Pachytene karyotypes of 17 species of birds

Background: To date less than 10% of bird species have been karyotyped. They are rather conservative with diploid chromosome numbers about 78-80 in most species examined. Immunostaining of meiotic chromosomes at pachytene stage enables more precise estimates of the number, morphology and variability of macro- and microchromosomes than conventional analysis of mitotic metaphase chromosomes does. Analysis of pachytene chromosomes led to discovery of germline-restricted chromosome (GRC) that was present in germline cells and absent in somatic cells in all 16 species of passerine birds examined. GRC has not been found in any non-passerine bird. Results: In this study, using immunolocalization of SYCP3, the main protein of the lateral elements of the synaptonemal complex (SC) and centromere proteins we examined male pachytene karyotypes of sixteen passerine species and one outgroup species the Common cuckoo Cuculus canorus and provided their idiograms and precise estimates of their diploid chromosome numbers and the numbers of chromosome arms. We provided the first description of the karyotypes of three species, corrected the published data on the karyotypes of ten species and confirmed them for four species. The pachytene cells of the Gouldian finch, Brambling and Common linnet contained heteromorphic bivalents indicating heterozygosity for inversions or centromere shifts. The European pied flycatcher, Gouldian finch and Domestic canary have extended centromeres in several macro- and microchromosomes. GRCs of various sizes and shapes were detected in all passerine species examined. Their chromatin was heavily labeled by anticentromere antibodies. The lateral elements of the GRC SC varied in their size from the largest to the smallest element of the pachytene karyotype. They also varied in shape from continuous to fragmented. Conclusions: All songbirds examined, except the Eurasian skylark, have highly conservative karyotypes, 2n=80-82+GRC with seven pairs of macrochromosomes and 33-34 pairs of microchromosomes. The interspecies differences concern the sizes of the macrochromosomes, morphology of the microchromosomes and sizes of the centromeres. GRC is present in all songbird species examined, varying in size, morphology and SC structure even between closely related species. This indicates its fast evolution.

High mRNA Expression of CENPL and Its Significance in Prognosis of Hepatocellular Carcinoma Patients

Centromere proteins (CENPs) are the main constituent proteins of kinetochore, which are essential for cell division. In recent years, several studies have revealed that several CENPs were aberrantly expressed in hepatocellular carcinoma (HCC). However, numerous centromere proteins have not been studied in HCC. In this study, we used databases of Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), the Kaplan-Meier Plotter, cBioPortal, the Human Protein Atlas (HPA), and TIMER (Tumor Immune Estimation Resource) and immunohistochemical staining of clinical specimens to investigate the expression of 15 major centromere proteins in HCC to evaluate their potential prognostic value. We found that the mRNA levels of 4 out of 15 centromere proteins (CENPL, CENPQ, CENPR, and CENPU) were significantly higher in HCC than in normal tissues, and their mRNA levels were associated with the tumor stages ( p values < 0.01). Patients with higher mRNA levels of CENPL had poorer overall survival, progression-free survival, relapse-free survival, and disease-specific survival ( p values < 0.05). Furthermore, the higher levels of CENPL mRNA were associated with worse overall survival in males without hepatitis virus infection ( p values < 0.05). The protein expression level of CENPL in human HCC tissue was higher than that in normal liver tissue. In addition, the expression of CENPL was positively correlated with the levels of the tumor-infiltrating lymphocytes. The results suggest that the high mRNA expression of CENPL may be a potential predictor of prognosis in HCC patients.

The protective role of MC1R in chromosome stability and centromeric integrity in melanocytes

Variants in the melanocortin-1 receptor (MC1R) gene, encoding a trimeric G-protein-coupled receptor and activated by α-melanocyte-stimulating hormone (α-MSH), are frequently associated with red or blonde hair, fair skin, freckling, and skin sensitivity to ultraviolet (UV) light. Several red hair color variants of MC1R are also associated with increased melanoma risk. MC1R variants affect melanoma risk independent of phenotype. Here, we demonstrated that MC1R is a critical factor in chromosome stability and centromere integrity in melanocytes. α-MSH/MC1R stimulation prevents melanocytes from UV radiation-induced damage of chromosome stability and centromere integrity. Mechanistic studies indicated that α-MSH/MC1R-controlled chromosome stability and centromeric integrity are mediated by microphthalmia-associated transcription factor (Mitf), a transcript factor needed for the α-MSH/MC1R signaling and a regulator in melanocyte development, viability, and pigment production. Mitf directly interacts with centromere proteins A in melanocytes. Given the connection among MC1R variants, red hair/fair skin phenotype, and melanoma development, these studies will help answer a question with clinical relevance “why red-haired individuals are so prone to developing melanoma”, and will lead to the identification of novel preventive and therapeutic strategies for melanomas, especially those with redheads.

