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Maintenance and Function of a Plant Chromosome in Human Cells Naoki Wada, Yasuhiro Kazuki, Kanako Kazuki, Toshiaki Inoue, Kiichi Fukui, and Mitsuo Oshimura ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00180 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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ACS Synthetic Biology

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Maintenance and Function of a Plant Chromosome in Human Cells

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Naoki Wada1,†, Yasuhiro Kazuki1,2, Kanako Kazuki2, Toshiaki Inoue2, Kiichi Fukui3,

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Mitsuo Oshimura1,2,✳

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Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction,

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Graduate School of Medical Science, Tottori University, Tottori, Japan

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Chromosome Engineering Research Center, Tottori University, Tottori, Japan

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Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka,

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Japan

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†

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Plant Bioengineering for Bioenergy Laboratory, Graduate School of Engineering, Osaka

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University, Osaka, Japan

Present address

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✳

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Email: [email protected]

Corresponding author: Mitsuo Oshimura

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ACS Paragon Plus Environment


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Abstract

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Replication, segregation, gene expression and inheritance are essential features of all

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eukaryotic chromosomes. To delineate the extent of conservation of chromosome functions

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between humans and plants during evolutionary history, we have generated the first human

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cell line containing an Arabidopsis chromosome. The Arabidopsis chromosome was

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mitotically stable in hybrid cells following cell division, and initially existed as a

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translocated chromosome. During culture, the translocated chromosomes then converted to

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two types of independent plant chromosomes without human DNA sequences, with the

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reproducibility. One pair of localization signals of CENP-A, a marker of functional

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centromeres was detected in the Arabidopsis genomic region in independent plant

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chromosomes. These results suggest that the chromosome maintenance system has been

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conserved between human and plants. Furthermore, the expression of plant endogenous

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genes were observed in the hybrid cells, implicating that plant chromosomal region existed

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as euchromatin in a human cell background and the gene expression system are conserved

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between two organisms. The present study suggests that the essential chromosome

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functions are conserved between evolutionarily distinct organisms such as humans and

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plants. Systematic analyses of hybrid cells may lead to the production of a shuttle vector

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between animal and plant, and a platform for the genome writing.

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For Table of Contents use only

Maintenance and Function of a Plant Chromosome in Human Cells Naoki Wada1,†, Yasuhiro Kazuki1,2, Kanako Kazuki2, Toshiaki Inoue2, Kiichi Fukui3, Mitsuo Oshimura1,2,✳

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Keywords

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Plant/animal chromosome, Chromosome functions, Hybrid cell, Evolutionary conservation,

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Centromere, Chromosomal shuttle vector

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Introduction

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A major challenge in biology is to understand the extent to which the chromosome functions

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including replication, segregation, gene expression and inheritance have been conserved

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during evolution. The hybrid cell line between different organisms would be an ideal tool to

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investigate the conservation of chromosome functions, gene expression systems, and

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chromosome evolution if the chromosomes themselves, not only each element, can be

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maintained in different cell environment 1-5. However, the successful production of

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proliferative hybrid cells was limited to sexually compatible or evolutionarily-close

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organisms1-5.

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Plants and animals were evolutionarily far-distant organisms that diverged from a

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common ancestor about 1.6 billion years ago6. This long evolutionary journey has given rise

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to the many diverse biological forms and processes seen within these kingdoms. Structural

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features of the chromosome are conserved between plants and humans. However, the extent

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to which the chromosome functions have been conserved during their evolution is largely

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unknown. The understanding of the conservation of chromosome functions is important for

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elucidating the underlying basic principles of evolutionary divergence. Several attempts

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were made between 1976-19847-9 to produce fusion between human and plant cells. In

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1976, Jones et al. reported the first interkingdom fusion between human (HeLa) cells and

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tobacco hybrid (GGLL) protoplasts7. However, the fused HeLa-GGLL cells were not viable

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beyond 6 days. Since the first trial, several attempts were made to produce fusion cells

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between human and plant cells, but the generation of proliferative hybrid cells has not been

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successful8, 9. Therefore, systematic analyses of chromosome functions between human and

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plants have never been performed so far.

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In this study, we challenged to establish the hybrid cells line by developing a new

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strategy for the production of hybrid cells between humans and plants. We established the

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novel partial hybrid cell line after 40 years since the first trial by Jones et al7. The hybrid cell

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line gives the first evidence that essential chromosome functions including replication,

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segregation, and gene expression are conserved between humans and plants. The hybrid cell

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line will be unique and ideal tool to experimentally analyze the conservation of plant

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chromosome functions in a human cell background.

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Furthermore, the plant derived chromosomes housed in human cells can be a

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starting material of a new chromosomal shuttle vector which will be a prominent tool for

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plant chromosome engineering. Recently, Boeke et al.10 have proposed the Genome

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Project-Write which requires the advance of genome-scale engineering technology and

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establishment of the ethical framework. The new chromosome shuttle vector will contribute

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to the project by developing the platform for genome-scale engineering of plant and

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mammalian genomes. The advanced technology will facilitate the production of the

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specialized chromosomes encoding one or several pathways for plant improvements.

