Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Interstitial deletion of proximal 8q including part of the centromere from unbalanced segregation of a paternal deletion/marker karyotype with neocentromere formation at 8p22.

BACKGROUND/AIMS: The 'McClintock mechanism' of chromosome breakage and centromere misdivision, in which a deleted chromosome with its concomitant excised marker or ring chromosome is formed, has been described in approximately one dozen reports. We report a case of a girl with short stature, developmental delay, and dysmorphic features. METHODS: Analysis was performed on the proband and father using cytogenetic chromosome analysis and the Affymetrix 6.0 SNP microarray. Fluorescence in situ hybridization (FISH) using a chromosome 8 alpha-satellite probe and immunofluorescence with antibodies to CENP-C were used to examine the centromere positions in these chromosomes. RESULTS: An abnormal chromosome 8 with a cytogenetically visible deletion was further defined by SNP array as a 10.6-Mb deletion from 8q11.1→q12.1. FISH with a chromosome 8 alpha-satellite probe demonstrated that the deletion removed a significant portion of the pericentromeric alpha-satellite repeat sequences and proximal q arm. The deleted chromosome 8 appeared to have a constriction at 8p22, suggesting the formation of a neocentromere, even though alpha-satellite sequences still appeared at the normal location. Chromosome analysis of the phenotypically normal father revealed the same deleted chromosome 8, as well as an apparently balancing mosaic marker chromosome 8. FISH studies revealed that the majority of the chromosome 8 alpha-satellite DNA resided in the marker chromosome. Immunofluorescence studies with antibodies to CENP-C, a kinetochore protein, proved the presence of a neocentromere at 8p22. The excision of the marker from the deleted chromosome 8 likely necessitated the formation of a new kinetochore at the 8p22 neocentromere to stabilize the chromosome during mitosis. CONCLUSION: This case clearly illustrates the utilization of classic cytogenetics, FISH, and array technologies to better characterize chromosomal abnormalities and provide information on recurrence risks. It also represents a rare case where a neocentromere can form even in the presence of existing alpha-satellite DNA.

A neocentromere derived from a supernumerary marker deleted from the long arm of chromosome 6.

Parental chromosome studies were referred to us after initial finding of a balanced translocation involving chromosomes 4 and 15 in their phenotypically abnormal male child (cytogenetic analysis was done at another laboratory). In addition to the same 4;15 translocation, the father also had an interstitial deletion of the long arm of one chromosome 6 and a marker chromosome. In this article, we report a neocentromere on this marker, which was determined to be composed of chromosome 6 material by FISH. The child's karyotype was re-interpreted to be unbalanced due to the presence of the abnormal chromosome 6, but without the marker. The clinical phenotype associated with the interstitial deletion of chromosome 6 is also reported.

Characterization of a neocentric supernumerary marker chromosome originating from the Xp distal region by FISH, CENP-C staining, and array CGH.

A small supernumerary marker chromosome (SMC) was observed in a girl with severe developmental delay. Her dysmorphism included prominent forehead, hypertelorism, down-slanting palpebral fissures, low-set/large ears, and flat nasal bridge with anteverted nares. This case also presented hypotonia, hypermobility of joints, congenital heart defect, umbilical hernia, failure to thrive, and seizures. The SMC originated from the distal region of Xp as identified by FISH with multiple DNA probes. Staining with antibodies to Centromere Protein C (CENP-C) demonstrated a neocentromere, while FISH with an alpha-satellite DNA probe showed no hybridization to the SMC. A karyotype was described as 47,XX,+neo(X)(pter-->p22.31::p22.31-->pter), indicating a partial tetrasomy of Xp22.31-->pter. This karyotype represents a functional trisomy for Xp22.31-->pter and a functional tetrasomy for the pseudoautosomal region given that there is no X-inactivation center in the marker chromosome. The SMC was further characterized by microarray-based comparative genomic hybridization (array CGH) as a duplicated DNA fragment of approximately 13 megabase pairs containing about 100 genes. We have described here a new neocentromere with discussion of its clinical significance.

Prenatal diagnosis of a karyotypically normal pregnancy in a mother with a supernumerary neocentric 13q21 -->13q22 chromosome and balancing reciprocal deletion.

