Biomedical subjects

K H Andy Choo

Publications and source records attributed to K H Andy Choo.

32 records · Page 2Linked to original sources

PubMed2004

Improved testing for CMT1A and HNPP using multiplex ligation-dependent probe amplification (MLPA) with rapid DNA preparations: comparison with the interphase FISH method.

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are the two most common peripheral neuropathies, with incidences of about 1 in 2,500. Several techniques can be used to detect the typical 1.5-Mb duplication or deletion associated with these respective conditions, but none combines simplicity with high sensitivity. MLPA is a new technique for measuring sequence dosage. We have assessed its performance for the detection of the specific 1.5-Mb duplication/deletion by prospectively testing 50 patients referred with differential diagnoses of CMT or HNPP. Probes were designed to evaluate the TEKT3, PMP22, and COX10 genes within the CMT1A/HNPP region. We have compared the results with our existing fluorescence in situ hybridization (FISH) assay, which was performed in parallel. There was concordance of results for 49 patients. Of note, one patient showed an intermediate multiplex ligation-dependent probe amplification (MLPA) result with an abnormal FISH result, which is consistent with mosaicism. The assay works equally well with either purified DNA or rapid DNA preparations made by direct cell lysis. The use of the latter significantly reduces the cost of the assay. MLPA is a sensitive, specific, robust, and cost-effective technique suitable for fast, high-throughput testing and offers distinct advantages over other testing methods.

Charcot-Marie-Tooth Disease↗

PubMed2004

Chromosome size and origin as determinants of the level of CENP-A incorporation into human centromeres.

We have expressed an EGFP-CENP-A fusion protein in human cells in order to quantitate the level of CENP-A incorporated into normal and variant human centromeres. The results revealed a 3.2-fold difference in the level of CENP-A incorporation into alpha-satellite repeat DNA-based centromeres, with the Y centromere showing the lowest level of all normal human chromosomes. Identification of individual chromosomes revealed a statistically significant, though not absolute, correlation between chromosome size and CENP-A incorporation. Analysis of three independent neocentromeres revealed a significantly reduced level of CENP-A compared to normal centromeres. Truncation of a neocentric marker chromosome to produce a minichromosome further reduced CENP-A levels, indicating a remodelling of centromeric chromatin. These results suggest a role for increased CENP-A incorporation in the faithful segregation of larger chromosomes and support a model of centromere evolution in which neocentromeres represent ancestral centromeres that, through adaptive evolution, acquire satellite repeats to facilitate the incorporation of higher numbers of CENP-A containing nucleosomes, thereby facilitating the assembly of larger kinetochore structures.

Autoantigens↗

PubMed2004

Building the centromere: from foundation proteins to 3D organization.

At each mitosis, accurate segregation of every chromosome is ensured by the assembly of a kinetochore at each centromeric locus. Six foundation kinetochore proteins that assemble hierarchically and co-dependently have been identified in vertebrates. CENP-A, Mis12, CENP-C, CENP-H and CENP-I localize to a core domain of centromeric chromatin. The sixth protein, CENP-B, although not essential in higher eukaryotes, has homologues in fission yeast that bind pericentric DNA and are essential for heterochromatin formation. Foundation kinetochore proteins have various roles and mutual interactions, and their associations with centromeric DNA and heterochromatin create structural domains that support the different functions of the centromere. Advances in molecular and microscopic techniques, coupled with rare centromere variants, have enabled us to gain fresh insights into the linear and 3D organization of centromeric chromatin.

Animals↗

PubMed2004

Evolutionary dynamics of transposable elements at the centromere.

Transposable elements are the single most abundant class of genetic material in higher eukaryotes. These elements show a genome-wide distribution but are found in disproportionate abundance at the centromeric and/or pericentric regions in a wide range of phylogenetic species. We propose at least three possible ways in which these elements could have directly contributed to the evolution of the architecture and function of the centromere in various organisms. An "extradition" mechanism also appears to have evolved, which enables the developing or established centromere to deal with the potentially disruptive effects of any subsequently arising transposable elements.

Centromere↗

PubMed2004

Centric fission--simple and complex mechanisms.

Centric fission describes a rather poorly molecularly defined process of the transverse division of a functional centromere to result in two new centric chromosomes. While centric fission occurs rarely in humans, this process has been attributed an important role in eukaryotic karyotype evolution. Recent studies have given insight into the complex molecular mechanisms that lead to apparent centric fission events, including evidence in support of a mechanism driven by centric preduplication. These studies suggest that the traditional definition of centric fission, based on gross cytogenetic and molecular cytogenetic observations, needs to be broadened. It is necessary to distinguish between simple centric fissions, that involve the direct transverse breakage of a functional centromere, and other more complex fission events at or around a centromere that may be preceded by chromosomal rearrangements.