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Results and Discussions

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Generation of a human/plant hybrid cell line

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In this study, we have established a partial hybrid cell line between human and plant

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(Arabidopsis thaliana) cells using a whole cell fusion technique. The plant Arabidopsis was

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first transformed with T-DNA containing the EGFP gene driven by the CAG promoter and

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the blasticidin S deaminase (Bsd) gene under the control of the human pGK promoter. The

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CAG and pGK promoter have high activity only in human cells, resulting in the strong

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EGFP expression only after the plant chromosome carrying marker genes was transferred

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into human cells. Protoplasts (plant cells without cell wall) were isolated from leaves of T3

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homozygous transgenic plants (referred to as ‘Arabidopsis T3 6-5′ hereafter) and used in

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cell fusion experiments.

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HT1080 cells were used as a human cell line in this study. It has been reported

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that de novo Human Artificial Chromosomes (HACs) are efficiently formed in HT1080

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cells due to its histone H3K9 acetyl/methyl balance11. A low level of heterochromatic

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modification of histone H3 in HT1080 cells allows the de novo kinetochore assembly. Thus,

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the HT1080 cells were expected to be a suitable human cell line as a host to maintain

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independent plant chromosomes.

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Based on a previous report of increased polyethylene glycol (PEG)-mediated

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fusion competence in mitotic cells of a mouse lymphoid cell line12 , the human HT1080

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cells were first synchronized at the G1/S phase by using a double thymidine block, then

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were released and incubated for 8 h to induce entry into M phase. The mitotic cells were

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harvested and fused via PEG-mediated cell fusion with the protoplasts, according to the

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protocol developed in this study (Figure 1, see Materials and Methods). After cell fusion,

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the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented

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with 10 % fetal calf serum and 6 µg/ml blasticidin. After selection, blasticidin-resistant

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cells expressing EGFP fluorescence were obtained (Figure 2a). The results indicated that

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the cell fusion was successful. The apparent morphology of the cells was not different from

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that of HT1080 cells and the cells are referred to as ‘hybrid cells’ hereafter. PCR analysis

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indicated that the hybrid cells maintained genes from both organisms: HPRT gene from

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human HT1080 cells and Bsd and EGFP genes from transgenic Arabidopsis T3 6-5

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protoplasts (Figure 2b).

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We have also successfully established a hybrid cell line between Arabidopsis and

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Chinese hamster ovary CHO cells, supporting that the cell fusion system established in this

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study is useful and applicable for other types of cells (Supplementary Figure S1).

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Generation of a human-plant chromosome with centromeres from both organisms

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We next analyzed the karyotyping of hybrid cells between human HT1080 and Arabidopsis

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T3 6-5 cells by multicolour fluorescence in situ hybridization (M-FISH) using a human

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M-FISH probe (Figure 2c). Aberrant signals were clearly present at the terminal of the long

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arm of human chromosome 15 (Figure 2c, arrow), suggesting that plant-derived

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chromosome existed as a translocated chromosome on human chromosome 15.

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To investigate the precise composition of human and plant-derived chromosomes,

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we performed FISH analysis using Arabidopsis DNAs, human Cot-1 DNA, and human

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centromere and telomere probes (Figure 3a). Human Cot-1 signals were detected along the

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entire region of all human chromosomes in human HT1080 cells. However, in the hybrid

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cells, human chromosome 15 had a partial region not stained by human Cot-1 (Figure 3a,

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upper row), supporting the notion that Arabidopsis genomic regions were translocated to

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human chromosome 15. Consistent with this notion, Arabidopsis DNA signals were

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detected in the region without human Cot-1 signals (Fig. 3a, middle row). The presence of

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the plant transformation vector was also confirmed (Supplementary Figure S2a, bottom

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row). Interestingly, we also detected Arabidopsis 180 bp centromeric repeats in the

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sub-terminal region of the chromosome (Figure 3a, AtCen). Human centromere signals

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were observed at the opposite side of the chromosome relative to the Arabidopsis 180 bp

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centromere repeats (Figure 3a, bottom row). Thus, the chromosome had two kinds of

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centromeric repeats, derived from human and Arabidopsis at two different locations. Strong

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telomere signals were observed at both sides of the chromosome. In addition, weak

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telomere signals were also often observed in the middle region (Supplementary Figure S2a

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upper row). These results demonstrated that the chromosome was formed by end-to-end

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fusion between human chromosome 15 and Arabidopsis chromosomes. This type of

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chromosome is hereafter referred to as “Plant Derived chromosome (PD chromosome)-type

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T (T stands for ‘Translocation’)”. Based on these observations, the structure of PD

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chromosome-type T is shown in Figure 3e.

