Neocentromeres: role in human disease,evolution, and centromere study

The centromere is essential for the proper segregation and inheritance of genetic information. Neocentromeres are ectopic centromeres that originate occasionally from noncentromeric regions of chromosomes. Despite the complete absence of normal centromeric alpha-satellite DNA, human neocentromeres are able to form a primary constriction and assemble a functional kinetochore. Since the discovery and characterization of the first case of a human neocentromere in our laboratory a decade ago, 60 examples of constitutional human neocentromeres distributed widely across the genome have been described. Typically, these are located on marker chromosomes that have been detected in children with developmental delay or congenital abnormalities. Neocentromeres have also been detected in at least two types of human cancer and have been experimentally induced in Drosophila. Current evidence from human and fly studies indicates that neocentromere activity is acquired epigenetically rather than by any alteration to the DNA sequence. Since human neocentromere formation is generally detrimental to the individual, its biological value must lie beyond the individual level, such as in karyotype evolution and speciation.     

Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres

Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha satellite DNA and permit analysis of centromeric DNA and chromatin organization. In this study, we present molecular cytogenetic and CENP-A chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique DNA and CENP-A chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated DNA. This is the first example of a neocentromere forming on repetitive DNA, as all other mapped neocentromeres have formed on single copy DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem DNA in centromere structure. The second neocentromere was found on a neocentric ring chromosome that contained the 8q21 tandemly repeated DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the ring chromosome. This neocentromere suggests that chromosomal rearrangement and DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.     

Neocentromeres: new insights into centromere structure, disease development, and karyotype evolution

Since the discovery of the first human neocentromere in 1993, these spontaneous, ectopic centromeres have been shown to be an astonishing example of epigenetic change within the genome. Recent research has focused on the role of neocentromeres in evolution and speciation, as well as in disease development and the understanding of the organization and epigenetic maintenance of the centromere. Here, we review recent progress in these areas of research and the significant insights gained.     

The activation of a neocentromere in Drosophila requires proximity to an endogenous centromere

The centromere is essential for proper segregation and inheritance of genetic information. Centromeres are generally regulated to occur exactly once per chromosome; failure to do so leads to chromosome loss or damage and loss of linked genetic material. The mechanism for faithful regulation of centromere activity and number is unknown. The presence of ectopic centromeres (neocentromeres) has allowed us to probe the requirements and characteristics of centromere activation, maintenance, and structure. We utilized chromosome derivatives that placed a 290-kilobase "test segment" in three different contexts within the Drosophila melanogaster genome--immediately adjacent to (1) centromeric chromatin, (2) centric heterochromatin, or (3) euchromatin. Using irradiation mutagenesis, we freed this test segment from the source chromosome and genetically assayed whether the liberated "test fragment" exhibited centromere activity. We observed that this test fragment behaved differently with respect to centromere activity when liberated from different chromosomal contexts, despite an apparent sequence identity. Test segments juxtaposed to an active centromere produced fragments with neocentromere activity, whereas test segments far from centromeres did not. Once established, neocentromere activity was stable. The imposition of neocentromere activity on juxtaposed DNA supports the hypothesis that centromere activity and identity is capable of spreading and is regulated epigenetically.     

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.     

Centromere identity: a challenge to be faced

The centromere is a genetic locus, required for faithful chromosome segregation, where spindle fibers attach to the chromosome through kinetochore. Loss of centromere or formation of multiple centromeres on a single chromosome leads to chromosome missegregation or chromosome breakage, respectively, which are detrimental for fitness and survival of a cell. Therefore, understanding the mechanism of centromere locus determination on the chromosome and perpetuation of such a locus in subsequent generation (known as centromere identity) is very fundamental to combat conditions like aneuploidy, spontaneous abortion, developmental defects, cell lethality and cancer. Recent studies have come up with different models to explain centromere identity. However, the exact mechanism still remains elusive. It has been observed that most eukaryotic centromeres are determined epigenetically rather than by a DNA sequence. The epigenetic marks that are instrumental in determining centromere identity are the histone H3 variant, CENP-A and the specialized posttranslational modification of the core histones. Here we will review the recent studies on the factors responsible for generating unique centromeric chromatin and how it perpetuates during cell division giving the present-day models. We will further focus on the probable mechanism of de novo centromere formation with an example of neocentromere. As a matter of similitude, this review will include marking extrachromosomal chromatin to be served as a partitioning locus by deposition of CENP-A homolog in budding yeast.     

