CENP-A de novo deposition and the importance of centromeric DNA recently uncovered with the added value from new ultra-long-read sequencing. We next review how to ensure the maintenance of CENP-A at the centromere throughout the cell cycle. Finally, we discuss the impact of disrupting CENP-A regulation on cancer and cell fate.

GRANT Motif Regulates CENP-A Incorporation and Restricts RNA Polymerase II Accessibility at Centromere.

Precise chromosome segregation is essential for maintaining genomic stability, and its proper execution centers on the centromere, a chromosomal locus that mounts the kinetochore complex to mediate attachment of chromosomes to the spindle microtubules. The location of the centromere is epigenetically determined by a centromere-specific histone H3 variant, CENP-A. Many human cancers exhibit overexpression of CENP-A, which correlates with occurrence of aneuploidy in these malignancies. Centromeric targeting of CENP-A depends on its histone fold, but recent studies showed that the N-terminal tail domain (NTD) also plays essential roles. Here, we investigated implications of NTD in conferring aneuploidy formation when CENP-A is overexpressed in fission yeast. A series of mutant genes progressively lacking one amino acid of the NTD have been constructed for overexpression in wild-type cells using the intermediate strength nmt41 promoter. Constructs hosting disrupted GRANT (Genomic stability-Regulating site within CENP-A N-Terminus) motif in NTD results in growth retardation, aneuploidy, increased localization to the centromere, upregulated RNA polymerase II accessibility and transcriptional derepression of the repressive centromeric chromatin, suggesting that GRANT residues fine-tune centromeric CENP-A incorporation and restrict RNA polymerase II accessibility. This work highlighted the importance of CENP-A NTD, particularly the GRANT motif, in aneuploidy formation of overexpressed CENP-A in fission yeast.

HJURP inhibits proliferation of ovarian cancer cells by regulating CENP-A/CENP-N.

OBJECTIVE: High expression of Holliday Junction-Recognizing Protein (HJURP) has been shown to be a marker of poor prognosis in ovarian cancer. The objective of this study was to investigate the molecular mechanisms of HJURP in ovarian cancer (OC) progression. PROCEDURES: Gene Expression Profiling Interactive Analysis (GEPIA) was used to analyze the gene expression profile. Real-time quantitative PCR (qRT-PCR) was used to detect the expression level and correlation of HJURP and centromere protein-A (CENP-A) in OC tissues and cell lines. CCK-8 assay was used to detect cell proliferation. The expression level of apoptosis-related proteins and cell cycle-related proteins were detected by western blotting. Cell cycle and mitochondrial content were determined by flow cytometry. RESULTS: The results showed that HJURP was up-regulated in OC tissues and cell lines, while the cell proliferation was inhibited after transfecting by si-HJURP. Knockdown of HJURP promoted cell apoptosis. Meanwhile, low-expression of HJURP could down-regulate cell replication cycle-related proteins (Cyclin-dependent kinase 2, cyclinD1 and Cyclin-dependent kinase 4) and make cell replication stay in the S phase. Moreover, further studies showed that HJURP was positively correlated with CENP-A in OC tissues. Finally, the rescue experiment further verified that HJURP targeted regulation of CENP-A in OC. CONCLUSIONS: The study indicated that HJURP plays a significant role in OC and could target CENP-A to regulate OC cell growth. These findings provide a clue to the diagnosis and treatment of OC.

Oncogenic lncRNAs alter epigenetic memory at a fragile chromosomal site in human cancer cells.

Chromosome instability is a critical event in cancer progression. Histone H3 variant CENP-A plays a fundamental role in defining centromere identity, structure, and function but is innately overexpressed in several types of solid cancers. In the cancer background, excess CENP-A is deposited ectopically on chromosome arms, including 8q24/cMYC locus, by invading transcription-coupled H3.3 chaperone pathways. Up-regulation of lncRNAs in many cancers correlates with poor prognosis and recurrence in patients. We report that transcription of 8q24-derived oncogenic lncRNAs plays an unanticipated role in altering the 8q24 chromatin landscape by H3.3 chaperone-mediated deposition of CENP-A-associated complexes. Furthermore, a transgene cassette carrying specific 8q24-derived lncRNA integrated into a naïve chromosome locus recruits CENP-A to the new location in a cis-acting manner. These data provide a plausible mechanistic link between locus-specific oncogenic lncRNAs, aberrant local chromatin structure, and the generation of new epigenetic memory at a fragile site in human cancer cells.

Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-AK124ub.

Functional tags are ubiquitous in cell biology, and for studies of one chromosomal locus, the centromere, tags have been remarkably useful. The centromere directs chromosome inheritance at cell division. The location of the centromere is defined by a histone H3 variant, CENP-A. The regulation of the chromatin assembly pathway essential for centromere inheritance and function includes posttranslational modification (PTM) of key components, including CENP-A itself. Others have recently called into question the use of functional tags, with the claim that at least two widely used tags obscured the essentiality of one particular PTM, CENP-AK124 ubiquitination (ub). Here, we employ three independent gene replacement strategies that eliminate large, lysine-containing tags to interrogate these claims. Using these approaches, we find no evidence to support an essential function of CENP-AK124ub. Our general methodology will be useful to validate discoveries permitted by powerful functional tagging schemes at the centromere and other cellular locations.

Prognostic model of lung adenocarcinoma constructed by the CENPA complex genes is closely related to immune infiltration.

BACKGROUND: Lung adenocarcinoma (LUAD) is still one of the primary malignant diseases leading to higher mortality worldwide. It has been previously reported that multiple genes in the CENPA-nucleosome associated complex (NAC) complex in lung cancer can be used as prognostic markers; however, there is lack of comprehensive research on the CENPA-NAC complex. METHODS: The hub genes of lung cancer were obtained by analyzing multiple gene expression omnibus (GEO) lung cancer datasets. The key genes of the CENPA-NAC complex in the evolution of LUAD were identified according to lung cancer data obtained from The Cancer Genome Atlas (TCGA) database, and the key genes were constructed as a survival prognostic model. The relationship between the model and immune cell infiltration was studied by the Tumor Immune Estimation Resource (TIMER) and single-sample gene set enrichment analysis (ssGSEA) studies.Droplet Digital polymerase chain reaction (ddPCR) was used to verify the effectiveness of the prognostic model to predict survival using clinical samples. RESULTS: A comprehensive study showed that CENPA, CENPH, CENPM, CENPN and CENPU were key genes in the development and evolution of LUAD. The constructed survival prognosis model was an independent risk factor for LUAD and can be used to assess the survival of LUAD patients. The risk score was closely related to the infiltration of multiple immune cells. The independent cohorts GSE31210 and GSE50081 further confirmed the validity of the prognostic model, and finally, the model was validated with clinical samples. CONCLUSIONS: In conclusion, the results of the present study showed that CENPA, CENPH, CENPM, CENPN, and CENPU are a group of potential prognostic markers in LUAD. The constructed model has been confirmed to be applicable in the clinical setting in evaluating the survival of patients with LUAD, and providing more evidence on immunotherapy for LUAD.

Genomic and Epigenetic Foundations of Neocentromere Formation.

Centromeres are essential to genome inheritance, serving as the site of kinetochore assembly and coordinating chromosome segregation during cell division. Abnormal centromere function is associated with birth defects, infertility, and cancer. Normally, centromeres are assembled and maintained at the same chromosomal location. However, ectopic centromeres form spontaneously at new genomic locations and contribute to genome instability and developmental defects as well as to acquired and congenital human disease. Studies in model organisms have suggested that certain regions of the genome, including pericentromeres, heterochromatin, and regions of open chromatin or active transcription, support neocentromere activation. However, there is no universal mechanism that explains neocentromere formation. This review focuses on recent technological and intellectual advances in neocentromere research and proposes future areas of study. Understanding neocentromere biology will provide a better perspective on chromosome and genome organization and functional context for information generated from the Human Genome Project, ENCODE, and other large genomics consortia.