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Conversion of translocated plant chromosomes to two types of independent

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chromosomes through the successive cell divisions

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PD chromosome-type T was relatively stable in the hybrid cells. FISH analysis indicated

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that 100 % of hybrid cells maintained a single copy of PD chromosome-type T after culture

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for 1 month. However, thereafter, we observed two types of structural change in PD

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chromosome-type T in a fraction of cells. These, we refer to as “PD chromosome-type S (S

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stands for ‘Small’)” and “PD chromosome-type A (A stands for ‘intrachromosomally

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Amplified’)” and both of the types existed as independent chromosomes unlike PD

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chromosome-type T as mentioned below (Figure 3b, c and Supplementary Figure S2b, c)

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FISH analysis using Arabidopsis centromeric repeats as a probe showed the

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emergence of a pair of small chromosome signals; PD chromosome-type S (Figure 3b,

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AtCen). PD chromosome-type S gave no signal when human Cot-1 DNA was used as a

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probe although all other human chromosomes were stained by human Cot-1 (Figure 3b,

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upper row). However, Arabidopsis genomic DNA produced signals over the entire region

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of PD chromosome-type S (Figure 3b, middle row). These results indicated that PD

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chromosome-type S was maintained without human DNAs in a human cell background.

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The absence of human centromeric repeats was also confirmed by FISH analysis using a

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FISH probe for human centromeres (Figure 3b, bottom row). PD chromosome-type S

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existed as a single copy or two copies and did not co-exist with PD chromosome-type T.

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This suggests a possibility that PD chromosome-type S was formed from PD

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chromosome-type T by a structural change during cell culture.

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Another change from PD chromosome-type T was observed during cell culture.

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The morphology of this altered chromosome, PD chromosome-type A, was similar to that

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of other human chromosomes (Figure 3c); however, FISH analysis using the Arabidopsis

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180 bp centromeric repeats as a probe showed amplified signals along the entire

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chromosomal region (Figure 3c, AtCen), while signals for human Cot-1 were not observed

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(Figure 3c, upper row). Arabidopsis genomic DNA signals were observed over the entire

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PD chromosome-type A (Figure 3c, middle row). These results indicated that PD

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chromosome-type A consisted of amplified Arabidopsis T3 6-5 genomic sequences without

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human genomic DNAs detectable by FISH analysis. The absence of human centromeric

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sequences was further confirmed using a FISH probe for human centromeric repeats

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(Figure 3c, bottom row). This result clearly indicated that PD chromosome-type A was

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maintained in the hybrid cells without a normal human centromere, as well as PD

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chromosome-type S. PD chromosome-type S and -type A did not co-exist with PD

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chromosome-type T, suggesting that both of them were generated by a structural change in

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PD chromosome-type T. Spontaneous reactivation of centromere of Arabidopsis genomic

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DNA and dicentric breakage might be one of the possible mechanisms.

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The localization of human CENP-A, a marker of functional centromeres, was

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investigated by simultaneous immunostaining and FISH (Figure 3d). PD chromosome-type

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T had only one pair of human CENP-A signals that did not co-localize with Arabidopsis 180

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bp centromeric repeats (Figure 3d, PD chromosome-type T). This observation supported the

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idea that only the human centromere was active in PD chromosome-type T. On the other

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hand, in PD chromosome-type S and- type A, one pair of CENP-A signals were detected in

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the Arabidopsis genomic region (Figure 3d, PD chromosome-type S, -type A). These results

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demonstrated that PD chromosome-type S and -type A were maintained by human CENP-A

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localized on Arabidopsis genomic region.

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Neocentromeres are ectopic centromeres that arise occasionally from

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noncentromeric regions of chromosomes and are functionally and structurally similar to

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endogenous centromeres. Neocentromeres arise near sites of former centromere function

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13-18

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epigenetic status is important for centromere activation. Our results demonstrate that the

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Arabidopsis genomic region has the potential to form new functional centromeres in the

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hybrid cells with a human cell background, although the precise mechanism remains to be

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clarified. By an analogy of a previous insight into neocentromeres, it is plausible that the

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changes of epigenetic status that allow the neocentromere formation occurred on the

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Arabidopsis genomic regions during the formation of PD chromosome-type S and- type A.

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High-resolution analysis is required to investigate the possibility.

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. DNA sequence itself is not an important factor for centromere function 13-18, whereas

Figure 4a shows the percentage of the cells containing each type of chromosomes

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during culture. After 30 days culture, all cells contained PD chromosome-type T, but not the

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other two types of chromosomes. After 60 days culture, the percentage of cells containing

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PD chromosome-type T decreased to 52.9 %, while that of PD chromosome-type S

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increased to 39.5 %. The percentage of cells containing PD chromosome-type S then

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decreased to 8.3 % at 90 days and 3.3 % at 120 days, while the percentage of cells

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containing PD chromosome-type A increased to 8.3 % at 60 days, 20.2 % at 90 days and

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28.3 % at 120 days. These results indicated that PD chromosome-type S was not stable

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during cell proliferation and that PD chromosome-type A was stable in the hybrid cells. The

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fact that PD chromosome-type S and -type A were not observed during the culture for the

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first 1 month implicates that the plant chromosomes require a given time period to convert

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from translocated type to independent type for change in the biological property such as

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epigenetics change. PD chromosome-type T was stably maintained after 60 days culture,

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with the percentage of cells containing the chromosome at 52.9 % at 60 days, 78 % at 90

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days and 63 % at 120 days. These results indicated that PD chromosomes could maintain

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chromosome functions, including duplication and segregation. These results also support

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the possibility that essential chromosome maintenance system is conserved between

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humans and plants.