Animals↗

PubMed2003

Analysis of mammalian proteins involved in chromatin modification reveals new metaphase centromeric proteins and distinct chromosomal distribution patterns.

We have examined the metaphase chromosomal localization of 15 proteins that have previously been described as involved in mammalian chromatin modification and/or transcriptional modulation. Immunofluorescence data indicate that all the proteins localize to human and mouse centromeres, a neocentromere, and the active centromere of a dicentric chromosome, with six of these proteins (Sin3A, PCAF, MYST, MBD2, ORC2, P300/CBP) being demonstrated at mammalian centromeres for the first time. Most of these proteins fall into two distinct chromosomal distribution patterns: (a) kinetochore-associated proteins (Sin3A, PCAF, MYST and BAF180), which colocalize with metaphase kinetochores, but not any of the pericentric and other major heterochromatic regions; and (b) heterochromatin-associated proteins (MeCP2, MBD1, MBD2, ATRX, HP1alpha, HDAC1, HDAC2, DNMT1 and DNMT3b), which colocalize with centromeric/pericentric heterochromatin and all other major heterochromatic sites. A heterogeneous third group (c) consists of the origin recognition complex subunit ORC2 and the histone acetyltransferase P300/CBP, which associate generally with kinetochores in humans and centromeric/pericentric heterochromatin in mouse, with some minor differences in localization. These observations indicate an extensive sharing of protein components involved in chromatin modification at gene loci, centromeres and various chromosomal heterochromatic landmarks. The definition of distinct patterns of chromosomal distribution for these proteins provides a useful basis for the further investigation of the broad-ranging roles of these proteins.

Animals↗

PubMed2003

Centromeric chromatin pliability and memory at a human neocentromere.

We show that Trichostatin A (TSA)-induced partial histone hyperacetylation causes a unidirectional shift in the position of a previously defined binding domain for the centromere-specific histone H3 homologue CENP-A at a human neocentromere. The shift of approximately 320 kb is fully reversible when TSA is removed, but is accompanied by an apparent reduction in the density of CENP-A per unit length of genomic DNA at the neocentromere. TSA treatment also instigates a reversible abolition of a previously defined major domain of differentially delayed replication timing that was originally established at the neocentromeric site. None of these changes has any measurable deleterious effects on mitosis or neocentromere function. The data suggest pliability of centromeric chromatin in response to epigenetic triggers, and the non-essential nature of the regions of delayed replication for centromere function. Reversibility of the CENP-A-binding position and the predominant region of delayed replication timing following removal of TSA suggest strong memory at the original site of neocentromeric chromatin formation.

Acetylation↗

PubMed2003

Transcription within a functional human centromere.

Recent data in yeast and Drosophila suggest a domain-like centromere structure with a modified chromatin core and flanking regions of heterochromatin. We have analyzed a functional human centromere and defined a region of increased chromosome scaffold/matrix attachment that overlaps three other distinct and nonoverlapping domains for constitutive centromere proteins CENP-A and CENP-H, and heterochromatin protein HP1. Transcriptional competency is intact throughout the S/MAR-enriched region and within the CENP-A- and CENP-H-associated chromatin. These results provide insights into the relationship between centromeric chromatin and transcriptional competency in vivo, highlighting the permissibility of transcription within the constitutively modified, nonheterochromatic chromatin of a functional eukaryotic centromere.

Animals↗

PubMed2003

Partially functional Cenpa-GFP fusion protein causes increased chromosome missegregation and apoptosis during mouse embryogenesis.

CENP-A is an essential histone H3-like protein that localizes to the centromeric region of eukaryotic chromosomes. Heterozygous and homozygous Cenpa-GFP fusion-protein mouse mutants, generated through targeted insertion of the green fluorescent protein (GFP) gene into the mouse Cenpa gene locus, show specific localized fluorescence at all the centromeres. Heterozygous mice are healthy and fertile. Cenpa-GFP homozygotes (Cenpag/g) undergo many cell divisions, giving rise to up to one million cells that show relatively accurate differentiation into distinct mouse embryonic tissues until day 10.5 when significant levels of chromosome missegregation, aneuploidy and apoptosis result in death. Cenpag/g embryos assemble functional kinetochores that bind to a host of centromere-specific structural and mitotic spindle checkpoint proteins (Cenpc, BubR1, Mad2 and Zw10). Examination of the nucleosomal phasing of centromeric minor and pericentromeric major satellite sequences indicates that the formation of Cenpag/g homotypic nucleosomes is not accompanied by any overt alteration to the overall size of the monomeric nucleosomal structure or the spacing of these structures. This study provides the first example of an essential centromeric protein gene variant in which subtle perturbation at the centromeric nucleosomal/chromatin level manifests in a significantly delayed lethality when compared with Cenpa null mice.

