Human Artificial Chromosomes that Bypass Centromeric DNA

Logsdon, Glennis A.; Gambogi, Craig W.; Liskovykh, Mikhail; Barrey, Evelyne J.; Larionov, Vladimir; Miga, Karen H.; Heun, Patrick; Black, Ben E.

doi:10.1016/j.cell.2019.06.006

Cited by 83 publications

(99 citation statements)

References 81 publications

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“…homolog , yet sequence features of X. laevis centromeric DNA may facilitate centromere function, as has been suggested for other species (Kasinathan and Henikoff 2018) . A recent study demonstrated that certain non-repetitive chromosomal fragments contain the ability to retain CENP-A after transient targeting of HJURP to deposit CENP-A (Logsdon et al 2019) suggesting that some non-alphoid sequences are competent for CENP-A retention. These studies challenge the notion that centromeres are specified purely by epigenetic factors and motivate the investigation of DNA sequence contributions into centromere maintenance in diverse model organisms.…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of centromeric sequences inXenopus laevis

Smith

Limouse

Fryer

et al. 2020

Preprint

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Centromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). CENP-A nucleosomes maintain the centromere by designating the site for new CENP-A assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells.Xenopus laevis egg extracts assemble centromeres in vitro , providing a system for studying centromeric DNA functions. However, centromeric sequences in X. laevis have not been extensively characterized. In this study we combine CENP-A ChIP-seq with a k-mer based analysis approach to identify the X. laevis centromere repeat sequences. By in situ hybridization we show that X. laevis centromeres contain diverse repeat sequences and we map the centromere position on each X. laevis chromosome using the distribution of centromere enriched k-mers. Our identification of X. laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

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Section: Discussionmentioning

confidence: 99%

Identification and characterization of centromeric sequences inXenopus laevis

Smith

Limouse

Fryer

et al. 2020

Preprint

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“…To determine the location of CENP-A relative to methylated DNA (specifically, 5-methylcytosines), we performed IF on stretched CHM13 chromatin fibers as previously described with modifications 82,83 . Briefly, CHM13 cells were swollen in a hypotonic buffer consisting of a 1:1:1 ratio of 75 mM KCl, 0.8% NaCitrate, and dH2O for 5 min.…”

Section: Immunofluorescence (If) and Fluorescence In Situ Hybridizatimentioning

confidence: 99%

The structure, function, and evolution of a complete human chromosome 8

Logsdon

Hsieh

Mao

et al. 2020

Preprint

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The complete assembly of each human chromosome is essential for understanding human biology and evolution. Using complementary long-read sequencing technologies, we complete the first linear assembly of a human autosome, chromosome 8. Our assembly resolves the sequence of five previously long-standing gaps, including a 2.08 Mbp centromeric α-satellite array, a 644 kbp defensin copy number polymorphism important for disease risk, and an 863 kbp variable number tandem repeat at chromosome 8q21.2 that can function as a neocentromere. We show that the centromeric α-satellite array is generally methylated except for a 73 kbp hypomethylated region of diverse higher-order α-satellite enriched with CENP-A nucleosomes, consistent with the location of the kinetochore. Using a dual long-read sequencing approach, we complete the assembly of the orthologous chromosome 8 centromeric regions in chimpanzee, orangutan, and macaque for the first time to reconstruct its evolutionary history. Comparative and phylogenetic analyses show that the higher-order α-satellite structure evolved specifically in the great ape ancestor, and the centromeric region evolved with a layered symmetry, with more ancient higher-order repeats located at the periphery adjacent to monomeric α-satellites. We estimate that the mutation rate of centromeric satellite DNA is accelerated at least 2.2-fold, and this acceleration extends beyond the higher-order α-satellite into the flanking sequence.

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“…HACs form in a complex and as-yet incompletely characterized process after transfection of seeding DNA into human cells. During this relatively lengthy period (typically ~3 weeks), the HAC-seeding DNA undergoes spontaneous multimerization and in at least some cases may pass through a stage when it is transiently inserted into the arm of an endogenous chromosome 3,22 . The multimerization presumably allows it to attain a threshold size required for stable chromosome segregation 23 .…”

Section: Introductionmentioning

confidence: 99%

Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

Pesenti

Liskovykh

Okazaki

et al. 2020

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AbstractHuman Artificial Chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN) and possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.

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Human Artificial Chromosomes that Bypass Centromeric DNA

Cited by 83 publications

References 81 publications

Identification and characterization of centromeric sequences inXenopus laevis

Identification and characterization of centromeric sequences inXenopus laevis

The structure, function, and evolution of a complete human chromosome 8

Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

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