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Further characterization of PD chromosomes

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For further structural analysis of the PD chromosomes, FISH analysis using Arabidopsis

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BAC DNAs was performed. At first, the insertion site of T-DNA containing selection

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marker genes in Arabidopsis T3 6-5 was investigated by POP-PCR analysis. The results

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indicated that the selection marker genes were integrated into Arabidopsis chromosome 3 at

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position 1,334,923 (Supplementary Figure S3). We chose the Arabidopsis BAC DNA

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containing this region (BAC T9J14 derived chromosome 3) as a probe for the FISH

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analysis. The other two BAC DNAs (BAC T1J8, T22P22 derived from chromosome 2, 5,

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respectively) were also chosen based on the results of microarray analysis of expressed

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genes from Arabidopsis as described below. The signals of each probe were detected in

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different positions on PD chromosome-type T (Figure 4b). The results indicated that PD

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chromosome had a complicated structure, consisting of the chromosomal regions from

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Arabidopsis chromosome 2 (BAC T1J8), 3 (BAC T9J14), and 5 (BAC T22P22).

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The human chromosome 15 containing PD chromosome-type T was also investigated because the results suggested that PD chromosome-type S and -type A were

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generated by a structural change in PD chromosome-type T. Arabidopsis genomic regions

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were not detected in the human chromosome 15 in the hybrid cells (Supplementary Figure

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S4). This result suggests that PD chromosome-type S and -type A are formed after

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separation from the human chromosome 15 and also supports the idea that PD

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chromosome-type S and -type A were converted from type T.

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Expression of Arabidopsis genes in hybrid cells; the conservation of gene expression

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system between humans and plants

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The conservation of gene expression systems and gene functions between different

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organisms has been investigated in several studies. Most of these studies are limited to

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in-silico comparisons among plants19 and animals 20-23. As a few examples to investigate

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the conservation of gene expression systems experimentally, Wilson et al. introduced a

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human chromosome 21 into mouse cells and analyzed the expression of the human genes in

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mice. They showed that, in equivalent mouse and human tissues, DNA sequences play a

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primary role in directing transcriptional programs 5. However, experimental studies are

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limited to the evolutionary close organisms. In order to show the feasibility of our hybrid

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cell line for gene expression analysis, we examined whether endogenous genes on the

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Arabidopsis chromosome were expressed in the hybrid cells using the Arabidopsis Oligo

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microarray. As a result, 462 Arabidopsis genes were detected to be expressed in hybrid cells

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only and not in human HT1080 cells, as shown in the heat map of Figure 5a (Yellow, red

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colors in Hybrid cells). The expressed genes were mapped mainly on chromosome 2, 3, and

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5 of Arabidopsis thaliana (Supplementary Figure S5). The genes whose expression was

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detected only in human HT1080 cells could be the human genes which cross-hybridized

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with the microarray (Blue color in Hybrid Cells).

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The reverse-transcription (RT)-PCR analysis of three highly expressed genes

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confirmed that several types of Arabidopsis genes were expressed in the hybrid cells

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(Figure 5b). For example, NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NADHU7)

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has homologues in human and Arabidopsis genomes. The expression of AtNADHU7 was

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detected in the hybrid cells and Arabidopsis protoplasts but not in human HT1080 cells.

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This demonstrated that AtNADHU7 was expressed from PD chromosomes in the human

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cell background. The Arabidopsis Aquaporin TIP1-1 (GAMMA TIP) gene also has a human

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homologue. However, for this gene, expression was detected only in the hybrid cells and

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not in Arabidopsis protoplasts or human HT1080 cells. This indicated the cell-type specific

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expression of AtGAMMA TIP from the Arabidopsis genomic region. The PATATIN-like

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protein-6 (PLP-6) gene was also selected as a representative gene that is present

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exclusively in the Arabidopsis genome; no homologue is present in the human genome.

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AtPLP-6 was expressed in the hybrid cells, suggesting that several kinds of

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Arabidopsis-specific genes could be expressed in a human cell background.

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The systematic humanization of yeast genes has also been reported recently and

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indicated that critical ancestral functions of many essential genes were conserved in a

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pathway-specific manner, despite differences in sequence, splicing, and protein interfaces24.

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Integrated information on the conservation of gene expression systems and gene functions

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would give valuable insight into understanding basic principles of living organisms. The

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hybrid cell line established in this study will be a new experimental system to

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systematically investigate the extent to which deeply divergent orthologues can stand in for

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each other like the systematic humanization of yeast genes.

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Conclusion

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Here we succeeded in the first development of hybrid cells, although partial one, between

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human HT1080 cells and plant Arabidopsis protoplasts. Using the hybrid cell line, we

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showed that the essential chromosome functions including replication, segregation, gene

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expression and inheritance are basically conserved between evolutionarily distinct

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organisms such as humans and plants. Based on the results, the hybrid cell line is expected

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to be a unique and powerful tool to examine functional conservation of chromosome

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functions, gene regulation system, and chromosome evolution over long evolutionary

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distances. The reunion of chromosomes derived from two evolutionarily far separated

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organisms, plants and humans, has the potential to open a new avenue for the better

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understanding of evolutionary alterations of chromosomes and will contribute to the

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engineering of living organisms by synthetic biology approaches. In future, systematic

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analyses of hybrid cells may lead to the production of a platform for the genome writing10.