Centromere drive and suppression by parallel pathways for recruiting microtubule destabilizers

SummarySelfish centromere DNA sequences bias their transmission to the egg in female meiosis. Evolutionary theory suggests that centromere proteins evolve to suppress costs of this “centromere drive”. In hybrid mouse models with genetically different maternal and paternal centromeres, selfish centromere DNA exploits a kinetochore pathway to recruit microtubule-destabilizing proteins that act as drive effectors. We show that such functional differences are suppressed by a parallel pathway for effector recruitment by heterochromatin, which is similar between centromeres in this system. Disrupting heterochromatin by CENP-B deletion amplifies functional differences between centromeres, whereas disrupting the kinetochore pathway with a divergent allele of CENP-C reduces the differences. Molecular evolution analyses using newly sequenced Murinae genomes identify adaptive evolution in proteins in both pathways. We propose that centromere proteins have recurrently evolved to minimize the kinetochore pathway, which is exploited by selfish DNA, relative to the heterochromatin pathway that equalizes centromeres, while maintaining essential functions.

Anti-centromere antibodies target centromere–kinetochore macrocomplex: a comprehensive autoantigen profiling

ObjectivesAnti-centromere antibodies (ACAs) are detected in patients with various autoimmune diseases such as Sjögren’s syndrome (SS), systemic sclerosis (SSc) and primary biliary cholangitis (PBC). However, the targeted antigens of ACAs are not fully elucidated despite the accumulating understanding of the molecular structure of the centromere. The aim of this study was to comprehensively reveal the autoantigenicity of centromere proteins.MethodsA centromere antigen library including 16 principal subcomplexes composed of 41 centromere proteins was constructed. Centromere protein/complex binding beads were used to detect serum ACAs in patients with SS, SSc and PBC. ACA-secreting cells in salivary glands obtained from patients with SS were detected with green fluorescent protein-fusion centromere antigens and semiquantified with confocal microscopy.ResultsA total of 241 individuals with SS, SSc or PBC and healthy controls were recruited for serum ACA profiling. A broad spectrum of serum autoantibodies was observed, and some of them had comparative frequency as anti-CENP-B antibody, which is the known major ACA. The prevalence of each antibody was shared across the three diseases. Immunostaining of SS salivary glands showed the accumulation of antibody-secreting cells (ASCs) specific for kinetochore, which is a part of the centromere, whereas little reactivity against CENP-B was seen.ConclusionsWe demonstrated that serum autoantibodies target the centromere–kinetochore macrocomplex in patients with SS, SSc and PBC. The specificity of ASCs in SS salivary glands suggests kinetochore complex-driven autoantibody selection, providing insight into the underlying mechanism of ACA acquisition.

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.

Bioinformatics analysis reveals Centromere protein K can serve as potential prognostic biomarker and therapeutic target for non-small cell lung cancer

Background: Non-small cell lung carcinoma (NSCLC) accounts for 80% of all lung cancers cases, which had been a leading cause of morbidity and mortality worldwide. Previous studies demonstrated that centromere proteins were dysregulated and involved in regulating the tumorigenesis and development of human cancers. However, the roles of centromere protein family members in NSCLC remained to be further elucidated Objective: The present study aimed to explore the roles of centromere protein family members in NSCLC. Method: GEPIA (http://gepia.cancer-pku.cn/) was used to analyze the target's expression between normal and human cancers. We explored the prognostic value of centromere proteins in NSCLC using the Kaplan–Meier plotter (http://kmplot.com). The protein-protein interaction among centromere proteins were determined using GeneMANIA (http://www.genemania.org). TISIDB (http://cis.hku.hk/TISIDB) database was used to detect the relationship between centromere proteins expression and clinical stages, lymphocytes, immunomodulators and chemokines in NSCLC. The DAVID database (https://david.ncifcrf.gov) was used to detect potential roles of CENPK using its co-expressing genes Results: The present study for the first time showed that centromere protein family members including CENPA, CENPF, CENPH, CENPI, CENPK, CENPM, CENPN, CENPO, CENPQ, CENPU were dysregulated and correlated to the poor prognosis of patients with LUAD. CENPK showed the greatest correlation with the prognosis of patients with NSCLC. We found that CENPK was significantly highly expressed in LUAD samples and overexpression of CENPK was remarkably correlated to the shorter OS and DFS on patients with different stage NSCLC. Of note, this study for the first time showed that CENPK was significantly correlated to the lymphocytes and immunomodulators using the TISIDB database Conclusion: Therefore, CENPK may prove to be a novel biomarker for the diagnosis of patients with NSCLC

THU0045 IDENTIFICATION OF NOVEL CENTROMERE AUTOANTIGENS IN SJÖGREN’S SYNDROME, SYSTEMIC SCLEROSIS AND PRIMARY BILIARY CHOLANGITIS.