Centromeric DNA characterization in the model grass Brachypodium distachyon provides insights on the evolution of the genus

Brachypodium distachyon is a well‐established model monocot plant, and its small and compact genome has been used as an accurate reference for the much larger and often polyploid genomes of cereals such as Avena sativa (oats), Hordeum vulgare (barley) and Triticum aestivum (wheat). Centromeres are indispensable functional units of chromosomes and they play a core role in genome polyploidization events during evolution. As the Brachypodium genus contains about 20 species that differ significantly in terms of their basic chromosome numbers, genome size, ploidy levels and life strategies, studying their centromeres may provide important insight into the structure and evolution of the genome in this interesting and important genus. In this study, we isolated the centromeric DNA of the B. distachyon reference line Bd21 and characterized its composition via the chromatin immunoprecipitation of the nucleosomes that contain the centromere‐specific histone CENH3. We revealed that the centromeres of Bd21 have the features of typical multicellular eukaryotic centromeres. Strikingly, these centromeres contain relatively few centromeric satellite DNAs; in particular, the centromere of chromosome 5 (Bd5) consists of only ~40 kb. Moreover, the centromeric retrotransposons in B. distachyon (CRBds) are evolutionarily young. These transposable elements are located both within and adjacent to the CENH3 binding domains, and have similar compositions. Moreover, based on the presence of CRBds in the centromeres, the species in this study can be grouped into two distinct lineages. This may provide new evidence regarding the phylogenetic relationships within the Brachypodium genus.     

Functional centromeres determine the activation time of pericentric origins of DNA replication in Saccharomyces cerevisiae

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.     

Epigenetically-Inherited Centromere and Neocentromere DNA Replicates Earliest in S-Phase

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.     

Molecular Characterization of a Maize B Chromosome Centric Sequence

Supernumerary chromosomes are widespread in the plant kingdom but little is known of their molecular nature or mechanism of origin. We report here the initial cloning of sequences from the maize B chromosome. Our analysis suggests that many sequences are highly repetitive and shared with the normal A chromosomes. However, all clones selected for B-specificity contain at least one copy of a particular repeat. Cytological mapping using B chromosome derivatives and in situ hybridization show that the B specific repeats are derived from the centric region of the chromosome. Sequence analysis of this repeat shows homology to motifs mapped to various plant and animal centromeres and to the maize neocentromere. A precise localization of these sequences among breakpoints within the B centromere and an homology to a facultative centromere, suggest a role for this sequence in centromere function.     

Evolutionary and clinical neocentromeres: two faces of the same coin?

It has been hypothesized that human clinical neocentromeres and evolutionary novel centromeres (ENC) represent two faces of the same phenomenon. However, there are only two reports of loci harboring both a novel centromere and a clinical neocentromere. We suggest that only the tip of the iceberg has been scratched because most neocentromerization events have a very low chance of being observed. In support of this view, we report here on a neocentromere at 9q33.1 that emerged in a ring chromosome of about 12 Mb. The ring was produced by a balanced rearrangement that was fortuitously discovered because of its malsegregation in the propositus. Chromatin-immunoprecipitation-on-chip experiments using anti-centromere protein (CENP)-A and anti-CENP-C antibodies strongly indicated that a novel centromeric domain was present in the ring, in a chromosomal domain where an ENC emerged in the ancestor to Old World monkeys.     

Phylogenomics of the genus Mus (Rodentia; Muridae): extensive genome repatterning is not restricted to the house mouse

The house mouse (Mus musculus) is universally adopted as the mammalian laboratory model, and it is involved in most studies of large-scale comparative genomics. Paradoxically, this taxon is rarely the index species for evolutionary analyses of genome architecture owing to its highly rearranged karyotype. To unravel the origin and nature of this extensive repatterning genome, we performed a multidirectional chromosome painting study of representative species within the genus Mus. However, the latter includes four extant subgenera (Mus, Coelomys, Nannomys and Pyromys) between which the phylogenetic relationships remain elusive despite the numerous molecular studies. Comparative genomic maps were established using chromosome-specific painting probes of the laboratory mouse and Nannomys minutoides. Hence, by integrating closely related species within Mus, this study allowed us to: (i) unambiguously resolve for the first time the long-standing controversial phylogeny, (ii) trace the evolution of genome organization in the house mouse, (iii) track rearrangements that necessitated new centromere locations, i.e. formation of neocentromere or reactivation of latent centromeres, (iv) reveal an extremely high rate of karyotypic evolution, with a 10- to 30-fold acceleration which was coincidental with subgeneric cladogenesis and (v) highlight genomic areas of interest for high-resolution studies on neocentromere formation and synteny breakpoints.     