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Furthermore, PD chromosomes in the hybrid cells can be used for developing a

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shuttle vector between plants and mammalians, if they replicate and segregate both in plant

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and mammalian cells. Multinuclei formed from clusters of mis-segregated uncondensed

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chromosome resulting from cytokinesis failure have been used for

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Microcell-Mediated-Chromosome Transfer (MMCT)25. Since the efficient multinuclei

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formation is not induced by the treatment of microtubule inhibitors such as colcemid in

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HT1080 cells, the CHO cells would be a good material for this purpose. The transfer of PD

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chromosomes into other types of animal cells is also expected to be useful to investigate

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whether PD chromosomes are stable in other type of animal cells, and it will allow more

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careful analysis of their structure and exclude the possibility that plant chromosomes gain

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some “human DNA sequences”. In addition, it has been reported that the chicken DT40

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cells have high homologous recombination efficiency and are useful for engineering of

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HACs1. The transfer of PD chromosomes into chicken DT40 cells will give unique

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opportunity to engineer the PD chromosomes by homologous recombination. Thus, the

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shuttle vector will make it possible to engineer plant chromosomes in animal cells and

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chicken DT40 cells in which chromosomes can be manipulated much more easily,

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efficiently, and freely than in plant cells. The shuttle vector system will expand the

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possibility of the production of useful plants in near future.

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Methods

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Plasmid construction

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pCAMBIA1305.1 (Cambia, Canberra, Australia) was digested with Kpn I and Sac I. The

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digested vector was ligated with a fragment containing both EGFP under the hCAG

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promoter and Bsd under the pGK promoter. The resulting plasmid, pCAM-Bsd-EGFP, was

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used to transform Arabidopsis plants. All restriction enzymes were purchased from New

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England Biolab Inc., Ipswich, MA, USA.

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Agrobacterium-mediated transformation of Arabidopsis by the floral dip method

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330

pCAM-Bsd-EGFP was transferred into Arabidopsis (Col) using the floral dip method

331

according to a protocol provided by RIKEN BioResource Center. T1 generation seeds were

332

sown on medium containing 20 µg/ml hygromycin. Germinated hygromycin-resistant seeds

333

were chosen and grown to obtain homozygous transgenic plants. A homozygous line

334

(referred to as ‘Arabidopsis T3 6-5′) was obtained from the T3 generation and used as the

335

parental plant for cell fusion experiments.

336 337

Cell culture

338

Human fibrosarcoma HT1080 cells were cultured in DMEM (Wako, Tokyo, Japan)

339

supplemented with 10 % fetal bovine serum (FBS) at 37°C in a 5 % CO2 atmosphere. The

340

hybrid cells between A. thaliana T3 6-5 protoplasts and human HT1080 cells were cultured

341

in DMEM supplemented with 10 % FBS and 6 µg/ml blasticidin (Funakoshi, Tokyo,

342

Japan).

343 344

Cell fusion

345

Human fibrosarcoma HT1080 cells and Arabidopsis T3 6-5 protoplasts were chosen as

346

parent cells for cell fusion experiments. Arabidopsis protoplasts were isolated according to

347

a previously reported protocol 26. HT1080 cells were synchronized by double thymidine

348

block. Cells (5×105 per 60 mm dish) were seeded and subjected to 2 mM thymidine

349

treatment after 1 day of culture. After 16 h, the medium was exchanged with DMEM

350

supplemented with 10 % FBS. The cells were cultured for 8 h and then cultured again for

351

16 h in the presence of 2 mM thymidine. The medium was then exchanged with DMEM

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

352

supplemented with 10 % FBS and synchronized cells were cultured for 8 h. Synchronized

353

cells were then collected by trypsin treatment and centrifugation. The 1×107 synchronized

354

cells and 1×106 Arabidopsis protoplasts were mixed and centrifuged at 270 × g for 3 min.

355

The supernatant was removed and the pellets were re-suspended in ca. 50 µl of serum-free

356

DMEM. Polyethylene glycol 1500 (Roche Diagnostics GmbH, Mannheim, Germany) was

357

then slowly added to the suspended cells followed by incubation at 37°C for 90 s. Nine

358

millilitres of DMEM supplemented with FBS were then added, and the cells were

359

transferred to 100 mm dishes and cultured overnight at 37°C in a 5 % CO2 atmosphere.

360

After 1 day of incubation, the cells were passaged to five 100 mm dishes. After 1 day of

361

culture, the medium was changed to the selection medium containing 6 µg/ml blasticidin

362

for the selection of hybrid cells. Colonies that showed blasticidin S resistance and GFP

363

expression were selected and used for the further analysis.

364 365

PCR analysis

366

Genomic DNAs were isolated from human and hybrid cells using the Gentra Puregene Cell

367

Kit (Qiagen) and from plants using the DNeasy Plant Mini Kit (Qiagen, Tokyo, Japan).

