Subscriber access provided by UNIVERSITY OF LEEDS
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Synthetic Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 37
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
ACS Synthetic Biology
1
Maintenance and Function of a Plant Chromosome in Human Cells
2
Naoki Wada1,†, Yasuhiro Kazuki1,2, Kanako Kazuki2, Toshiaki Inoue2, Kiichi Fukui3,
3
Mitsuo Oshimura1,2,✳
4 5
1
Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction,
6
Graduate School of Medical Science, Tottori University, Tottori, Japan
7
2
Chromosome Engineering Research Center, Tottori University, Tottori, Japan
8
3
Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka,
9
Japan
10 11
†
12
Plant Bioengineering for Bioenergy Laboratory, Graduate School of Engineering, Osaka
13
University, Osaka, Japan
Present address
14 15
✳
16
Email:
[email protected] Corresponding author: Mitsuo Oshimura
17 18 19 20 21 22
1
ACS Paragon Plus Environment
ACS Synthetic Biology
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
23
Abstract
24
Replication, segregation, gene expression and inheritance are essential features of all
25
eukaryotic chromosomes. To delineate the extent of conservation of chromosome functions
26
between humans and plants during evolutionary history, we have generated the first human
27
cell line containing an Arabidopsis chromosome. The Arabidopsis chromosome was
28
mitotically stable in hybrid cells following cell division, and initially existed as a
29
translocated chromosome. During culture, the translocated chromosomes then converted to
30
two types of independent plant chromosomes without human DNA sequences, with the
31
reproducibility. One pair of localization signals of CENP-A, a marker of functional
32
centromeres was detected in the Arabidopsis genomic region in independent plant
33
chromosomes. These results suggest that the chromosome maintenance system has been
34
conserved between human and plants. Furthermore, the expression of plant endogenous
35
genes were observed in the hybrid cells, implicating that plant chromosomal region existed
36
as euchromatin in a human cell background and the gene expression system are conserved
37
between two organisms. The present study suggests that the essential chromosome
38
functions are conserved between evolutionarily distinct organisms such as humans and
39
plants. Systematic analyses of hybrid cells may lead to the production of a shuttle vector
40
between animal and plant, and a platform for the genome writing.
2
ACS Paragon Plus Environment
Page 2 of 37
Page 3 of 37
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
ACS Synthetic Biology
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,✳
41 42 43 44
3
ACS Paragon Plus Environment
ACS Synthetic Biology
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
45
Keywords
46
Plant/animal chromosome, Chromosome functions, Hybrid cell, Evolutionary conservation,
47
Centromere, Chromosomal shuttle vector
48 49
Introduction
50
A major challenge in biology is to understand the extent to which the chromosome functions
51
including replication, segregation, gene expression and inheritance have been conserved
52
during evolution. The hybrid cell line between different organisms would be an ideal tool to
53
investigate the conservation of chromosome functions, gene expression systems, and
54
chromosome evolution if the chromosomes themselves, not only each element, can be
55
maintained in different cell environment 1-5. However, the successful production of
56
proliferative hybrid cells was limited to sexually compatible or evolutionarily-close
57
organisms1-5.
58
Plants and animals were evolutionarily far-distant organisms that diverged from a
59
common ancestor about 1.6 billion years ago6. This long evolutionary journey has given rise
60
to the many diverse biological forms and processes seen within these kingdoms. Structural
61
features of the chromosome are conserved between plants and humans. However, the extent
62
to which the chromosome functions have been conserved during their evolution is largely
63
unknown. The understanding of the conservation of chromosome functions is important for
64
elucidating the underlying basic principles of evolutionary divergence. Several attempts
65
were made between 1976-19847-9 to produce fusion between human and plant cells. In
66
1976, Jones et al. reported the first interkingdom fusion between human (HeLa) cells and
4
ACS Paragon Plus Environment
Page 4 of 37
Page 5 of 37
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
ACS Synthetic Biology
67
tobacco hybrid (GGLL) protoplasts7. However, the fused HeLa-GGLL cells were not viable
68
beyond 6 days. Since the first trial, several attempts were made to produce fusion cells
69
between human and plant cells, but the generation of proliferative hybrid cells has not been
70
successful8, 9. Therefore, systematic analyses of chromosome functions between human and
71
plants have never been performed so far.