A satellite-like sequence,representing a “clone gap” in the human genome,was likely involved in the seeding of a novel centromere in macaque

Although the human genome sequence is generally considered “finished”, the latest assembly (NCBI Build 36.1) still presents a number of gaps. Some of them are defined as “clone gaps” because they separate neighboring contigs. Evolutionary new centromeres are centromeres that repositioned along the chromosome, without marker order variation, during evolution. We have found that one human “clone gap” at 18q21.2 corresponds to an evolutionary new centromere in Old World Monkeys (OWM). The partially sequenced gap revealed a satellite-like structure. DNA stretches of the same satellite were found in the macaque (flanking the chromosome 18 centromere) and in the marmoset (New World Monkey), which was used as an outgroup. These findings strongly suggested that the repeat was present at the time of novel centromere seeding in OWM ancestor. We have provided, therefore, the first instance of a specific sequence hypothesized to have played a role in triggering the emergence of an evolutionary new centromere. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Birth of the Centromere

The centromere is the region of the eukaryotic chromosome that determines kinetochore formation and sister chromatid cohesion. Centromeres interact with spindle microtubules to ensure chromatid segregation during mitosis and homologous chromosome segregation during meiosis I. In recent years, the overall organization of centromeres in several eukaryotic species has been described, yet the mechanisms of centromere definition remain elusive. Understanding the evolutionary origin of the centromere may well elucidate aspects of its function. With such intention, we hypothesize that centromeres were derived from telomeres during the evolution of the eukaryotic chromosome. We propose that the proto-eukaryotic cell could not have evolved a nucleus without concurrently evolving a new tubulin-based cytoskeleton, the microtubules, and a specific chromosomal region that enabled the chromosome-microtubule interaction, the centromere. The repetitive nature of the subtelomeric regions that gave rise to the centromeres forced the concerted evolution of the centromeres. Although this implies the absence of a conserved primary sequence, a conserved centromere-specific structural motif could still exist and determine where in the chromosome the centromere is to be formed.To support the “centromeres-from-telomeres” hypothesis, we discuss several situations, in meiosis and mitosis, where telomeric regions took over centromeric roles. The recently discovered phenomenon of centromere repositioning is also discussed because it has revealed new insights into how neocentromeres evolve.     

First prenatally detected small supernumerary neocentromeric derivative chromosome 13 resulting in a non-mosaic partial tetrasomy 13q

Neocentromeres are functional centromeres located in non-centromeric euchromatic regions of chromosomes. The formation of neocentromeres results in conferring mitotic stability to chromosome fragments that do not contain centromeric alpha satellite DNA. We present a report of a prenatal diagnosis referred to cytogenetic studies due to ultrasound malformations such as large cisterna magna, no renal differentiation, hypotelorism and ventriculomegaly. Cytogenetic analysis of GTG-banded chromosomes from amniotic fluid cells and fetal blood cells revealed a de novo small supernumerary marker chromosome. Molecular cytogenetic studies using fluorescence in situ hybridization and comparative genomic hybridization showed this marker to be an inverted duplication of the distal portion of chromosome 13q which did not contain detectable alpha satellite DNA. The neocentromeric constriction was located at band 13q31. The presence of a functional neocentromere on this marker chromosome was confirmed by immunofluorescence with antibodies to centromere protein-C. The anatomopathologic study revealed a female fetus with facial dysmorphisms, low set ears and renal dysplasia. Ten small supernumerary neocentromeric chromosomes originating from the distal region of chromosome 13q have been reported to date. There are only three additional cases described with the location of the neocentromere in band 13q31. This is the first reported case detected prenatally.     

A centromere-specific retroviral element associated with breaks of synteny in macropodine marsupials

Studies of chromosome evolution have focused heavily on the evolution of conserved syntenic, gene-rich domains. It is obvious, however, that the centromere plays an equally important role in chromosome evolution, through its involvement in fissions, centric fusions, translocations, inversions and centric shifts. It is unclear how the centromere, either as a functioning unit of the chromosome or as a DNA sequence motif, has been involved in these processes. Marsupials of the family Macropodidae (kangaroos, wallabies, rat kangaroos and potoroos) offer unique insights into current theories expositing centromere emergence during karyotypic diversification and speciation. Tracing the genomic distribution of centromeric sequences in a model macropodine (subfamily Macropodinae: kangaroos and wallabies) species, Macropus eugenii (tammar wallaby), indicates these sequences have played an important role in chromosome evolution through possible segmental duplications associated with phylogenetically conserved breaks of synteny, pericentromeric and subtelomeric regions. Hybrids between different kangaroo species provide evidence that the centromere is unstable within this group of mammals and is involved in a large number of chromosome aberrations. A better understanding of the genetic and epigenetic factors that define centromeres and how centromeres may mediate changes in chromosome architecture are critical not only to our understanding of basic cellular functioning but also to our understanding of the process of speciation.