Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization

Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. The organization of the interface between the kinetochore and the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, remains incompletely understood. A 16-subunit complex, the constitutive centromere associated network (CCAN), bridges CENP-A to the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has also important implications for the mechanism of specific recognition of the CENP-A nucleosome. A supported model depicts the interaction as fuzzy and identifies the disordered CCAN subunit CENP-C as only determinant of specificity. A more speculative model identifies both CENP-C and CENP-N as specificity determinants, but implies CENP-A may be in a hemisome rather than in a classical octamer.

Computer-aided molecular modeling and structural analysis of the human centromere protein-HIKM complex

Protein-peptide and protein-protein interactions play an essential role in different functional and structural cellular organizational aspects. While X-ray crystallography generates the most complete structural characterization, most biological interactions exist in biomolecular complexes that are neither compliant nor responsive to direct experimental analysis. The development of computational docking approaches is therefore necessary. This starts from component protein structures to the prediction of their complexes, preferentially with precision close to complex structures generated by X-ray crystallography. To guarantee faithful chromosomal segregation, there must be a proper assembling of the kinetochore (a protein complex with multiple subunits) at the centromere during the process of cell division. As an important member of the inner kinetochore, defects in any of the subunits making up the CENP-HIKM complex leads to kinetochore dysfunction and an eventual chromosomal mis-segregation and cell death. Previous studies in an attempt to understand the assembly and mechanism devised by the CENP-HIKM in promoting functionality of the kinetochore, have reconstituted the protein complex from different organisms including fungi and yeast. Here, we present a detailed computational model of the physical interactions that exist between each component of the human CENP-HIKM, while validating each modeled structure using orthologs with existing crystal structures from the protein data bank. Results from this study substantiates the existing hypothesis that the human CENP-HIK complex share a similar architecture with its fungal and yeast orthologs, and likewise validates the binding mode of CENP-M to the C-terminus of the human CENP-I based on existing experimental reports.

Immunization with CENP-C Causes Aberrant Chromosome Segregation during Oocyte Meiosis in Mice

Anticentromere antibodies (ACA) were associated with lower oocyte maturation rates and cleavage rates, while the mechanism was not clear. Aims of this study were to examine whether active immunization with centromere protein C could elicit the CENP-C autoantibody in mice and the impacts of the CENP-C autoantibody on oocyte meiosis. Mice were divided into two groups, one was the experimental group immunized with human centromere protein C and Freund’s adjuvant (CFA), and the other was the control group injected with CFA only. Serum and oocytes of BALB/c mice immunized with human centromere protein C (CENP-C) in complete Freund’s adjuvant (CFA) or injected with only CFA were studied for the development of the CENP-C antibody. Rates of germinal vesicle breakdown (GVBD), first polar body (Pb1) extrusion, abnormal spindle morphology, and chromosome misalignment were compared between the experimental group and the control group. The CENP-C antibody was only observed in serum and oocytes of mice immunized with the centromere protein C antigen. The first polar body (Pb1) extrusion rate was lower in the experimental group ( P < 0.01 ). A higher percentage of spindle defects and chromosome congression failure were also detected in the experimental group (spindle defects: 64.67 ± 1.16 % vs. 9.27 ± 2.28 % control; chromosome misalignment: 50.80 ± 2.40 % vs. 8.30 ± 1.16 % control; P < 0.01 for both). Oocyte meiosis was severely impaired by the CENP-C antibody, which may be the main mechanism of adverse reproductive outcomes for ACA-positive women who have no clinical symptoms of any autoimmune diseases.

Knockdown of CENPF Inhibits the Progression of Lung Adenocarcinoma Mediated by ERβ2/5 Pathway

The signal transduction pathways of estrogen receptors (ER) mainly includes gene pathway and non-gene pathway. Studies have shown that the gene pathway of ER is related with the expression of nuclear proteins, and this is the key issue for our current research. With the GEO database analysis, Human centromere protein F (CENPF) is highly expressed in adenocarcinoma of lung (LUAD), and the co-expression of CENPF and ER&beta; was found in the nucleus of LUAD cells. Meanwhile, CENPF and ER&beta;2/5 were related with T stage and poor prognosis (P&lt;0.05). Knockdown of CENPF gene significantly inhibited the biological effects of LUAD cells, the tumor growth of mice and the expression of ER&beta;2/5 (P&lt;0.05). Further, group experiments showed that knockdown CENPF inhibits biological effects of LUAD cells mediated by ER&beta; pathway. All the results indicated that both CENPF and ER&beta;2/5 play important roles in the progression of LUAD, and knockdown of CENPF can inhibit the progression of LUAD by inhibiting the expression of ER2/5. Thus, the development of inhibitors against ER&beta;2/5 subtype and CENPF remained more effective in improving the therapeutic effect of LUAD.