72
In this study, we challenged to establish the hybrid cells line by developing a new
73
strategy for the production of hybrid cells between humans and plants. We established the
74
novel partial hybrid cell line after 40 years since the first trial by Jones et al7. The hybrid cell
75
line gives the first evidence that essential chromosome functions including replication,
76
segregation, and gene expression are conserved between humans and plants. The hybrid cell
77
line will be unique and ideal tool to experimentally analyze the conservation of plant
78
chromosome functions in a human cell background.
79
Furthermore, the plant derived chromosomes housed in human cells can be a
80
starting material of a new chromosomal shuttle vector which will be a prominent tool for
81
plant chromosome engineering. Recently, Boeke et al.10 have proposed the Genome
82
Project-Write which requires the advance of genome-scale engineering technology and
83
establishment of the ethical framework. The new chromosome shuttle vector will contribute
84
to the project by developing the platform for genome-scale engineering of plant and
85
mammalian genomes. The advanced technology will facilitate the production of the
86
specialized chromosomes encoding one or several pathways for plant improvements.
87
5
ACS Paragon Plus Environment
ACS Synthetic Biology
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
88
Results and Discussions
89
Generation of a human/plant hybrid cell line
90
In this study, we have established a partial hybrid cell line between human and plant
91
(Arabidopsis thaliana) cells using a whole cell fusion technique. The plant Arabidopsis was
92
first transformed with T-DNA containing the EGFP gene driven by the CAG promoter and
93
the blasticidin S deaminase (Bsd) gene under the control of the human pGK promoter. The
94
CAG and pGK promoter have high activity only in human cells, resulting in the strong
95
EGFP expression only after the plant chromosome carrying marker genes was transferred
96
into human cells. Protoplasts (plant cells without cell wall) were isolated from leaves of T3
97
homozygous transgenic plants (referred to as ‘Arabidopsis T3 6-5′ hereafter) and used in
98
cell fusion experiments.
99
HT1080 cells were used as a human cell line in this study. It has been reported
100
that de novo Human Artificial Chromosomes (HACs) are efficiently formed in HT1080
101
cells due to its histone H3K9 acetyl/methyl balance11. A low level of heterochromatic
102
modification of histone H3 in HT1080 cells allows the de novo kinetochore assembly. Thus,
103
the HT1080 cells were expected to be a suitable human cell line as a host to maintain
104
independent plant chromosomes.
105
Based on a previous report of increased polyethylene glycol (PEG)-mediated
106
fusion competence in mitotic cells of a mouse lymphoid cell line12 , the human HT1080
107
cells were first synchronized at the G1/S phase by using a double thymidine block, then
108
were released and incubated for 8 h to induce entry into M phase. The mitotic cells were
109
harvested and fused via PEG-mediated cell fusion with the protoplasts, according to the
6
ACS Paragon Plus Environment
Page 6 of 37
Page 7 of 37
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
ACS Synthetic Biology
110
protocol developed in this study (Figure 1, see Materials and Methods). After cell fusion,
111
the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
112
with 10 % fetal calf serum and 6 µg/ml blasticidin. After selection, blasticidin-resistant
113
cells expressing EGFP fluorescence were obtained (Figure 2a). The results indicated that
114
the cell fusion was successful. The apparent morphology of the cells was not different from
115
that of HT1080 cells and the cells are referred to as ‘hybrid cells’ hereafter. PCR analysis
116
indicated that the hybrid cells maintained genes from both organisms: HPRT gene from
117
human HT1080 cells and Bsd and EGFP genes from transgenic Arabidopsis T3 6-5
118
protoplasts (Figure 2b).
119
We have also successfully established a hybrid cell line between Arabidopsis and
120
Chinese hamster ovary CHO cells, supporting that the cell fusion system established in this
121
study is useful and applicable for other types of cells (Supplementary Figure S1).
122 123
Generation of a human-plant chromosome with centromeres from both organisms
124
We next analyzed the karyotyping of hybrid cells between human HT1080 and Arabidopsis
125
T3 6-5 cells by multicolour fluorescence in situ hybridization (M-FISH) using a human
126
M-FISH probe (Figure 2c). Aberrant signals were clearly present at the terminal of the long
127
arm of human chromosome 15 (Figure 2c, arrow), suggesting that plant-derived
128
chromosome existed as a translocated chromosome on human chromosome 15.