An adult female patient with a history of miscarriages was found to be carrying a stable supernumerary chromosome. The patient also carried a reciprocal paracentric deletion in chromosome 13q21/22. Microdissection and reverse fluorescence in situ hybridization FISH revealed that this supernumerary chromosome was derived from region 13q21 --> 13q22. The presence of a neocentromere on this supernumerary chromosome was confirmed by the absence of detectable alpha satellite DNA using FISH and the presence of centromere proteins CENP-C and CENP-A using immunofluorescence. The absence of telomere sequences suggests that the marker is a ring chromosome (r(13)). FISH using ordered BACs from the chromosome region 13q21 --> 13q31 permitted the precise positioning of the r(13) chromosome and the corresponding deletion to chromosome bands 13q21.32 --> 13q22.2. BAC 280J7 from within the r(13) was used as a FISH probe for the prenatal analysis of amniocytes at 16 weeks of gestation, which revealed a normal karyotype for the fetus. This r(13) chromosome represents the first description of chromosome 13 of the rarer class of neocentric chromosomes that are derived from interstitial deletions. It represents the first example of prenatal diagnosis in a phenotypically normal female that was ascertained to carry a neocentric marker. The presence of such a neocentric marker/deletion karyotype in a parent presents unique possible karyotypic outcomes for conceptions and unusual challenges for genetic counseling.

An unstable dicentric Robertsonian translocation in a markedly discordant twin.

Karyotypes from independent amniocenteses reflected a rare, unstable, functionally dicentric Robertsonian translocation chromosome in most cells in male Twin B who grew more slowly than the chromosomally normal female sib (Twin A). Twin B's balanced de novo Robertsonian translocation dic(13;14)(p11.1;p11.1), present in 81% of cells, underwent recurrent centromeric fission in 6 out of 30 independent colonies that explains a balanced 46,XY,-13,+fis(13)(p11.1),-14,+fis(14)(p11.1) karyotype. Aneuploidy for chromosomes 13q or 14q was present in 5% of cells. Instability of the Robertsonian translocation was evident because nine of the 30 colonies (30%) grown from single amniocytes had metaphase cells with more than one chromosome complement. Although uniparental disomy was excluded and a targeted ultrasound was normal, the couple was advised of the uncertain but real risk of abnormalities in Twin B and the risk to Twin A of terminating Twin B. The pregnancy proceeded and at 31 weeks gestation Twin A was in the 33rd percentile for size and Twin B in the 1st percentile. At 32 weeks, chromosome analysis revealed a balanced 45,XY,dic(13;14)(p11.1;p11.1) karyotype in all of Twin B's newborn cord blood cells with no evidence of fission or aneuploidy. Selection against unbalanced mitotic products of the unstable, functionally dicentric chromosome in early fetal development is proposed to result in Twin B's highly discordant small birth size.

Chromosome engineering: prospects for gene therapy.

Recent advances in chromosome engineering and the potential for downstream applications in gene therapy were presented at the Artificial Chromosome Session of Genome Medicine: Gene Therapy for the Millennium in Rome, Italy in September 2001. This session concentrated primarily on the structure and function of human centromeres and the ongoing challenge of equipping human artificial chromosomes (HACs) with centromeres to ensure their mitotic stability. Advances in the 'bottom up' construction of HACs included the transfer into HT1080 cells of circular PACs containing alpha satellite DNA, and the correction of HPRT deficiency in cells using HACs. Advances in the 'top down' construction of HACs using telomere associated chromosome fragmentation in DT40 cells included the formation of HACs that are less than a megabase in size and transfer of HACs through the mouse germline. Significant progress has also been made in the use of human minichromosomes for stable trans-gene expression. While many obstacles remain towards the use of HACs for gene therapy, this session provided an optimistic outlook for future success.

Epigenetic analysis of kinetochore assembly on variant human centromeres.

Human centromere formation involves the assembly of the mitotic kinetochore onto chromosomal locations that contain the interphase prekinetochore. Immunofluorescent analysis of two functionally converse human centromere variants, neocentromeres and inactive centromeres, has been used to evaluate the functional significance of over 24 CENTROMERE proteins, providing important insight into the epigenetics of centromere formation and kinetochore assembly.

Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere--kinetochore complexes.

Centromere and kinetochore proteins have a pivotal role in centromere structure, kinetochore formation and sister chromatid separation. However, the molecular architecture and the precise dynamic function of the centromere-kinetochore complex during mitosis remain poorly understood. Here we report the isolation and characterization of human CENP-H. Confocal microscopic analyses of HeLa cells with anti-human CENP-H-specific antibody demonstrated that CENP-H colocalizes with inner kinetochore plate proteins CENP-A and CENP-C in both interphase and metaphase. CENP-H was present outside centromeric heterochromatin, where CENP-B is localized, and inside the kinetochore corona, where CENP-E is localized during prometaphase. Furthermore, CENP-H was detected at neocentromeres, but not at inactive centromeres in stable dicentric chromosomes. In vitro binding assays of human CENP-H with centromere-kinetochore proteins suggest that the CENP-H binds to itself and MCAK, but not to CENP-A, CENP-B or CENP-C. CENP-H multimers were observed in cells in which both FLAG-tagged CENP-H and hemagglutinin-tagged CENP-H were expressed. These results suggest that CENP-H multimers localize constitutively to the inner kinetochore plate and play an important fundamental role in organization and function of the active human centromere-kinetochore complex.

A neocentromere in the DAZ region of the human Y chromosome.

We describe a novel rearranged human Y chromosome consisting of an inverted duplication of the long arm heterochromatin and a small amount of euchromatin: rea(Y)(qter-q11.2::q11.2-qter). The normal centromere has been deleted and a neocentromere containing CENP-A, -C, -E and Mad2 but not CENP-B has formed close to the breakpoint. A 2.7 Mb yeast artificial chromosome contig spanning the breakpoint was constructed and the breakpoint was localised to a region of <120 kb close to the DAZ gene cluster. Combined immunofluorescence and fluorescence in situ hybridisation showed that the centromeric protein-binding domain of the neocentromere was located near the breakpoint and within the DAZ cluster.

Molecular cytogenetic analysis of eight inversion duplications of human chromosome 13q that each contain a neocentromere.

Neocentromeres are fully functional centromeres that have arisen in previously noncentromeric chromosomal locations on rearranged chromosomes. The formation of neocentromeres results in the mitotic stability of chromosomal fragments that do not contain endogenous centromeres and that would normally be lost. Here we describe a unique collection of eight independent patient-derived cell lines, each of which contains a neocentromere on a supernumerary inversion duplication of a portion of human chromosome 13q. Findings in these patients reveal insight into the clinical manifestations associated with polysomy for portions of chromosome 13q. The results of FISH and immunofluorescent analysis of the neocentromeres in these chromosomes confirm the lack of alpha-satellite DNA and the presence of CENtromere proteins (CENP)-C, -E, and hMAD2. The positions of the inversion breakpoints in these chromosomes have been placed onto the physical map of chromosome 13, by means of FISH mapping with cosmid probes. These cell lines define, within chromosome 13q, at least three distinct locations where neocentromeres have formed, with five independent neocentromeres in band 13q32, two in band 13q21, and one in band 13q31. The results of examination of the set of 40 neocentromere-containing chromosomes that have thus far been described, including the 8 neocentromere-containing chromosomes from chromosome 13q that are described in the present study, suggest that chromosome 13q has an increased propensity for neocentromere formation, relative to some other human chromosomes. These neocentromeres will provide the means for testing hypotheses about sequence requirements for human centromere formation.

Making CENs of mammalian artificial chromosomes.

Mammalian artificial chromosomes (MACs) hold the promise of providing autonomous vectors for gene therapy in dividing cells. They would not require insertion into the genome and could include sufficient genomic sequences that surround the therapeutic gene to ensure proper tissue-specific and temporal regulation. Several groups have reported successful formation of MACs in human cells using transfection strategies that included alpha satellite DNA, the primary DNA found at normal human centromeres. These results, although extremely encouraging, have limitations such as unpredictable chromosome formation and success thus far in only one transformed human cell line. Examination of other cells where alpha satellite DNA has integrated into ectopic chromosomal locations, as well as naturally occurring dicentric and neocentromere-containing cell lines, suggests that alpha satellite DNA may not be necessary or sufficient for centromere formation. Overall, these results suggest that epigenetic modifications of centromeric DNA are required for efficient centromere formation. Models for this centromere-specific epigenetic modification include a specialized chromatin structure and differential replication timing of centromeric DNA. Thus, further investigation of these centromere-specific epigenetic modifications may suggest strategies for increasing the efficiency of generating human artificial chromosomes for use as gene therapy vectors.

