Comparative study of artificial chromosome centromeres in human and murine cells

Moralli, Daniela; Jefferson, Ann; Volpi, Emanuela V.; Monaco, Zoia Larin

doi:10.1038/ejhg.2012.296

Cited by 3 publications

(3 citation statements)

References 23 publications

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“…Previous molecular analyses have revealed that HACs are formed by amplification of the input DNA that may be accompanied by structural rearrangements, generating complex structures containing alternating blocks of α-satellite DNA and vector backbone. ,,, Because we used a hybrid α-satellite array as input DNA for HAC formation, it was possible that the resulting alphoid hybrid -HAC could have lost one of the two original arrays as a consequence of DNA reorganization in HT1080 cells. Therefore, we used oligo-FISH with labeled oligonucleotides that specifically recognize the tetO, lacO, and gal4 sequences to examine whether the HAC contains both α-satellite arrays.…”

Section: Resultsmentioning

confidence: 99%

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies

et al. 2018

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It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoidtetO-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoidhybrid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.

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Section: Resultsmentioning

confidence: 99%

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies

et al. 2018

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“…Heterochromatin itself may act as a large chromatin boundary between the core centromere on alpha satellite and the chromosome arms, since depletion or removal of heterochromatin allows centrochromatin to spread and/or chromatin domains to reposition on alpha satellite (Mravinac et al, 2009, Sullivan et al, 2011, Sullivan et al, 2016). Similar to endogenous centromeres, HAC centromeres are assembled into centrochromatin that is flanked by heterochromatin (Lam et al, 2006, Nakano et al, 2008, Ohzeki et al, 2012, Moralli et al, 2013) (Fig. 5).…”

Section: Introductionmentioning

confidence: 99%

Alpha satellite DNA biology: finding function in the recesses of the genome

2018

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Repetitive DNA, formerly referred to by the misnomer "junk DNA," comprises a majority of the human genome. One class of this DNA, alpha satellite, comprises up to 10% of the genome. Alpha satellite is enriched at all human centromere regions and is competent for de novo centromere assembly. Because of the highly repetitive nature of alpha satellite, it has been difficult to achieve genome assemblies at centromeres using traditional next-generation sequencing approaches, and thus, centromeres represent gaps in the current human genome assembly. Moreover, alpha satellite DNA is transcribed into repetitive noncoding RNA and contributes to a large portion of the transcriptome. Recent efforts to characterize these transcripts and their function have uncovered pivotal roles for satellite RNA in genome stability, including silencing "selfish" DNA elements and recruiting centromere and kinetochore proteins. This review will describe the genomic and epigenetic features of alpha satellite DNA, discuss recent findings of noncoding transcripts produced from distinct alpha satellite arrays, and address current progress in the functional understanding of this oft-neglected repetitive sequence. We will discuss unique challenges of studying human satellite DNAs and RNAs and point toward new technologies that will continue to advance our understanding of this largely untapped portion of the genome.

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“…HAC containing the entire human genomic hypoxanthine phosphoribosyltransferase (HPRT) region fully complemented the HPRT deficiency in cultured human fibrosarcoma HT1080 cells (Mejía et al 2001 ). The de novo HAC structure was relatively simple compared to normal chromosomes (Mejía et al 2001 ; Moralli et al 2009 , 2013 ) and allowed easy characterisation of their composition and chromatin environment.…”

Section: Introductionmentioning

confidence: 99%

Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology

Moralli

Monaco

2015

Chromosome Res

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De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

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Comparative study of artificial chromosome centromeres in human and murine cells

Cited by 3 publications

References 23 publications

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies

Alpha satellite DNA biology: finding function in the recesses of the genome

Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology

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