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PubMed2026

Dynamic Centromeres Under Epigenetic Constraint.

Centromeres are essential chromosomal loci specified epigenetically by CENP-A chromatin, yet they undergo rapid sequence turnover, structural remodeling, and occasional repositioning. In this review, we integrate recent advances enabled by long-read genome assemblies and high-resolution chromatin mapping to synthesize current understanding of centromere organization across taxa. We examine how satellite repeats, transposable elements, molecular drive, and meiotic conflict generate extreme centromere diversity. We further explore how DNA methylation and H3K9me3 heterochromatin constrain CENP-A positioning, stabilize centromeric domains, and shape boundary dynamics during centromere drift, duplication, and de novo formation. Together, these perspectives show how centromeres accommodate evolutionary change while preserving the stringent requirements of faithful chromosome segregation.

Journal Article↗

PubMed1993

Cytogenetic survey in systemic sclerosis: correlation of aneuploidy with the presence of anticentromere antibodies.

Previous cytogenetic studies of patients with systemic sclerosis have obtained conflicting results regarding the presence of chromosomal anomalies. We studied 38 patients and 15 controls to determine whether these inconsistencies were due to differences in the subgroups of patients who were studied. Because many patients with systemic sclerosis produce autoantibodies to protein antigens that have been implicated in chromosome structure and function, we further hypothesized that the presence of these autoantibodies might correlate with the presence of chromosomal anomalies. Patients were classified into clinical subgroups based on the extent of their disease. Their sera were assayed for autoantibodies to topoisomerase I and centromere proteins (CENP-A, CENP-B, and CENP-C) by immunoblotting. Cytogenetic analyses for aneuploidy and chromosome breaks were performed. Anticentromere antibody positive (ACA+) patients had significantly more aneuploidy than either ACA negative (ACA-) patients or controls (P = 0.041). Although the patient group, when considered as a whole, had significantly greater aneuploidy than the control group (P < 0.005), patients who were ACA--did not have more aneuploidy than the controls had. Patients with Type I disease (sclerodactyly), the majority of whom were ACA+, also had significantly more aneuploidy than did either the controls or patients with Type III (diffuse) disease, most of whom were ACA- (P < 0.005). ACA+ patients also had more chromatid breaks than the controls had (P < 0.05). The correlation between the presence of ACAs and chromosomal aneuploidy suggests that aneuploidy may be the result of nondisjunction secondary to centromeric dysfunction. In support of this hypothesis, the ACA+ patients who had antibodies to CENP-C exhibited more chromosomal aneuploidy than did either anti-CENP-A or anti-CENP-B positive patients (P < 0.048). Unlike CENP-A and CENP-B, which are present at both functional and inactivated centromeres, CENP-C is present at the kinetochore of functional centromeres.

Adult↗

PubMed1988

Proteins responsible for anticentromere activity found in the sera of patients with CREST-associated Raynaud's phenomenon.

Anticentromere antibodies (ACA) present in a high percentage of patients with complete or incomplete CREST scleroderma, and which are presently used in the diagnosis of this disease, also appear in some primary Raynaud's phenomenon patients. Three principal centromeric antigens, CENP-A, CENP-B and CENP-C, have been described as reacting with the sera of these individuals. We attempt to determine whether or not a correlation between the presence of ACA and serum reactivity against one or more of these peptides could be established, and have observed that CENP-A, but not CENP-B or CENP-C, is specifically recognized by all patients sera tested. The fact that this reactivity is clearly detectable at very high serum dilutions, thus eliminating other non-specific interference, suggests that anti-CENP-A activity might be useful in the diagnosis of patients with CREST-associated Raynaud's phenomenon.

Adult↗

PubMed1991

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.

Anaphase↗

PubMed1985

Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome.

We developed an aqueous spreading procedure that permits simultaneous analysis of human chromosomes by Q-banding and indirect immunofluorescence. Using this methodology and anticentromere antibodies from an autoimmune patient we compared the active and inactive centromeres of an isodicentric X chromosome. We show that a family of structurally related human centromere proteins (CENP-A, CENP-B, and CENP-C) is detectable only at the active centromere. These antigens therefore may be regarded both as morphological and functional markers for active centromeres.

Cell Transformation, Viral↗

PubMed1988

Kinetochore appearance during meiosis, fertilization and mitosis in mouse oocytes and zygotes.

