Human Artificial Chromosomes with Alpha Satellite-Based De Novo Centromeres Show Increased Frequency of Nondisjunction and Anaphase Lag

ABSTRACT Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.

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Partial deletion of alpha satellite DNA associated with reduced amounts of the centromere protein CENP-B in a mitotically stable human chromosome rearrangement.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6374 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6374-6380 ◽

Cited By ~ 34

Author(s):

R Wevrick ◽

W C Earnshaw ◽

P N Howard-Peebles ◽

H F Willard

Keyword(s):

Human Chromosome ◽

Satellite Dna ◽

Centromeric Region ◽

Chromosome Rearrangement ◽

Specific Antigen ◽

Chromosome 17 ◽

Alpha Satellite ◽

Protein Antigens ◽

Alpha Satellite Dna ◽

Human Chromosome 17

A familial, constitutionally rearranged human chromosome 17 is deleted for much of the DNA in its centromeric region but retains full mitotic centromere activity. Fluorescence in situ hybridization, pulsed-field gel electrophoresis, and Southern blot analysis of the residual centromeric region revealed a approximately 700-kb centromeric array of tandemly repeated alpha satellite DNA that was only approximately 20 to 30% as large as a normal array. This deletion was associated with a reduction in the amount of the centromere-specific antigen CENP-B detected by indirect immunofluorescence. The coincidence of the primary constriction, the small residual array of alpha satellite DNA, and the reduced amount of detectable CENP-B support the hypothesis that CENP-B is associated with alpha satellite DNA. Furthermore, the finding that both the deleted chromosome 17 and its derivative supernumerary fragment retained mitotic function and possess centromeric protein antigens suggests that human centromeres are structurally and functionally repetitive.

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Detection of an i(17q) chromosome by fluorescent in situ hybridization with a chromosome 17 alpha satellite DNA probe

Cancer Genetics and Cytogenetics ◽

10.1016/0165-4608(92)90252-4 ◽

1992 ◽

Vol 62 (2) ◽

pp. 140-143 ◽

Cited By ~ 8

Author(s):

Hitoshi Nakagawa ◽

Johji Inazawa ◽

Shinichi Misawa ◽

Shinji Tanaka ◽

Teruyuki Takashima ◽

...

Keyword(s):

In Situ Hybridization ◽

Satellite Dna ◽

Fluorescent In Situ Hybridization ◽

Dna Probe ◽

Chromosome 17 ◽

Alpha Satellite ◽

Alpha Satellite Dna

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Alpha satellite insertions and the evolutionary landscape of centromeres

10.1101/2021.03.10.434819 ◽

2021 ◽

Author(s):

Giuliana Giannuzzi ◽

Glennis A. Logsdon ◽

Nicolas Chatron ◽

Danny E. Miller ◽

Julie Reversat ◽

...

Keyword(s):

Satellite Dna ◽

Adaptive Sampling ◽

De Novo ◽

Regulatory Element ◽

Higher Order ◽

Alpha Satellite ◽

Chromosome 18 ◽

Alpha Satellite Dna ◽

Repeat Structure ◽

Human Genome Assembly

AbstractHuman centromeres are composed of alpha satellite DNA hierarchically organized as higher-order repeats and epigenetically specified by CENP-A binding. Current evolutionary models assert that new centromeres are first epigenetically established and subsequently acquire an alphoid array. We identified during routine prenatal aneuploidy diagnosis by FISH a de novo insertion of alpha satellite DNA array (~50-300 kbp) from the centromere of chromosome 18 (D18Z1) into chromosome 15q26 euchromatin. Although bound by CENP-B, this locus did not acquire centromeric functionality as demonstrated by lack of constriction and absence of CENP-A binding. We characterized the rearrangement by FISH and sequencing using Illumina, PacBio, and Nanopore adaptive sampling which revealed that the insertion was associated with a 2.8 kbp deletion and likely occurred in the paternal germline. Notably, the site was located ~10 Mbp distal from the location where a centromere was ancestrally seeded and then became inactive sometime between 20 and 25 million years ago (Mya), in the common ancestor of humans and apes. Long reads spanning either junction showed that the organization of the alphoid insertion followed the 12-mer higher-order repeat structure of the D18Z1 array. Mapping to the CHM13 human genome assembly revealed that the satellite segment transposed from a specific location of chromosome 18 centromere. The rearrangement did not directly disrupt any gene or predicted regulatory element and did not alter the epigenetic status of the surrounding region, consistent with the absence of phenotypic consequences in the carrier. This case demonstrates a likely rare but new class of structural variation that we name ‘alpha satellite insertion’. It also expands our knowledge about the evolutionary life cycle of centromeres, conveying the possibility that alphoid arrays can relocate near vestigial centromeric sites.

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