Recent insights into mechanisms preventing ectopic centromere formation.

The centromere is a specialized chromosomal structure essential for chromosome segregation. Centromere dysfunction leads to chromosome segregation errors and genome instability. In most eukaryotes, centromere identity is specified epigenetically by CENP-A, a centromere-specific histone H3 variant. CENP-A replaces histone H3 in centromeres, and nucleates the assembly of the kinetochore complex. Mislocalization of CENP-A to non-centromeric regions causes ectopic assembly of CENP-A chromatin, which has a devastating impact on chromosome segregation and has been linked to a variety of human cancers. How non-centromeric regions are protected from CENP-A misincorporation in normal cells is largely unexplored. Here, we review the most recent advances on the mechanisms underlying the prevention of ectopic centromere formation, and discuss the implications in human disease.

Centromeres Transcription and Transcripts for Better and for Worse.

Centromeres are chromosomal regions that are essential for the faithful transmission of genetic material through each cell division. They represent the chromosomal platform on which assembles a protein complex, the kinetochore, which mediates attachment to the mitotic spindle. In most organisms, centromeres assemble on large arrays of tandem satellite repeats, although their DNA sequences and organization are highly divergent among species. It has become evident that centromeres are not defined by underlying DNA sequences, but are instead epigenetically defined by the deposition of the centromere-specific histone H3 variant, CENP-A. In addition, and although long regarded as silent chromosomal loci, centromeres are in fact transcriptionally competent in most species, yet at low levels in normal somatic cells, but where the resulting transcripts participate in centromere architecture, identity, and function. In this chapter, we discuss the various roles proposed for centromere transcription and their transcripts, and the potential molecular mechanisms involved. We also discuss pathological cases in which unscheduled transcription of centromeric repeats or aberrant accumulation of their transcripts are pathological signatures of chromosomal instability diseases. In sum, tight regulation of centromeric satellite repeats transcription is critical for healthy development and tissue homeostasis, and thus prevents the emergence of disease states.

Reduced gene dosage of histone H4 prevents CENP-A mislocalization and chromosomal instability in Saccharomyces cerevisiae.

Mislocalization of the centromeric histone H3 variant (Cse4 in budding yeast, CID in flies, CENP-A in humans) to noncentromeric regions contributes to chromosomal instability (CIN) in yeast, fly, and human cells. Overexpression and mislocalization of CENP-A have been observed in cancers, however, the mechanisms that facilitate the mislocalization of overexpressed CENP-A have not been fully explored. Defects in proteolysis of overexpressed Cse4 (GALCSE4) lead to its mislocalization and synthetic dosage lethality (SDL) in mutants for E3 ubiquitin ligases (Psh1, Slx5, SCFMet30, and SCFCdc4), Doa1, Hir2, and Cdc7. In contrast, defects in sumoylation of overexpressed cse4K215/216/A/R prevent its mislocalization and do not cause SDL in a psh1Δ strain. Here, we used a genome-wide screen to identify factors that facilitate the mislocalization of overexpressed Cse4 by characterizing suppressors of the psh1Δ GALCSE4 SDL. Deletions of histone H4 alleles (HHF1 or HHF2), which were among the most prominent suppressors, also suppress slx5Δ, cdc4-1, doa1Δ, hir2Δ, and cdc7-4 GALCSE4 SDL. Reduced dosage of H4 leads to defects in sumoylation and reduced mislocalization of overexpressed Cse4, which contributes to suppression of CIN when Cse4 is overexpressed. We determined that the hhf1-20, cse4-102, and cse4-111 mutants, which are defective in the Cse4-H4 interaction, also exhibit reduced sumoylation of Cse4 and do not display psh1Δ GALCSE4 SDL. In summary, we have identified genes that contribute to the mislocalization of overexpressed Cse4 and defined a role for the gene dosage of H4 in facilitating Cse4 sumoylation and mislocalization to noncentromeric regions, leading to CIN when Cse4 is overexpressed.