The events of mammalian fertilization overlap with the completion of meiosis and first mitosis; the pro-nuclei never fuse, instead the parental genomes first intermix at the mitotic spindle equator at metaphase. Since kinetochores are essential for the attachment of chromosomes to spindle microtubules, this study explores their appearance and behavior in mouse oocytes, zygotes and embryos undergoing the completion of meiosis, fertilization and mitoses. Kinetochores are traced with immunofluorescence microscopy using autoimmune sera from patients with CREST (CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) scleroderma. These sera cross-react with the 17 kDa centromere protein (CENP-A) and the 80 kDa centromere protein (CENP-B) found at the kinetochores in human cell cultures. The unfertilized oocyte is ovulated arrested at second meiotic metaphase and kinetochores are detectable as paired structures aligned at the spindle equator. At meiotic anaphase, the kinetochores separate and remain aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis is lost. The female pronucleus and the second polar body nucleus each receive a detectable complement of kinetochores. Mature sperm have neither detectable centrosomes nor detectable kinetochores, and shortly after sperm incorporation kinetochores become detectable in the decondensing male pronucleus. In pronuclei, the kinetochores are initially distributed randomly and later found in apposition with nucleoli. At mitosis, the kinetochores behave in a pattern similar to that observed at meiosis or mitosis in somatic cells: irregular distribution at prophase, alignment at metaphase, separation at anaphase and redistribution at telophase. They are also detectable in later stage embryos. Colcemid treatment disrupts the meiotic spindle and results in the dispersion of the meiotic chromosomes along the oocyte cortex; the chromosomes remain condensed with detectable kinetochores. Fertilization of Colcemid-treated oocytes results in the incorporation of a sperm which is unable to decondense into a male pronucleus. Remarkably kinetochores become detectable at 5 h post-insemination, suggesting that the emergence of the paternal kinetochores is not strictly dependent on male pronuclear decondensation.

Animals↗

PubMed1987

Mammalian kinetochore/centromere composition: a 50 kDa antigen is present in the mammalian kinetochore/centromere.

The composition of the mammalian kinetochore/centromere was studied by indirect immunofluorescence and immunoblotting protocols using serum from a patient with the CREST variant of scleroderma. The results of these studies suggest that a protein with a molecular weight of 50 kDa is localized at the surface of the primary constrictions (the kinetochore region) of both human and Indian muntjac chromosomes. In addition, we were able to verify the presence of a 19.5 kDa antigen (CENP-A), previously detected in human centromeres, within the kinetochore region of the Indian muntjac. These data suggest that the composition of the kinetochore region of the primary constriction is complex and that there is conservation in composition within the mammals. These features may reflect the important role of this unique chromosomal domain in the maintenance of ploidy.

Animals↗

PubMed1994

Analysis of centromere structure in the fly Megaselia scalaris (Phoridae, Diptera) using CREST sera, anti-histone antibodies, and a repetitive DNA probe.

We have used CREST anti-centromere sera, rabbit anti-histone antibodies, and repetitive DNA analysis to study centromere structure in the fly Megaselia scalaris (Phoridae). In a panel of eight CREST sera, four were positive in immunofluorescence experiments for prekinetochores, ie centromeres in interphase nuclei. The access of the antibodies to CREST antigens may be compromised in the condensed state, since centromeres of prometaphase and metaphase chromosomes remained unstained. When Western blots of embryonic nuclei were probed with these four CREST sera, three of them showed a 17 kDa band. Human CENP-A, likewise recognized by the CREST sera, is a 17 kDa protein. The remaining sera were negative for centromeres although some detected centrosomes and non-histone chromosomal proteins not confined to the centromeres. The use of antibodies generated against histone H4 acetylated at four different sites of the N-terminal domain revealed that heterochromatic regions of M scalaris mitotic chromosomes, ie pericentric and NOR-associated segments, are hyperacetylated. This is at variance with a variety of other systems, where transcriptionally active chromatin is hyperacetylated. Finally, a repetitive 165 base pair fragment was isolated from genomic DNA of the fly and sequenced. An oligonucleotide from this sequence mapped to the centromere region of interphase nuclei and the pericentric regions of condensed chromosomes.

Animals↗

PubMed2025

DIS3 licenses B cells for plasma cell differentiation in humans.