368

Genomic DNA samples were subjected to PCR analysis using Ex Taq polymerase (Takara

369

Bio Inc., Shiga, Japan). The primer sequences for each primer set are provided as

370

Supplementary Table S2. The resulting PCR products were subjected to electrophoresis and

371

visualized with ethidium bromide.

372 373

Partially Overlapping Primer-Based PCR (POP-PCR)

18


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374

POP-PCR was performed according to the method previously described27. The primers

375

designated by authors are listed in Supplementary Table S2. The PCR products were cloned

376

using TArget Clone -Plus- Kit (TOYOBO, Tokyo, Japan) and sequenced using ABI PRISM

377

3100 DNA Sequencer (Applied Biosystems, CA, USA) and BigDye terminator ver. 3.1

378

(Applied Biosystems). Sequence analysis was performed using BLASTn analysis

379

(http://www.ncbi.nlm.nih.gov/blast) to compare the isolated sequences with the Arabidopsis

380

thaliana genome sequences.

381 382

Fluorescent in situ hybridization (FISH) and Genomic in situ hybridization (GISH)

383

The preparation of chromosome spreads and FISH analysis were performed according to a

384

previously described protocol. Briefly, human HT1080 cells and hybrid cells were

385

synchronized to M phase by 0.05 µg/ml colcemid treatment for 1.5 h. The synchronized

386

cells were trypsinized and collected by centrifugation. The cells were suspended in 0.075 M

387

KCl and incubated for 15 min at room temperature. The hypotonized cell suspensions were

388

centrifuged and fixed with Carnoy’s solution (methanol/acetic acid = 3/1). Chromosome

389

spreads were prepared by dropping fixed cells onto glass slides followed by exposure to hot

390

steam. The probes were labelled with digoxigenin (11093088910, Roche Diagnostics) or

391

biotin (#11093070910, Roche Diagnostics) using a Nick Translation Kit (Roche

392

Diagnostics) or PCR labelling. The following commercial probes were used in this study:

393

Star*FISH ©Human Chromosome Pan-Centromeric Probe consisting of alpha-satellite

394

DNA repeat sequences highly conserved on centromeric region of all human chromosomes

395

(#1695, Cambio, UK) and Human Cot-1 DNA (#15279-011, Life Technologies, Carlsbad,

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

396

CA, USA). For GISH analysis, 5 µg of Arabidopsis genomic DNA was prepared from

397

Arabidopsis leaves using a DNeasy Plant Mini kit (Qiagen). The genomic DNAs were

398

labelled with biotin using a Nick Translation Kit (Roche Diagnostics) and used for staining

399

chromosome spreads. For the FISH analysis using BAC DNAs, the BAC DNAs (provided

400

from Arabidopsis Biological Resource Center) were prepared using the NucleoBond Xtra

401

Maxi Kit (Takara Bio Inc.) and labelled with digoxigenin (Roche Diagnostics) or biotin

402

(Roche Diagnostics) using a Nick Translation Kit (Roche Diagnostics). Excess amount of

403

AtCen repeats, 5S rDNA, 18S-5.8S-25S rDNA sequences were added. The detection of

404

signals was performed using the following antibodies: anti-digoxigenin-Rhodamine, Fab

405

fragment (#1-207-750, Roche Diagnostics), Avidin-FITC (#100205, Roche Diagnostics),

406

biotinylated anti-Avidin D (#BA-0300, Vector laboratories, Burlingame, CA, USA).

407

Chromosomal DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI;

408

Sigma-Aldrich, Tokyo, Japan). The chromosome spreads were observed by fluorescence

409

microscopy (Axio Imager-Z2, Carl Zeiss, Germany). For the FISH using BAC DNAs as

410

probes, images were acquired with DeltaVision OMX ver. 3 (GE Healthcare) using a 1.4

411

NA PlanApo 100× oil-immersion objective (Olympus), immersion oil (refractive index

412

1.518); and a Cascade II:512 camera (Photometrics). The z-section distance was 200 nm

413

and the total z-section thickness was set at 4–6 µm. To reconstruct high-resolution images,

414

raw images were computationally processed by softWoRx 6.0 Beta 19, using custom

415

optical transfer functions for each wavelength with Wiener filter constants from 0.002 to

416

0.012. Channel alignment was used to correct for chromatic shift. Images were adjusted

417

using the Brightness/Contrast command of ImageJ (National Institutes of Health, USA).

20


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418 419

Multicolour FISH analysis (M-FISH)

420

Multicolour FISH analysis was performed as previously described 28. Human M-FISH

421

probes were purchased from MetaSystems GmbH (Altlussheim, Germany). Microscopy

422

analysis was performed using an AxioImagerZ2 fluorescence microscope (Carl Zeiss) with

423

an HBO-103 mercury lamp and filter sets for FITC, Cy3, Texas Red, Cy5, DEAC, and

424

DAPI. Metaphase images were captured with a CoolCube I CCD camera. Digital images

425

were obtained using mFISH software (MetaSystems).

426 427

Combined Immunostaining and FISH

428

Chromosome spreads were prepared using Shadon Cytospin 4 (Thermo Scientific Japan,

429

Tokyo, Japan). Combined immunostaining and FISH was performed according to published

430

protocols with minor modifications 29, 30. Briefly, hybrid cells were incubated with 0.05

431

µg/ml colcemid for 1.5 h. Synchronized cells were collected by shaking and tapping dishes.