Long-read Data Revealed Structural Diversity in Human Centromere Sequences

ABSTRACTCentromeres invariably serve as the loci of kinetochore assembly in all eukaryotic cells, but their underlying DNA sequences evolve rapidly. Human centromeres are characterized by their extremely repetitive structures, i.e., higher-order repeats, rendering the region one of the most difficult parts of the genome to assess. Consequently, our understanding of centromere sequence variations across human populations is limited. Here, we analyzed chromosomes 11, 17, and X using long sequencing reads of two European and two Asian genomes, and our results show that human centromere sequences exhibit substantial structural diversity, harboring many novel variant higher-order repeats specific to individuals, while frequent single-nucleotide variants are largely conserved. Our findings add another dimension to our knowledge of centromeres, challenging the notion of stable human centromeres. The discovery of such diversity prompts further deep sequencing of human populations to understand the true nature of sequence evolution in human centromeres.

Next-Generation Sequencing Enables Spatiotemporal Resolution of Human Centromere Replication Timing

Centromeres serve a critical function in preserving genome integrity across sequential cell divisions, by mediating symmetric chromosome segregation. The repetitive, heterochromatic nature of centromeres is thought to be inhibitory to DNA replication, but has also led to their underrepresentation in human reference genome assemblies. Consequently, centromeres have been excluded from genomic replication timing analyses, leaving their time of replication unresolved. However, the most recent human reference genome, hg38, included models of centromere sequences. To establish the experimental requirements for achieving replication timing profiles for centromeres, we sequenced G1- and S-phase cells from five human cell lines, and aligned the sequence reads to hg38. We were able to infer DNA replication timing profiles for the centromeres in each of the five cell lines, which showed that centromere replication occurs in mid-to-late S phase. Furthermore, we found that replication timing was more variable between cell lines in the centromere regions than expected, given the distribution of variation in replication timing genome-wide. These results suggest the potential of these, and future, sequence models to enable high-resolution studies of replication in centromeres and other heterochromatic regions.

Repetitive Fragile Sites: Centromere Satellite DNA As a Source of Genome Instability in Human Diseases

Maintenance of an intact genome is essential for cellular and organismal homeostasis. The centromere is a specialized chromosomal locus required for faithful genome inheritance at each round of cell division. Human centromeres are composed of large tandem arrays of repetitive alpha-satellite DNA, which are often sites of aberrant rearrangements that may lead to chromosome fusions and genetic abnormalities. While the centromere has an essential role in chromosome segregation during mitosis, the long and repetitive nature of the highly identical repeats has greatly hindered in-depth genetic studies, and complete annotation of all human centromeres is still lacking. Here, we review our current understanding of human centromere genetics and epigenetics as well as recent investigations into the role of centromere DNA in disease, with a special focus on cancer, aging, and human immunodeficiency–centromeric instability–facial anomalies (ICF) syndrome. We also highlight the causes and consequences of genomic instability at these large repetitive arrays and describe the possible sources of centromere fragility. The novel connection between alpha-satellite DNA instability and human pathological conditions emphasizes the importance of obtaining a truly complete human genome assembly and accelerating our understanding of centromere repeats’ role in physiology and beyond.

DNA replication-mediated error correction of ectopic CENP-A deposition maintains centromere identity

Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A is shown in early G1 to be assembled into nucleosomes within megabase, repetitive α-satellite DNAs at each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. Here we identify that DNA replication acts as an error correction mechanism to sustain centromere identity through the removal of the sites of CENP-A loading on the chromosome arms, while maintaining centromere-bound CENP-A with the same DNA sequence preferences as in its initial loading.