Background:Anti-centromere antibodies (ACA) are detected in the serum of patients with various autoimmune diseases including Sjögren’s syndrome (SjS), systemic sclerosis (SSc) and primary biliary cholangitis (PBC). ACA positivity is correlated with clinical manifestations such as Raynaud’s phenomenon and sclerodactyly and these features are commonly seen across diseases. Although CENPB is thought to be the major antigen against ACA, autoimmune features of other centromere proteins have not been fully evaluated.Objectives:The aim of this study is to elucidate centromere autoantigens comprehensively and clarify their association with pathogenesis of SjS, SSc and PBC.Methods:A centromere protein library was created by cloning 6 single proteins and 10 complexes consisting of 35 proteins belonging to human centromere region. The centromere antigens were immobilized on beads and incubated in the serum of patients with SjS (n = 86), SSc (n = 35), PBC (n = 10), patients with two or more diseases above (n = 44), and healthy volunteers (n = 68). Autoantibodies to each centromere protein were analyzed by flow cytometry.Results:Patients had a wide variety of antibodies against most of centromere antigens including 4 newly identified autoantigens. The hierarchical clustering of each antigen distinguished 2 antigen clusters. The reactivity of autoantibodies against a centromere protein of one cluster was mutually correlated regardless of disease types, indicating that these proteins/protein complexes might be the target of ACA. In addition, our method enabled us to detect sera reacted against multiple centromere antigens in some of the ACA-negative patients with existing methods.Conclusion:We identified 4 novel centromere autoantigens and our data suggested that the main target of ACA was the protein complex rather than a single specific antigen in SjS, SSc and PBC patients. Using the combination of centromere proteins may be useful to detect ACA with higher sensitivity.References:[1]Fritzler MJ, Rattner JB, Luft LM, Edworthy SM, Casiano CA, Peebles C, Mahler M. Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F. Autoimmun Rev. 2011;10:194-200.Disclosure of Interests:Nobuhiko Kajio: None declared, Masaru Takeshita: None declared, Katsuya Suzuki: None declared, Tsutomu Takeuchi Grant/research support from: Eisai Co., Ltd, Astellas Pharma Inc., AbbVie GK, Asahi Kasei Pharma Corporation, Nippon Kayaku Co., Ltd, Takeda Pharmaceutical Company Ltd, UCB Pharma, Shionogi & Co., Ltd., Mitsubishi-Tanabe Pharma Corp., Daiichi Sankyo Co., Ltd., Chugai Pharmaceutical Co. Ltd., Consultant of: Chugai Pharmaceutical Co Ltd, Astellas Pharma Inc., Eli Lilly Japan KK, Speakers bureau: AbbVie GK, Eisai Co., Ltd, Mitsubishi-Tanabe Pharma Corporation, Chugai Pharmaceutical Co Ltd, Bristol-Myers Squibb Company, AYUMI Pharmaceutical Corp., Eisai Co., Ltd, Daiichi Sankyo Co., Ltd., Gilead Sciences, Inc., Novartis Pharma K.K., Pfizer Japan Inc., Sanofi K.K., Dainippon Sumitomo Co., Ltd.

Alpha-satellite RNA transcripts are repressed by centromere-nucleolus associations

Centromeres play a fundamental role in chromosome segregation. Although originally thought to be silent chromosomal regions, centromeres are actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA smFISH foci fluctuate in their levels over the cell cycle and do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Our results demonstrate that alpha-satellite expression occurs through RNA Polymerase II-dependent transcription, but does not require centromere proteins and other cell division components. Instead, our work implicates centromere-nucleolar associations as the major factor regulating alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels, explaining variations in alpha-satellite RNA between cell lines. In addition, alpha-satellite transcript levels increase substantially when the nucleolus is disrupted. Together, our results are inconsistent with a direct, physical role for alpha-satellite transcripts in cell division processes, and instead support a role for ongoing transcription in promoting centromere chromatin dynamics. The control of alpha-satellite transcription by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A–CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A–binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A–CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.