Transmission of a fully functional human neocentromere through three generations.

An unusual Y chromosome with a primary constriction inside the long-arm heterochromatin was found in the amniocytes of a 38-year-old woman. The same Y chromosome was found in her husband and brother-in-law, thus proving that it was already present in the father. FISH with alphoid DNA showed hybridization signals at the usual position of the Y centromere but not at the primary constriction. Centromere proteins (CENP)-A, CENP-C, and CENP-E could not be detected at the site of the canonic centromere but were present at the new constriction, whereas CENP-B was not detected on this Y chromosome. Experiments with 82 Y-specific loci distributed throughout the chromosome confirmed that no gross deletion or rearrangement had taken place, and that the Y chromosome belonged to a haplogroup whose members have a mean alphoid array of 770 kb (range 430-1,600 kb), whereas that of this case was approximately 250 kb. Thus, this Y chromosome appeared to be deleted for part of the alphoid DNA. It seems likely that this deletion was responsible for the silencing of the normal centromere and that the activation of the neocentromere prevented the loss of this chromosome. Alternatively, neocentromere activation could have occurred first and stimulated inactivation of the normal centromere by partial deletion. Whatever the mechanism, the presence of this chromosome in three generations demonstrates that it functions sufficiently well in mitosis for male sex determination and fertility and that neocentromeres can be transmitted normally at meiosis.

Hamster chromosomes containing amplified human alpha-satellite DNA show delayed sister chromatid separation in the absence of de novo kinetochore formation.

The centromeres of human chromosomes contain large amounts of the tandemly repeated alpha-satellite DNA family. Previous studies have shown that integration of alpha-satellite DNA into ectopic locations in mammalian chromosomes can result in the de novo formation of several features of centromeric function. Here we further examine the possible centromeric properties of alpha-satellite DNA by introducing it into hamster chromosomes. A large amplified region of ectopic alpha-satellite DNA was shown to direct binding of anticentromere antibodies (ACAs) and centromere protein B (CENP-B). The chromosome containing these ectopic arrays showed a high frequency of formation of anaphase bridges. Owing to the favourable morphology of these chromosomes, we were able to determine that this bridging was due to delayed sister chromatid disjunction at the location of the ectopic alpha-satellite, and not due to de novo formation of a fully functional kinetochore. A separate hamster cell line containing large tandemly repeated amplicons including the DHFR gene also displayed similar behaviour during anaphase. These results may support a role for alpha-satellite DNA in sister chromatid cohesion at centromeres. However, other repetitive DNA in favourable configurations appears to be capable of mimicking this behaviour during anaphase.

Centromeres, CENP-B and Tigger too.

The highly conserved centromere-associated protein CENP-B is a common feature of mammalian centromeres. Binding sites for CENP-B, so-called 'CENP-B boxes', are present in the otherwise unrelated centromeric satellite DNAs of humans, Mus musculus, Mus caroli, ferrets, giant pandas, tree shrews and gerbils, suggesting a role for CENP-B in centromere function. However, CENP-B and its binding sites are not detected at the centromeres of mammalian Y chromosomes and few, if any, binding sites seem present on African green monkey chromosomes. There is extensive sequence similarity between CENP-B and transposase proteins encoded by the pogo superfamily of transposable elements, which includes the human Tigger elements. Intriguingly, Tigger 2 has an almost perfect match to the CENP-B-binding site within its terminal inverted repeat. Comparison of the amino acid sequence of CENP-B with related proteins raises the possibility that CENP-B might share the ability to cause single-stranded DNA breaks. Such nicks could promote recombination, as has been suggested for the Charcot-Marie-Tooth disease duplication where a recombination hotspot exists close to a mariner-like element. We suggest that by promoting nicks adjacent to CENP-B boxes, CENP-B might facilitate the evolution and maintenance of satellite sequence arrays, rather than have a direct role in centromere function.

