Inactivation of a Human Kinetochore by Specific Targeting of Chromatin Modifiers

Nakano, Mimako; Cardinale, Stefano; Noskov, Vladimir N.; Gassmann, Reto; Vagnarelli, Paola; Kandels‐Lewis, Stefanie; Larionov, Vladimir; Earnshaw, William C.; Masumoto, Hiroshi

doi:10.1016/j.devcel.2008.02.001

Cited by 244 publications

(470 citation statements)

References 62 publications

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“…2B). As expected from previous studies (Cardinale et al, 2009;Nakano et al, 2008), in parallel control experiments, interphase HACs targeted by tetR-EYFP retained robust CENP-A staining for at least 2 days after transfection ( Fig. 2A).…”

Section: Acetylated Chromatin Is Compatible With the Integrity Of Thesupporting

confidence: 63%

“…Our experimental system enables us to engineer changes directly in the chromatin of a stable artificial chromosome (the alphoid tetO HAC), whose centromeric DNA contains an array of alpha-satellite repeats containing tetracycline operators (Nakano et al, 2008). Using this system, we previously found that the specific removal of the histone mark H3K4me2 from kinetochore chromatin resulted in a concomitant decrease in the level of transcription of this chromatin in human cells (Bergmann et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

“…This special chromatin, which has been termedx 'centrochromatin' (Sullivan and Karpen, 2004), suggests a functional link between the local chromatin environment and kinetochore function. Indeed, previous work from our laboratories indicates that the local chromatin state is crucial for the maintenance of a functional kinetochore structure (Bergmann et al, 2011;Cardinale et al, 2009;Nakano et al, 2008;Nakano et al, 2003;Nakashima et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…This human artificial chromosome (HAC) carrying the alphoid array with the tetO sequence replacing every second CENP-B box (hereafter referred to as the alphoid tetO HAC) exhibits a mitotic stability that is comparable to that of native chromosomes in cultured human cells (Nakano et al, 2008). We have since successfully used the alphoid tetO HAC to manipulate the chromatin environment underlying its functional kinetochore by the specific targeting of tet repressor (tetR) fusion proteins (Cardinale et al, 2009;Nakano et al, 2008). Using this approach, we recently showed that H3 dimethylated on lysine 4 (H3K4me2) is required for the long-term maintenance of the kinetochore (Bergmann et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

“…The generation of an artificial chromosome carrying a synthetic, higher-order alpha-satellite repeat array with a tet operator (tetO) sequence in every other alphoid monomer has been described previously (Nakano et al, 2008). This human artificial chromosome (HAC) carrying the alphoid array with the tetO sequence replacing every second CENP-B box (hereafter referred to as the alphoid tetO HAC) exhibits a mitotic stability that is comparable to that of native chromosomes in cultured human cells (Nakano et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

Bergmann

Jakubsche

Martins

et al. 2012

Journal of Cell Science

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103

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SummaryHuman kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-kB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ,10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ,150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity -kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

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Section: Acetylated Chromatin Is Compatible With the Integrity Of Thesupporting

confidence: 63%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

Bergmann

Jakubsche

Martins

et al. 2012

Journal of Cell Science

Self Cite

103

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Yeast ( YAC ) and Human ( HAC ) Artificial Chromosome Clones

