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

Rudd, M. Katharine; Mays, Robert W.; Schwartz, Stuart; Willard, Huntington F.

doi:10.1128/mcb.23.21.7689-7697.2003

Cited by 51 publications

(67 citation statements)

References 74 publications

(125 reference statements)

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“…To test these models and to investigate the role of genomic structure in centromere organization, we studied two different human artificial chromosomes, X4 and X5, generated from X-chromosome-derived ␣-satellite sequences (DXZ1) (21,22). Extended chromatin fibers from X4 and X5 were stained with antibodies to CENP-A.…”

Section: Resultsmentioning

confidence: 99%

“…Chromosome-specific ␣-satellite DNA was identified by using PCR-cloned products (37). Vector DNA on human artificial chromosomes was detected by using pPAC4 or pPAC4⌬oriP (21,38). Probes were labeled with biotin-16-dUTP (Roche), digoxygenin-11-dUTP (Roche), or AlexaFluor dUTPs (Molecular Probes).…”

Section: Immunofluorescence (If)-fishmentioning

confidence: 99%

“…Here, we report histone modification patterns at human centromeres and on human artificial chromosomes by using chromatin immunoprecipitation (ChIP) with a panel of antibodies that recognize specific methylated lysine residues on histone H3. We also used extended chromatin fibers to compare the arrangement of CEN chromatin at endogenous centromeres and on de novo human artificial chromosomes (21,22) that contain interrupted blocks of ␣-satellite sequences. Finally, we show that CEN chromatin is assembled on noncentromeric sequences and does not silence gene expression within the context of a functional centromere and that centromere domain organization is disrupted when the dosage of CENP-A is altered.…”

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confidence: 99%

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Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA

Lam

Boivin²,

Bonney³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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130

141

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Human centromeres are specialized chromatin domains containing the centromeric histone H3 variant CENP-A. CENP-A nucleosomes are interspersed with nucleosomes containing histone H3 dimethylated at lysine 4, distinguishing centromeric chromatin (CEN chromatin) from flanking heterochromatin that is defined by H3 lysine 9 methylation. To understand the relationship between chromatin organization and the genomic structure of human centromeres, we compared molecular profiles of three endogenous human centromeres, defined by uninterrupted higher-order α-satellite DNA, with human artificial chromosomes that contain discontinuous blocks of higher-order α-satellite DNA and noncentromeric DNA. The underlying sequence did not correlate with chromatin states, because both higher-order α-satellite DNA and noncentromeric DNA were enriched for modifications that define CEN chromatin, euchromatin, and heterochromatin. Human artificial chromosomes were also organized into distinct domains. CENP-A and heterochromatin were assembled over noncentromeric DNA, including the gene blasticidin, into nonoverlapping domains. Blasticidin transcripts were enriched at sites of CENP-A binding but not at H3 methylated at lysine 9, indicating that formation of CEN chromatin within a repetitive DNA environment does not preclude gene expression. Finally, we tested the role of centric heterochromatin as a centromeric boundary by increasing CENP-A dosage to expand the CEN domain. In response, H3 lysine 9 dimethylation, but not trimethylation, was markedly decreased at all centromeres examined. We propose that human centromere regions normally exist in a dynamic state in which a regional boundary, defined by H3 lysine 9 dimethylation, separates CEN chromatin from constitutive heterochromatin.

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Section: Resultsmentioning

confidence: 99%

Section: Immunofluorescence (If)-fishmentioning

confidence: 99%

mentioning

confidence: 99%

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Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA

Lam

Boivin²,

Bonney³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

130

141

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“…Two types of errors have been mainly observed: nondisjunction and anaphase lag; the frequency of these defects are statistically different between natural and artificial chromosomes suggesting that the latter responds differently to the mechanisms causing segregation errors. 30 Although MCs have not been tested for segregation errors, analysis of their mitotic stability revealed a decreased stability of small MCs with respect to larger molecules of the same origin, suggesting the requirements of an optimal size for full chromosome function.…”

Section: Artificial Chromosomes Can Be Obtained By In Vitro Chromosommentioning

confidence: 99%

Gene Therapy Progress and Prospects: Episomally maintained self-replicating systems

Conese¹,

Auriche

Ascenzioni

2004

Gene Ther

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The use of nonviral gene therapy vectors has been hampered by low level of transfection efficiency and lack of sustained gene expression. Episomal self-replicating systems may overcome these hurdles through their large packaging capacity, stability and reduced toxicity. This article reviews three classes of episomal molecules that have been tested with possible therapeutic genes: (1) self-replicating circular vectors, containing the Epstein-Barr virus (EBV) elements oriP and EBNA1; (2) small circular vectors containing scaffold/matrix attachment regions (S/MARs) as cis-acting elements to maintain the episomal status of the vector; (3) chromosomal vectors, based on the functional elements of the natural chromosomes. The studies reported validate the use of episomal vectors to obtain stable and prolonged gene expression, although reveal some limitations that necessitate additional work.

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“…As a positive control, we also tested constructs containing DXZ1 sequences shown previously to interact with CENP-A both at endogenous human centromeres and in previous human artificial chromosome studies (7,43). Constructs to be tested were transfected into HT1080 cells (9), and 10 to 30 drug-resistant colonies were isolated for each construct.…”

Section: Assessment Of Alpha Satellite Sequence Variation In An Indivmentioning

confidence: 99%

Sequences Associated with Centromere Competency in the Human Genome

Hayden

Strome

Merrett

et al. 2013

Molecular and Cellular Biology

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124

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Centromeres, the sites of spindle attachment during mitosis and meiosis, are located in specific positions in the human genome, normally coincident with diverse subsets of alpha satellite DNA. While there is strong evidence supporting the association of some subfamilies of alpha satellite with centromere function, the basis for establishing whether a given alpha satellite sequence is or is not designated a functional centromere is unknown, and attempts to understand the role of particular sequence features in establishing centromere identity have been limited by the near identity and repetitive nature of satellite sequences. Utilizing a broadly applicable experimental approach to test sequence competency for centromere specification, we have carried out a genomic and epigenetic functional analysis of endogenous human centromere sequences available in the current human genome assembly. The data support a model in which functionally competent sequences confer an opportunity for centromere specification, integrating genomic and epigenetic signals and promoting the concept of context-dependent centromere inheritance.

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

Cited by 51 publications

References 74 publications

Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA

Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA

Gene Therapy Progress and Prospects: Episomally maintained self-replicating systems

Sequences Associated with Centromere Competency in the Human Genome

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