Untangling the role of DNA topoisomerase II in mitotic chromosome structure and function.

DNA topoisomerase II (topo II) is involved in chromosome structure and function, although its exact location and role in mitosis are somewhat controversial. This is due in part to the varied reports of its localization on mitotic chromosomes, which has been described at different times as uniformly distributed, axial on the chromosome arms and predominantly centromeric. These disparate results are probably due to several factors, including use of different preparation and fixation techniques, species differences and changes in distribution during the cell cycle. Recently, several papers have re-investigated the distribution of topo II on chromosomes as a function of cell cycle and species(1-3). The new studies suggest that Topo II has a dynamic pattern of distribution on the chromosomes, in general becoming axial as chromosomes condense during prophase and then concentrating at centromeres during metaphase. These experiments suggest a novel role for topo II in centromere structure and function.

Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres.

The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood [1]. The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly [2], but whose molecular role in kinetochore structure and function is unknown. Here we have raised for the first time monospecific antisera to CENP-A [3], a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 [4,5] and that resembles the yeast centromeric component CSE4 [6]. We have found by simultaneous immunofluorescence with centromere antigens of known ultrastructural location that CENP-A is concentrated in the region of the inner kinetochore plate at active centromeres. Because CENP-A was previously shown to co-purify with nucleosomes [7], our data suggest a specific nucleosomal substructure for the kinetochore. In human cells, these kinetochore-specific nucleosomes are enriched in alpha-satellite DNA [8]. However, the association of CENP-A with neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition. We speculate that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres.

Oligonucleotide probes for alpha satellite DNA variants can distinguish homologous chromosomes by FISH.

Chromosomal heteromorphisms have been used extensively to mark individual chromosomes. However, classical banding techniques used to identify these structural variants are imprecise and difficult to quantify. Different chromosomes 17 from the human population are characterized by distinct haplotypes of alpha satellite DNA. We have used these sequence variants to construct oligonuoleotide probes for fluorescence in situ hybridization (FISH). These oligomers are the first reported FISH probes that can discriminate between cytogenetically indistinguishable chromosome homologues. They have been used to follow the transmission of a single chromosome 17 through a pedigree, similar to a typical polymorphic marker. Furthermore, extended chromatin fiber techniques reveal the presence of discrete domains of different sequence variants within individual centromeres. Extension of this strategy to create a battery of other variant-specific oligoprobes should provide a powerful diagnostic tool for parent of origin effects in the study of aneuploidy, imprinting and cancer cytogenetics.

Surprising deficiency of CENP-B binding sites in African green monkey alpha-satellite DNA: implications for CENP-B function at centromeres.

Centromeres of mammalian chromosomes are rich in repetitive DNAs that are packaged into specialized nucleoprotein structures called heterochromatin. In humans, the major centromeric repetitive DNA, alpha-satellite DNA, has been extensively sequenced and shown to contain binding sites for CENP-B, an 80-kDa centromeric autoantigen. The present report reveals that African green monkey (AGM) cells, which contain extensive alpha-satellite arrays at centromeres, appear to lack the well-characterized CENP-B binding site (the CENP-B box). We show that AGM cells express a functional CENP-B homolog that binds to the CENP-B box and is recognized by several independent anti-CENP-B antibodies. However, three independent assays fail to reveal CENP-B binding sites in AGM DNA. Methods used include a gel mobility shift competition assay using purified AGM alpha-satellite, a novel kinetic electrophoretic mobility shift assay competition protocol using bulk genomic DNA, and bulk sequencing of 76 AGM alpha-satellite monomers. Immunofluorescence studies reveal the presence of significant levels of CENP-B antigen dispersed diffusely throughout the nuclei of interphase cells. These experiments reveal a paradox. CENP-B is highly conserved among mammals, yet its DNA binding site is conserved in human and mouse genomes but not in the AGM genome. One interpretation of these findings is that the role of CENP-B may be in the maintenance and/or organization of centromeric satellite DNA arrays rather than a more direct involvement in centromere structure.