432

The collected cells were centrifuged at 450 g for 5 min. The precipitate was suspended in

433

10 µl/ml cytochalasin B and mixed by pipetting. The cells were centrifuged again at 450 g

434

for 5 min. The supernatant was removed and the cell density was adjusted to 1–2 × 105

435

cells/ml. The suspended cells were incubated at room temperature for 15 min and cytospun

436

onto MAS-coated glass slides (Matsunami glass Ind., Osaka, Japan) and air-dried. The

437

chromosome spreads were fixed with 4 % paraformaldehyde (PFA) for 15 min at room

438

temperature. The slides were washed with phosphate-buffered saline (PBS) three times and

439

incubated with 0.2 % Triton X-100 for 15 min. Blocking was performed with 3 % BSA in

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

440

PBS for 1 h at room temperature in a moist box. A mouse anti-CENP-A antibody (ab13939,

441

Abcam, Cambridge, MA, USA) was diluted 50 times in PBS and applied with 1 % BSA

442

onto the slides. The slides were kept at 4°C overnight in a moist box. The slides were

443

washed with PBST (0.05 % Tween 20 in PBS) three times, then fixed with 4 % PFA for 15

444

min at room temperature. After washing with PBS three times, the chromosomes were

445

denatured at 72°C for 2 min. Then the slides were hybridized with FISH probes at 37°C

446

overnight. The preparation and denaturation of FISH probes were performed as mentioned

447

above. Then, the slides were washed in wash buffer (50 % formamide/2×SSC) for 15 min at

448

37°C, followed by incubation in 2×SSC for 15 min and then washed in PBS for 5 min three

449

times. The goat anti-mouse IgG (H+L) secondary antibody conjugated with Alexa Flour®

450

488 (Life Technologies) was diluted 200 times in PBS and applied with 1 % BSA and

451

anti-digoxigenin-Rhodamine (Roche Diagnostics) and incubated for 1 h at 37°C. The slides

452

were washed with PBST for 5 min three times and chromosomal DNA was counterstained

453

with DAPI (Sigma-Aldrich). The slides were observed under a laser scanning confocal

454

fluorescence microscope (model LSM 780, Carl Zeiss).

455 456

Classification of chromosomes types

457

Based on the results of FISH analysis, each type of chromosome was defined as below. PD

458

chromosome-type T was defined by two characteristics: (1) the existence of human

459

Cot-1-positive and -negative regions on a chromosome; and (2) the presence of Arabidopsis

460

DNA on the chromosome. PD chromosome-type S was defined by three characteristics: (1)

461

the absence of human Cot-1 DNA signals; (2) the presence of Arabidopsis DNA; and (3) its

22


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462

small size compared to other chromosomes. PD chromosome-type A was defined by two

463

characteristics: (1) the presence of amplified Arabidopsis DNA; and (2) the absence of

464

human DNA. One hundred metaphases were classified at each time point (30, 60, 90 and

465

120 days) and counted.

466 467

Microarray analysis

468

The microarray experiment was performed using Arabidopsis Oligo DNA microarray ver.4

469

(Agilent Technologies, Santa Clara, CA, USA). Total RNAs were extracted from HT1080

470

cells and hybrid cells using the RNeasy Mini Kit (Qiagen). Gene expression profiles were

471

analyzed by Dragon Genomics Center (Takara Bio Inc.). The genes whose expression

472

changed more than 2-fold between hybrid cells and human HT1080 cells were picked up.

473

Three most highly expressed genes were selected and subjected to RT-PCR analysis to

474

confirm expression in hybrid cells. Total RNAs from Arabidopsis protoplasts were

475

extracted using the RNeasy Plant Mini kit (Qiagen). The extracted RNAs were subjected to

476

DNase treatment using the TURBO DNA-free kit (Life Technologies) followed by first

477

strand cDNA synthesis using a First strand cDNA synthesis Kit (GE Healthcare UK Ltd.,

478

Buckinghamshire, UK). The resulting cDNAs were used as a template for RT-PCR

479

analysis.

480 481

Supporting Information

482

Supplementary Figure S1. FISH analysis of CHO K1× ×Arabidopsis T3 6-5 hybrid cells.

483

The hybrid cells were cultured and analyzed by FISH analysis using the Arabidopsis 180

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

484

bp centromeric DNA as a probe. The left figure shows the CHO K1. The right figure shows

485

the hybrid cell between CHO K1 and Arabidopsis T3 6-5. The red color indicates the

486

Arabidopsis 180 bp centromeric repeats .Scale bar: 5 µm.

487 488

Supplementary Figure S2. FISH analysis of PD chromosomes. The vertebrate type

489

telomere repeats (TTAGGG)n and pCAM-Bsd-EGFP plasmids was used as probes for

490

FISH analysis of PD chromosomes. Red color indicates the Arabidopsis 180 bp

491

centromeric repeats (AtCen) (a, b). Green color indicates the vertebrate type telomere

492

repeats (a), pCAM-Bsd-EGFP sequence (b), respectively. Scale bar: 3 µm.