DIS3 is the main catalytic subunit of the nuclear RNA exosome, a complex playing a crucial role in RNA processing and the degradation of various noncoding RNA substrates. In mice, DIS3 is essential for genomic rearrangements during B cell development, but its role in terminal plasma cell (PC) differentiation has not been explored. Although DIS3 gene alterations are frequent in multiple myeloma (MM), a PC malignancy, their molecular impact remains poorly understood. In this study, we developed an antisense oligonucleotide strategy to knock down DIS3 expression in a well-characterized model of human PC differentiation. Reducing DIS3 expression systematically led to decreased B cell proliferation and impaired PC differentiation with lower levels of switched immunoglobulin secretion. Transcriptome analyses confirmed alterations in the proliferation and differentiation programs, alongside an accumulation of noncoding RNAs. Notably, centromere-associated noncoding RNAs were highly sensitive to DIS3 activity, and their accumulation in DIS3-deficient cells, either as transcripts or DNA-associated RNAs, correlated with the mislocalization of the centromere-specific histone variant CENP-A. We finally observed reduced physiological DNA recombination and somatic hypermutation but increased genomic instability in DIS3-deficient cells, in agreement with the higher levels of IGH translocations observed in our large cohort of DIS3-mutant MM patients. Together, these results underscore the essential role of DIS3 in regulating B cell proliferation, DNA recombination, and physiological or malignant PC differentiation in humans.

Humans↗

PubMed1990

Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis.

We have used autoantibodies to probe the function of three human centromere proteins in mitosis. These antibodies recognize three human polypeptides in immunoblots: CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD). Purified anticentromere antibodies (ACA-IgG) disrupt mitosis when introduced into tissue culture cells during interphase. We have identified two execution points for antibody inhibition. Antibodies injected into the nucleus greater than or equal to 3 h before mitosis prevent the chromosomes from undergoing normal prometaphase movements in the subsequent mitosis. Antibodies injected in the nucleus during late G2 cause cells to arrest in metaphase. Surprisingly, antibodies introduced subsequent to the beginning of prophase do not block mitosis. These results suggest that the CENP antigens are involved in two essential interphase events that are required for centromere action in mitosis. These may include centromere assembly coordinate with the replication of alpha-satellite DNA at the end of S phase and the structural maturation of the kinetochore that begins at prophase.

Anaphase↗

PubMed1994

Human centromere protein C (CENP-C) is a DNA-binding protein which possesses a novel DNA-binding motif.

Mammalian centromere proteins (CENPs) can be divided into those that translocate from centromere to midzone in the progress of mitosis, and those that remain at the centromere throughout the cell cycle. The latter including CENP-A, CENP-B, and CENP-C is the candidate for DNA-binding protein. CENP-B has been shown previously to possess the specific DNA-binding activity to 17-base pair sequences dispersed on human centromeric alphoid repeats. In this study, we examined DNA-binding property of CENP-C that is localized to inner kinetochore plate of the metaphase chromosome. We independently isolated a full-length cDNA encoding human CENP-C and expressed it as the polypeptide tagged with histidine oligomer in Escherichia coli. After affinity purification with Ni(2+)-chelated resin, DNA-binding activity of the recombinant CENP-C renatured on the membrane was demonstrated by using human genomic DNA and an alphoid subfamily in South-Western-type blotting analysis. By constructing a series of truncated products, the DNA-binding domain was located at an internal 101-amino-acid stretch with no apparent homology to any other DNA-binding proteins. This may suggest that CENP-C is directly involved in formation of kinetochore chromatin fibers.

Amino Acid Sequence↗

PubMed1995

A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis.

The centromere, a differentiated region of the eukaryotic chromosome, mediates the segregation of sister chromatids at mitosis. In this study, a Saccharomyces cerevisiae chromosome mis-segregation mutant, cse4-1, has been isolated and shown to increase the nondisjunction frequency of a chromosome bearing a mutant centromere DNA sequence. In addition, at elevated temperatures the cse4-1 allele causes a mitosis-specific arrest with a predominance of large budded cells containing single G2 nuclei and short bipolar mitotic spindles. The wild-type gene, CSE4, is essential for cell division and encodes a protein containing a domain that is 64% identical to the highly conserved chromatin protein, histone H3. Biochemical experiments demonstrate that CSE4p has similar DNA-binding characteristics as those of histone H3 and might form a specialized nucleosome structure in vivo. Interestingly, the human centromere protein, CENP-A, also contains this H3-like domain. Data presented here indicate that CSE4p is required for proper kinetochore function in yeast and may represent an evolutionarily conserved protein necessary for assembly of the unique chromatin structure associated with the eukaryotic centromere.