129
To investigate the precise composition of human and plant-derived chromosomes,
130
we performed FISH analysis using Arabidopsis DNAs, human Cot-1 DNA, and human
131
centromere and telomere probes (Figure 3a). Human Cot-1 signals were detected along the
7
ACS Paragon Plus Environment
ACS Synthetic Biology
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
132
entire region of all human chromosomes in human HT1080 cells. However, in the hybrid
133
cells, human chromosome 15 had a partial region not stained by human Cot-1 (Figure 3a,
134
upper row), supporting the notion that Arabidopsis genomic regions were translocated to
135
human chromosome 15. Consistent with this notion, Arabidopsis DNA signals were
136
detected in the region without human Cot-1 signals (Fig. 3a, middle row). The presence of
137
the plant transformation vector was also confirmed (Supplementary Figure S2a, bottom
138
row). Interestingly, we also detected Arabidopsis 180 bp centromeric repeats in the
139
sub-terminal region of the chromosome (Figure 3a, AtCen). Human centromere signals
140
were observed at the opposite side of the chromosome relative to the Arabidopsis 180 bp
141
centromere repeats (Figure 3a, bottom row). Thus, the chromosome had two kinds of
142
centromeric repeats, derived from human and Arabidopsis at two different locations. Strong
143
telomere signals were observed at both sides of the chromosome. In addition, weak
144
telomere signals were also often observed in the middle region (Supplementary Figure S2a
145
upper row). These results demonstrated that the chromosome was formed by end-to-end
146
fusion between human chromosome 15 and Arabidopsis chromosomes. This type of
147
chromosome is hereafter referred to as “Plant Derived chromosome (PD chromosome)-type
148
T (T stands for ‘Translocation’)”. Based on these observations, the structure of PD
149
chromosome-type T is shown in Figure 3e.
150 151
Conversion of translocated plant chromosomes to two types of independent
152
chromosomes through the successive cell divisions
153
PD chromosome-type T was relatively stable in the hybrid cells. FISH analysis indicated
8
ACS Paragon Plus Environment
Page 8 of 37
Page 9 of 37
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
ACS Synthetic Biology
154
that 100 % of hybrid cells maintained a single copy of PD chromosome-type T after culture
155
for 1 month. However, thereafter, we observed two types of structural change in PD
156
chromosome-type T in a fraction of cells. These, we refer to as “PD chromosome-type S (S
157
stands for ‘Small’)” and “PD chromosome-type A (A stands for ‘intrachromosomally
158
Amplified’)” and both of the types existed as independent chromosomes unlike PD
159
chromosome-type T as mentioned below (Figure 3b, c and Supplementary Figure S2b, c)
160
FISH analysis using Arabidopsis centromeric repeats as a probe showed the
161
emergence of a pair of small chromosome signals; PD chromosome-type S (Figure 3b,
162
AtCen). PD chromosome-type S gave no signal when human Cot-1 DNA was used as a
163
probe although all other human chromosomes were stained by human Cot-1 (Figure 3b,
164
upper row). However, Arabidopsis genomic DNA produced signals over the entire region
165
of PD chromosome-type S (Figure 3b, middle row). These results indicated that PD
166
chromosome-type S was maintained without human DNAs in a human cell background.
167
The absence of human centromeric repeats was also confirmed by FISH analysis using a
168
FISH probe for human centromeres (Figure 3b, bottom row). PD chromosome-type S
169
existed as a single copy or two copies and did not co-exist with PD chromosome-type T.
170
This suggests a possibility that PD chromosome-type S was formed from PD
171
chromosome-type T by a structural change during cell culture.
172
Another change from PD chromosome-type T was observed during cell culture.