Animals↗

PubMed2003

A rapid method of genomic array analysis of scaffold/matrix attachment regions (S/MARs) identifies a 2.5-Mb region of enhanced scaffold/matrix attachment at a human neocentromere.

Human neocentromeres are fully functional centromeres that arise at previously noncentromeric regions of the genome. We have tested a rapid procedure of genomic array analysis of chromosome scaffold/matrix attachment regions (S/MARs), involving the isolation of S/MAR DNA and hybridization of this DNA to a genomic BAC/PAC array. Using this procedure, we have defined a 2.5-Mb domain of S/MAR-enriched chromatin that fully encompasses a previously mapped centromere protein-A (CENP-A)-associated domain at a human neocentromere. We have independently verified this procedure using a previously established fluorescence in situ hybridization method on salt-treated metaphase chromosomes. In silico sequence analysis of the S/MAR-enriched and surrounding regions has revealed no outstanding sequence-related predisposition. This study defines the S/MAR-enriched domain of a higher eukaryotic centromere and provides a method that has broad application for the mapping of S/MAR attachment sites over large genomic regions or throughout a genome.

Cell Line, Transformed↗

PubMed2002

Poly(ADP-ribose) polymerase 2 localizes to mammalian active centromeres and interacts with PARP-1, Cenpa, Cenpb and Bub3, but not Cenpc.

Poly(ADP-ribose) polymerase 2 (PARP-2) is a newly discovered member of the PARP family. We report the association of PARP-2 with mammalian centromeres in a cell-cycle-dependent manner, accumulating at centromeres during prometaphase and metaphase, disassociating during anaphase, and disappearing from the centromeres by telophase. Analysis of a pseudodicentric chromosome and a human neocentromere indicates that PARP-2 binding occurs only at active centromeres in a sequence-independent manner. Centromere binding peaks at the outer centromere region, and is significantly enhanced upon treatment with microtubule-inhibiting drugs. Co-immunoprecipitation assay demonstrates interaction between PARP-2 and its functional homolog PARP-1, constitutive centromere proteins Cenpa and Cenpb, and spindle checkpoint protein Bub3, but not with a third constitutive centromere protein Cenpc. These results, together with our previous demonstration that PARP-1 displays an identical binding pattern with Cenpa, Cenpb and Bub3, but not Cenpc, and that all three proteins undergo significant poly(ADP-ribosyl)ation upon gamma-irradiation of cells, point to possible diverse roles of PARP-2 and PARP-1 in modulating the structure and checkpoint functions of the mammalian centromere, in particular during radiation-induced DNA damage.

Animals↗

PubMed2002

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.

Animals↗

PubMed2002

Centromere proteins Cenpa, Cenpb, and Bub3 interact with poly(ADP-ribose) polymerase-1 protein and are poly(ADP-ribosyl)ated.

Poly(ADP-ribose) polymerase-1 (PARP-1) is activated by DNA strand breaks during cellular genotoxic stress response and catalyzes poly(ADP-ribosyl)ation of acceptor proteins. These acceptor proteins include those involved in modulation of chromatin structure, DNA synthesis, DNA repair, transcription, and cell cycle control. Thus, PARP-1 is believed to play a pivotal role in maintaining genome integrity through modulation of protein-protein and protein-DNA interactions. We previously described the association of PARP-1 with normal mammalian centromeres and human neocentromeres by affinity purification and immunofluorescence. Here we investigated the interaction of this protein with, and poly(ADP-ribosyl)ation of, three constitutive centromere proteins, Cenpa, Cenpb, and Cenpc, and a spindle checkpoint protein, Bub3. Immunoprecipitation and Western blot analyses demonstrate that Cenpa, Cenpb, and Bub3, but not Cenpc, interacted with PARP-1, and are poly(ADP-ribosyl)ated following induction of DNA damage. The results suggest a role of PARP-1 in centromere assembly/disassembly and checkpoint control. Demonstration of PARP-1-binding and poly(ADP-ribosyl)ation in three of the four proteins tested further suggests that many more centromere proteins may behave similarly and implicates PARP-1 as an important regulator of diverse centromere function.

Amino Acid Motifs↗

PubMedDate not supplied

Strategies for engineering human chromosomes with therapeutic potential.

Human engineered chromosomes (HECs) have several potential advantages over currently used vectors for gene therapy applications. Firstly, there is no upper size limit to DNA that can be cloned in these vectors. Secondly, their extrachromosomal nature ensures that introduced genes are neither disruptive to, nor affected by, the genome of the host cell. Finally, being solely human in origin, HEC vectors should not evoke adverse host immunogenic responses. Recent advances have produced a variety of HECs via several different approaches. This review focuses on the current methodologies for making HEC vectors, the advantages and problems associated with each strategy, and discusses the outlook for HEC vectors as ex vivo therapeutic agents.

Chromosomes, Artificial, Human↗