ABSTRACT
The centromere is an essential chromatin domain required for kinetochore recruitment and chromosome segregation in eukaryotes. To perform this role, centro-chromatin adopts a unique structure that provides access to kinetochore proteins and maintains stability under tension during mitosis. This is achieved by the presence of nucleosomes containing the H3 variant CENP-A, which also acts as the epigenetic mark defining the centromere. In this review, we discuss the role of CENP-A on the structure and dynamics of centromeric chromatin. We further discuss the impact of the CENP-A binding proteins CENP-C, CENP-N, and CENP-B on modulating centro-chromatin structure. Based on these findings we provide an overview of the higher order structure of the centromere.
Subject(s)
Centromere Protein A/chemistry , Centromere Protein B/chemistry , Centromere/ultrastructure , Chromatin/ultrastructure , Chromosomal Proteins, Non-Histone/chemistry , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Centromere/chemistry , Centromere/metabolism , Centromere Protein A/genetics , Centromere Protein A/metabolism , Centromere Protein B/genetics , Centromere Protein B/metabolism , Chromatin/chemistry , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epigenesis, Genetic , Humans , Mitosis , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Nucleic Acid Conformation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , CohesinsABSTRACT
We combined classical salt fractionation with chromatin immunoprecipitation to recover human centromeric chromatin under native conditions. We found that >85% of the total centromeric chromatin is insoluble under conditions typically used for native chromatin extraction. To map both soluble and insoluble chromatin in situ, we combined CUT&RUN (cleavage under targets and release using nuclease), a targeted nuclease method, with salt fractionation. Using this approach, we observed unexpected structural and conformational variations of centromere protein A (CENP-A)-containing complexes on different α-satellite dimeric units within highly homogenous arrays. Our results suggest that slight α-satellite sequence differences control the structure and occupancy of the associated centromeric chromatin complex.
Subject(s)
Centromere Protein A/chemistry , Centromere/chemistry , Chromatin/chemistry , Centromere Protein A/isolation & purification , Centromere Protein A/metabolism , Centromere Protein B/chemistry , Centromere Protein B/metabolism , Chemical Fractionation , Chromatin/isolation & purification , Chromatin Immunoprecipitation , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , DNA, Satellite/chemistry , Humans , K562 Cells , SolubilityABSTRACT
The centromere is a chromatin region that is required for accurate inheritance of eukaryotic chromosomes during cell divisions. Among the different centromere-associated proteins (CENP) identified, CENP-B has been independently domesticated from a pogo-like transposase twice: Once in mammals and once in fission yeast. Recently, a third independent domestication restricted to holocentric lepidoptera has been described. In this work, we take advantage of the high-quality genome sequence and the wealth of functional information available for Drosophila melanogaster to further investigate the possibility of additional independent domestications of pogo-like transposases into host CENP-B related proteins. Our results showed that CENP-B related genes are not restricted to holocentric insects. Furthermore, we showed that at least three independent domestications of pogo-like transposases have occurred in metazoans. Our results highlight the importance of transposable elements as raw material for the recurrent evolution of important cellular functions.
Subject(s)
Centromere Protein B/genetics , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Evolution, Molecular , Transposases/genetics , Animals , Centromere Protein B/chemistry , DNA Transposable Elements , DNA-Binding Proteins/chemistry , Drosophila Proteins/chemistry , Drosophila melanogaster/chemistry , Genes, Insect , Humans , Models, Molecular , Phylogeny , Protein Structure, Tertiary , Transposases/chemistryABSTRACT
CENP-B is a highly conserved protein that facilitates the assembly of specific centromere structures both in interphase nuclei and on mitotic chromosomes. INMAP is a conserved protein that localizes at nucleus in interphase cells and at mitotic apparatus in mitotic cells. Our previous results showed that INMAP over-expression leads to spindle defects, mitotic arrest and formation of polycentrosomal and multinuclear cells, indicating that INMAP may modulate the function of (a) key protein(s) in mitotic apparatus. In this study, we demonstrate that INMAP interacts with CENP-B and promotes cleavage of the N-terminal DNA binding domain from CENP-B. The cleaved CENP-B cannot associate with centromeres and thus lose its centromere-related functions. Consistent with these results, CENP-B in INMAP knockdown cells becomes more diffused around kinetochores. Although INMAP knockdown cells do not exhibit gross defects in mitotic spindle formation, these cells go through mitosis, especially prophase and metaphase, with different relative timing, indicating subtle abnormality. These results identify INMAP as a model regulator of CENP-B and support the notion that INMAP regulates mitosis through modulating CENP-B-mediated centromere organization.
Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Centromere Protein B/metabolism , Centromere/metabolism , Gene Knockdown Techniques , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Active Transport, Cell Nucleus , Cell Cycle Proteins/deficiency , Centromere Protein B/chemistry , DNA/metabolism , HeLa Cells , Humans , Nuclear Proteins/deficiency , Protein Binding , Protein Structure, Tertiary , ProteolysisABSTRACT
The eukaryotic centromere is an essential chromatin region required for accurate segregation of sister chromatids during cell division. Centromere protein B (CENP-B) is a highly conserved protein which can bind to the 17-bp CENP-B box on the centromeric DNA. In this study, we found that CENP-B could be α-N-methylated in human cells. We also showed that the level of the α-N-methylation was stimulated in cells in response to a variety of extracellular stimuli, including increased cell density, heat shock, and arsenite treatment, although the methylation level was not altered upon metaphase arrest. We identified N-terminal RCC1 methyltransferase (NRMT) as a major enzyme required for the CENP-B methylation. Additionally, we found that chromatin-bound CENP-B was primarily trimethylated and α-N-trimethylation could enhance CENP-B's binding to CENP-B box in cells. Our study also expands the function of protein α-N-methylation that has been known for decades and whose function remains largely unexplored.
Subject(s)
Centromere Protein B/metabolism , Centromere/metabolism , DNA/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Centromere Protein B/chemistry , Gene Expression , HEK293 Cells , Humans , Methylation , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Protein Binding , Protein Structure, TertiaryABSTRACT
CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1.
Subject(s)
Centromere Protein B/metabolism , Nucleosomes/metabolism , Proteins/metabolism , Autoantigens/chemistry , Autoantigens/metabolism , Centromere/metabolism , Centromere Protein A , Centromere Protein B/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Human/metabolism , DNA, Satellite/chemistry , Electrophoretic Mobility Shift Assay , Histones/chemistry , Histones/metabolism , Humans , Microtubule-Associated Proteins , Neoplasm Proteins , Nuclear Proteins/metabolism , Nucleosomes/chemistry , Protein Binding , Protein Structure, Tertiary , Protein Transport , Proteins/chemistry , tRNA MethyltransferasesABSTRACT
Centromeres are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A (CENP-A nucleosome) and their inheritance is probably dictated by the architecture of the centromeric nucleosome. We previously determined the crystal structure of the human CENP-A nucleosome. CENP-A forms a histone octamer containing two each of histones H2A, H2B, H4 and CENP-A and the DNA is left-handedly wrapped around the histone octamer, as in canonical nucleosomes containing histone H3. In the CENP-A nucleosome structure, 13 base pairs of the DNA ends are detached from the histone surface and two CENP-A regions, the αN helix and loop 1, adopt different structures from those in the H3 nucleosome. In this Extra View article, we provide a detailed structural comparison between CENP-A and H3 in nucleosomes and describe their distinctions and similarities.
Subject(s)
Autoantigens/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Histones/chemistry , Nucleosomes/chemistry , Autoantigens/metabolism , Centromere Protein A , Centromere Protein B/chemistry , Centromere Protein B/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA/chemistry , DNA/metabolism , Histones/metabolism , Humans , Models, Molecular , Nucleic Acid Conformation , Nucleosomes/metabolism , Protein ConformationABSTRACT
Faithful chromatin segregation is mediated and controlled by the kinetochore protein network which assembles at centromeres. In this study, the neighbourhood relations of inner kinetochore and nucleosome-associated complex (NAC) proteins were analysed in living human interphase cells by acceptor photobleaching FRET. The data indicate that CENP-U is in close vicinity to CENP-I as well as to CENP-B and that CENP-M is close to CENP-T.
Subject(s)
Kinetochores/chemistry , Nuclear Proteins/chemistry , Bacterial Proteins/genetics , Blotting, Western , Cell Line, Tumor , Centromere Protein B/chemistry , Centromere Protein B/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Fluorescence Resonance Energy Transfer/methods , Green Fluorescent Proteins/genetics , Humans , Interphase/physiology , Kinetochores/metabolism , Luminescent Proteins/genetics , Nuclear Proteins/metabolism , Photobleaching , TransfectionABSTRACT
By using a quantitative immunoblotting technique on protein extracts of human macrovascular and microvascular endothelial cells, we have analyzed the self-reactive repertoires of IgG from 20 patients with limited cutaneous SSc, 40 patients with diffuse SSc and 60 age- and sex-matched healthy controls. Serum IgG from 15/20 patients with limited cutaneous SSc and anti-centromere antibodies bound to at least one of the two 75- and 85-kDa protein bands in the different endothelial cell extracts, whereas IgG from healthy controls or patients with diffuse SSc did not. N-terminal sequencing of the 75- and 85-kDa bands identified CENP-B as the sole antigen in both bands. Moreover, IgG from all of the SSc patients who recognized the 75- and/or 85-kDa bands bound to a full-length recombinant CENP-B protein as assessed by ELISA, whereas IgG from other SSc patients did not. The main target of anti-endothelial cell antibodies in patients with limited cutaneous SSc is the nuclear and ubiquitous protein CENP-B.
Subject(s)
Antigens, Surface/immunology , Autoantibodies/metabolism , Centromere Protein B/immunology , Endothelial Cells/immunology , Scleroderma, Diffuse/immunology , Scleroderma, Limited/immunology , Antibody Specificity , Antigens, Surface/chemistry , Autoantibodies/blood , Autoantibodies/chemistry , Cells, Cultured , Centromere Protein B/chemistry , Centromere Protein B/genetics , Endothelial Cells/chemistry , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Male , Middle Aged , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.