Nagaraja¹,

Kouprina²,

Larionov³

et al. 2013

Encyclopedia of Life Sciences

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Artificial chromosome vector systems in yeast and bacteria have facilitated the increasingly rapid pace of mapping and sequencing of complex genomes. Traditional bacterial cloning systems have remained important for the study of relatively short clones, but for the cloning of very large deoxyribonucleic acid (DNA) segments yeast artificial chromosomes (YACs) have completely replaced earlier bacterial systems, including lambda phage‐based cosmids. YACs comprise cloned DNA fragments ranging from 50 kb to more than 1 million base pairs, along with sequences that render them capable of growth in yeast or bacteria. YACs provide the additional advantage of permitting direct isolation of a targeted genomic region as a circular molecule from complex genomes by transformation‐associated recombination. Moreover, YAC cloning has allowed the propagation of large tandem repeat arrays and entire bacterial genomes. By taking advantage of the efficient yeast recombination system, YAC clones can be further modified and used for functional studies of full‐length genes and for the study of huge centromeric DNA repeat regions that are not yet analysed or included in the genome assemblies of human and other organisms. Key Concepts: Basic structure of vectors used in the construction of artificial chromosomes that can replicate in yeast, bacteria and human cells. Utilities of artificial chromosomes. Isolation of complete genes and large DNA segments, including repetitive chromosomal regions. Targeted recombination‐based cloning of genes and highly repetitive regions. Reconstruction of large DNA regions by recombination among multiple YAC clones.

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New Vectors for Gene Delivery: Human and Mouse Artificial Chromosomes

Oshimura

Kazuki

Iida

et al. 2013

Encyclopedia of Life Sciences

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Human artificial chromosomes (HACs) have been generated mainly by either a ‘top‐down approach’ (engineered creation) or a ‘bottom‐up approach’ ( de novo creation). HACs with acceptor sites exhibit several characteristics required by an ideal gene delivery vector, including stable episomal maintenance and capacity to carry large genomic loci plus their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. Mouse artificial chromosomes (MACs) with acceptor sites were also created from a native mouse chromosome. The microcell‐mediated chromosome transfer (MMCT) technique for manipulating HACs and MACs in donor cells in order to deliver them to recipient cells is required for each approach. This review describes the lessons learned and prospects identified from studies on the construction of HACs and MACs and their ability to drive exogenous gene expression in cultured cells and transgenic animals via MMCT. The recent emergence of stem cell‐based tissue engineering has opened up new avenues for gene and cell therapies, and possible applications for medical use of HACs and MACs are also proposed. Key Concepts: The microcell‐mediated chromosome transfer method can transfer an intact chromosome or chromosome fragment from donor cell to recipient cell. Human artificial chromosome (HAC), which is an episomal vector derived from a native chromosome, has several advantages: it can contain the entire genome sequences of interest including its regulatory regions with long‐term stable maintenance and gene expression in human cells. The de novo HAC (bottom‐up approach) has been developed with alphoid DNA in HT1080 cells under the condition with lower H3K9me3 methylated alphoid DNA concerning cell‐type‐specific barrier for de novo CENP‐A assembly. The tet‐O HAC is a kind of de novo HAC with a conditional centromere which is constructed with alphoid DNA, and tetracycline operator sequence can be inactivated by expression of tet‐repressor fusion protein for loss of the tet‐O HAC. Mouse artificial chromosome (MAC) is a useful tool for the animal transgenesis because it can be maintained stably in mouse cells and transchromosomic mice through germline transmission. HAC/MAC have a loxP site and/or five recombination platforms to insert circular vectors like bacterial artificial chromosome or make a translocation of the targeting region from other chromosomes. HACs could be used for the complete correction of iPS cells from a patient with Duchenne muscular dystrophy. An HAC which contains Klf4, Sox2, Oct4, c‐Myc and siRNA expression against p53 could reprogramme mouse embryonic fibroblast to pluripotent stem cells. Humanised model mice using human artificial chromosomes, which contain the human CYP3A cluster and the human immunoglobulins including whole 700kb or 1Mb genomic region, have been produced. HACs/MACs might be used for a wide variety of purposes including medical and pharmaceutical applications and even for synthetic biology in sophisticated control.

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Inactivation of a Human Kinetochore by Specific Targeting of Chromatin Modifiers

Cited by 244 publications

References 62 publications

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

Yeast ( YAC ) and Human ( HAC ) Artificial Chromosome Clones

New Vectors for Gene Delivery: Human and Mouse Artificial Chromosomes

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