Amino Acid Sequence↗

PubMed2025

Two CENH3 paralogs in the green alga Chlamydomonas reinhardtii have a redundantly essential function and associate with ZeppL-LINE1 elements.

Centromeres in eukaryotes are defined by the presence of histone H3 variant CENP-A/CENH3. Chlamydomonas encodes two predicted CENH3 paralogs, CENH3.1 and CENH3.2, that have not been previously characterized. We generated peptide antibodies to unique N-terminal epitopes for each of the two predicted Chlamydomonas CENH3 paralogs as well as an antibody against a shared CENH3 epitope. All three CENH3 antibodies recognized proteins of the expected size on immunoblots and had punctate nuclear immunofluorescence staining patterns. These results are consistent with both paralogs being expressed and localized to centromeres. CRISPR-Cas9-mediated insertional mutagenesis was used to generate predicted null mutations in either CENH3.1 or CENH3.2. Single mutants were viable but cenh3.1 cenh3.2 double mutants were not recovered, confirming that the function of CENH3 is essential. We sequenced and assembled two chromosome-scale Chlamydomonas genomes from strains CC-400 and UL-1690 (a derivative of CC-1690) with complete centromere sequences for 17/17 and 14/17 chromosomes respectively, enabling us to compare centromere evolution across four isolates with near complete assemblies. These data revealed significant changes across isolates between homologous centromeres including mobility and degeneration of ZeppL-LINE1 (ZeppL) transposons that comprise the major centromere repeat sequence in Chlamydomonas. We used cleavage under targets and tagmentation (CUT&Tag) to purify and map CENH3-bound genomic sequences and found enrichment of CENH3-binding almost exclusively at predicted centromere regions. An interesting exception was chromosome 2 in UL-1690, which had enrichment at its genetically mapped centromere repeat region as well as a second, distal location, centered around a single recently acquired ZeppL insertion. The CENH3-bound regions of the 17 Chlamydomonas centromeres ranged from 63.5 kb (average lower estimate) to 175 kb (average upper estimate). The relatively small size of its centromeres suggests that Chlamydomonas may be a useful organism for testing and deploying artificial chromosome technologies.

Chlamydomonas reinhardtii↗

PubMed1996

A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission.

The histone proteins are essential for the assembly and function of th e eukaryotic chromosome. Here we report the first isolation of a temperature-sensitive lethal histone H4 mutant defective in mitotic chromosome transmission Saccharomyces cerevisiae. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, was found to suppress the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation.

Chromatin↗

PubMed1993

Anticentromere antibodies in subjects with no apparent connective tissue disease.

OBJECTIVES: To study the association of anticentromere antibodies (ACA) in various diseases. METHODS: A total of 4800 consecutive serum samples were tested for ACA by indirect immunofluorescence using HEp-2 cells as substrates and by immunoblotting of Molt-4 cell mitotic chromosomal antigens and recombinant CENP-B protein. RESULTS: Anticentromere antibodies were identified in the serum samples of 24 subjects, including eight without apparent connective tissue diseases, six with primary biliary cirrhosis, two with diffuse scleroderma, one with pulmonary hypertension, one with primary Raynaud's phenomenon, one with CREST syndrome (calcinosis, Raynaud's phenomenon, oesophageal dysmotility, sclerodactyly, telangiectasia), and five with other connective tissue diseases. By immunoblotting using Molt-4 cells mitotic chromosomal antigens three centromere antigens were recognised by these serum samples. These were: CENP-A (17 kilodalton recognised by 22 of 24 ACA positive serum samples); CENP-B (80 kilodalton recognised by 22 of 24 ACA positive serum samples); and CENP-C (140 kilodalton recognised by 19 of 24 ACA positive serum samples). There was no specific pattern for serum samples from patients with different groups of diseases on immunoblotting. Recombinant CENP-B proteins were all recognised by these samples. Patients without apparent connective tissue disease often had a lower ACA titre than patients with primary biliary cirrhosis. CONCLUSIONS: These data suggest that a positive result for ACA does not always indicate the presence of a connective tissue disease.