493 494

Figure S3. BLASTn result of T-DNA flanking sequences in Arabidopsis T3 6-5. The

495

isolated flanking sequence was used as a query sequence against Arabidopsis thaliana

496

genome sequences and the alignment was shown.

497 498

Figure S4. FISH analysis of PD chromosomes and human chromosome 15. The PD

499

chromosomes were stained by Arabidopsis 180 bp centromeric repeat probe (AtCen, Red).

500

The human chromosome 15 was stained by 15SAT7/8 probe31 (a) PD chromosome-type T.

501

(b) PD chromosome-type A and human chromosome 15. Scale bars: 3 µm.

502 503

Figure S5. Mapping of expressed genes from PD chromosomes in the hybrid cells on the

504

Arabidopsis chromosomes.

505

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506

Supplementary Table S1. The percentage of the cells containing each type of PD

507

chromosomes after 50 passages of newly isolated clone from the hybrid cell line.

508 509

Supplementary Table S2. Primer sequences used in this study 32, 33.

510 511

Author Information

512

Corresponding Author

513

Mitsuo Oshimura. Email: [email protected]

514 515

Author Contributions

516

N.W. performed experimental design, all experiments except M-FISH analysis, analyzed

517

data and wrote the paper. Y.K. and M.O were involved in experimental design, analyzed

518

data. K.K performed M-FISH analysis. All authors discussed the results and commented on

519

the manuscript.

520

The authors declare no competing financial interest.

521 522

Acknowledgements

523

The work was supported by a Grant-in-Aid for JSPS fellows 23-7429 from JSPS

524

KAKENHI.

525 526

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527

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628

Figure captions

629

Figure 1. Strategy for the production of hybrid cells. Protoplasts were isolated from

630

transgenic plants T3 6-5 with EGFP under control of the CAG promoter and Bsd under

631

control of the hpGK promoter. The isolated protoplasts were fused with synchronized

632

human HT1080 cells by PEG treatment. The hybrid cells were cultured in DMEM

633

supplemented with 10 % FBS and 6 µg/ml of blasticidin. The blasticidin resistant cells

634

expressing GFP fluorescence can be obtained only after the plant chromosome with GFP

635

and Bsd genes were transferred into human cells.

636 637

Figure 2. Generation of human-plant hybrid cells. (a) EGFP-positive and

638

blasticidin-resistant hybrid cells. (b) PCR analysis of hybrid cells. The hybrid cells

639

maintained the genes of both human cells (HPRT gene) and Arabidopsis T3 6-5 plants

640

(EGFP and Bsd genes). (c) Karyotype analysis of hybrid cells by M-FISH. Arrow shows

641

the human chromosome 15 with the Arabidopsis chromosomal region (PD

642

chromosome-type T). Right panels show enlarged images of human chromosome 15 and

643

PD chromosome-type T, the obtained spectrum, and their ideograms.

644 645

Figure 3. FISH analysis of PD chromosomes. FISH analysis of PD chromosome-type T

646

(A), -type S (B), -type A (C). The probes used were as follows: Arabidopsis 180 bp

647

centromeric repeats (AtCen, Red), human Cot-1, Arabidopsis DNAs, human centromere

648

(Green). Right figures show the summary of FISH results. Scale bar: 3 µm. (d) Combined

649

immunostaining and FISH analysis of PD chromosome-type T, -type-S and -type A. Red

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

650

and green indicate the Arabidopsis 180 bp centromeric repeats and human CENP-A protein,

651

respectively Scale bar: 5 µm. The enlarged image of PD chromosome-type S is also shown.

652

Scale bar: 1 µm. (e) The structures of each types of PD chromosomes were shown: PD

653

chromosome-type T (upper), -type S (middle), -type A (bottom).

654 655

Figure 4. Changes of chromosome structure during cell culture and human CENP-A

656

localization on each type of chromosome. (a) Changes of chromosome structure during

657

cell culture. The chromosome types were characterized at each time point. Chromosomes

658

with Arabidopsis regions were classified into three types based on the results of FISH

659

analysis: PD chromosome-type T, -type S and -type A. More than 100 chromosome sets

660

were counted at each time point. (b) FISH analysis of PD chromosome-type T using BAC

661

DNAs as probes. The probes used were as follows: Arabidopsis 180 bp centromeric repeats

662

(AtCen), BAC T1J8 (from Arabidopsis chromosome 2), BAC T9J14 (from Arabidopsis

663

chromosome 3), and BAC T22P22 (from Arabidopsis chromosome 5). Scale bars: 3 µm.

664 665

Figure 5. Gene expression analysis of Arabidopsis genes in the hybrid cells. (a) Heat

666

map of microarray data arranged according to a hierarchical clustering method. (b) RT-PCR

667

analysis of the three most highly expressed Arabidopsis genes according to microarray

668

analysis. The selected genes were NADH dehydrogenase (ubiquinone) Fe-S protein 7

669

(NADHU 7), Aquaporin TIP1-1 (GAMMA TIP) and PLP-6.

670

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Figures

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Maintenance and Function of a Plant Chromosome ... - ACS Publications

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