Adult↗

PubMed1989

Proteins of the inner and outer centromere of mitotic chromosomes.

We have used immunocytochemistry and molecular cloning methods to identify and characterize structural polypeptides of the centromere. These studies permit us to resolve two distinct regions: the inner and outer centromere. (i) Components of the outer centromere: autoantibodies from certain patients with rheumatic disease identify a family of three immunologically related polypeptides that we have designated CENP-A (17 kDa), CENP-B (80 kDa), and CENP-C (140 kDa). CENP-B has been cloned and sequenced. DNA sequence analysis indicates that this polypeptide possesses two large regions with extraordinary concentrations of acidic residues (region I: 61 residues with 79% glu + asp; region II: 31 residues with 87% glu + asp). Despite this concentration of negative charge, immunocytochemical experiments suggest that CENP-B may be a DNA binding protein. In these experiments, the levels of CENP-B are seen to vary reproducibly from chromosome to chromosome. The role of CENP-B in vivo is unknown. However, it is unlikely to bind directly to the spindle microtubules since it is found at an inactive centromere that apparently does not attach to the spindle. (ii) Components of the inner centromere: we have injected mice with the whole chromosome scaffold fraction to elicit production of monoclonal antibodies. One such antibody identifies two structurally related polypeptides (the INCENP antigens, 135 and 155 kDa) that are preferentially located between the sister chromatids at the centromere. The INCENP antigens undergo dramatic movements from the chromosomes to the central spindle during mitosis. They are ultimately sequestered in the midbody and discarded. Several lines of evidence suggest that the INCENP polypeptides may be involved in the regulation of sister chromatid separation at the metaphase-anaphase transition.

Amino Acid Sequence↗

PubMed1986

Three human chromosomal autoantigens are recognized by sera from patients with anti-centromere antibodies.

We have identified 39 individuals with anti-centromere antibodies (ACA) in our patient population, all of whom have Raynaud's syndrome or disease. We have used sera from the ACA-positive patients and from 123 controls (22 normal individuals and 101 additional patients with either Raynaud's disease or Raynaud's syndrome plus an associated connective tissue disease) to screen the proteins of highly purified human (HeLa) mitotic chromosomes by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. Three antigens were recognized by the sera from the ACA-positive patients. These were centromere protein (CENP)-B (80,000 mol wt--recognized by all ACA-positive sera), CENP-A (17,000 mol wt--recognized by 38 of 39 ACA-positive sera), and CENP-C (140,000 mol wt--recognized by 37 of 39 ACA-positive sera). None of these antigens were recognized by any of the 123 control sera, although binding was occasionally seen to other chromosomal antigens. Therefore the ACA response is highly uniform in our patient population. Antibody to CENP-B shows a 100% correlation with anti-centromere staining by indirect immunofluorescence.

Adult↗

PubMed1992

Anti-histone autoantibodies recognize centromeric heterochromatin in metaphase chromosomes and hidden epitopes in interphase cells.

IgM autoantibodies from a subset of patients with undifferentiated rheumatic disease syndromes stained mouse kidney nuclei with a distinctive variable large-speckled (VLS) indirect immunofluorescence (IIF) pattern. However, these antibodies did not stain nuclei of tissue culture cells prepared with conventional fixation. These sera were shown to react with histone H3 by an enzyme-linked immunosorbent assay (ELISA), and adsorption with H3 reduced or eliminated the IIF reaction. Sera yielding a VLS-IIF pattern reacted in ELISA with all three H3 variants as well as the native (H3-H4)2 tetramer, but the reactive determinants were unavailable for binding when chromatin was the substrate. By IIF assay, the epitopes were exposed after treatment of tissue culture cells with 0.5 M NaCl, and were removed by 1.5 M NaCl. These sera also stained the centromeric region of metaphase chromosomes. These observations suggest that the VLS-IIF pattern is due to antibodies that recognize epitopes on constitutive heterochromatin near the centromere. The epitopes are exposed in differentiated cells but hidden in dividing cells. Histone in heterochromatin, or CENP-A, a histone-like protein in the centromere with a sequence similarity to histone H3, are candidates for the target antigen.

Autoantibodies↗