173
The morphology of this altered chromosome, PD chromosome-type A, was similar to that
174
of other human chromosomes (Figure 3c); however, FISH analysis using the Arabidopsis
175
180 bp centromeric repeats as a probe showed amplified signals along the entire
9
ACS Paragon Plus Environment
ACS Synthetic Biology
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
176
chromosomal region (Figure 3c, AtCen), while signals for human Cot-1 were not observed
177
(Figure 3c, upper row). Arabidopsis genomic DNA signals were observed over the entire
178
PD chromosome-type A (Figure 3c, middle row). These results indicated that PD
179
chromosome-type A consisted of amplified Arabidopsis T3 6-5 genomic sequences without
180
human genomic DNAs detectable by FISH analysis. The absence of human centromeric
181
sequences was further confirmed using a FISH probe for human centromeric repeats
182
(Figure 3c, bottom row). This result clearly indicated that PD chromosome-type A was
183
maintained in the hybrid cells without a normal human centromere, as well as PD
184
chromosome-type S. PD chromosome-type S and -type A did not co-exist with PD
185
chromosome-type T, suggesting that both of them were generated by a structural change in
186
PD chromosome-type T. Spontaneous reactivation of centromere of Arabidopsis genomic
187
DNA and dicentric breakage might be one of the possible mechanisms.
188
The localization of human CENP-A, a marker of functional centromeres, was
189
investigated by simultaneous immunostaining and FISH (Figure 3d). PD chromosome-type
190
T had only one pair of human CENP-A signals that did not co-localize with Arabidopsis 180
191
bp centromeric repeats (Figure 3d, PD chromosome-type T). This observation supported the
192
idea that only the human centromere was active in PD chromosome-type T. On the other
193
hand, in PD chromosome-type S and- type A, one pair of CENP-A signals were detected in
194
the Arabidopsis genomic region (Figure 3d, PD chromosome-type S, -type A). These results
195
demonstrated that PD chromosome-type S and -type A were maintained by human CENP-A
196
localized on Arabidopsis genomic region.
197
Neocentromeres are ectopic centromeres that arise occasionally from
10
ACS Paragon Plus Environment
Page 10 of 37
Page 11 of 37
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
ACS Synthetic Biology
198
noncentromeric regions of chromosomes and are functionally and structurally similar to
199
endogenous centromeres. Neocentromeres arise near sites of former centromere function
200
13-18
201
epigenetic status is important for centromere activation. Our results demonstrate that the
202
Arabidopsis genomic region has the potential to form new functional centromeres in the
203
hybrid cells with a human cell background, although the precise mechanism remains to be
204
clarified. By an analogy of a previous insight into neocentromeres, it is plausible that the
205
changes of epigenetic status that allow the neocentromere formation occurred on the
206
Arabidopsis genomic regions during the formation of PD chromosome-type S and- type A.
207
High-resolution analysis is required to investigate the possibility.
208
. 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
209
during culture. After 30 days culture, all cells contained PD chromosome-type T, but not the
210
other two types of chromosomes. After 60 days culture, the percentage of cells containing
211
PD chromosome-type T decreased to 52.9 %, while that of PD chromosome-type S
212
increased to 39.5 %. The percentage of cells containing PD chromosome-type S then
213
decreased to 8.3 % at 90 days and 3.3 % at 120 days, while the percentage of cells
214
containing PD chromosome-type A increased to 8.3 % at 60 days, 20.2 % at 90 days and
215
28.3 % at 120 days. These results indicated that PD chromosome-type S was not stable
216
during cell proliferation and that PD chromosome-type A was stable in the hybrid cells. The
217
fact that PD chromosome-type S and -type A were not observed during the culture for the
218
first 1 month implicates that the plant chromosomes require a given time period to convert
219
from translocated type to independent type for change in the biological property such as
11
ACS Paragon Plus Environment
ACS Synthetic Biology
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
220
epigenetics change. PD chromosome-type T was stably maintained after 60 days culture,
221
with the percentage of cells containing the chromosome at 52.9 % at 60 days, 78 % at 90
222
days and 63 % at 120 days. These results indicated that PD chromosomes could maintain
223
chromosome functions, including duplication and segregation. These results also support
224
the possibility that essential chromosome maintenance system is conserved between
225
humans and plants.
226 227
Further characterization of PD chromosomes
228
For further structural analysis of the PD chromosomes, FISH analysis using Arabidopsis
229
BAC DNAs was performed. At first, the insertion site of T-DNA containing selection
230
marker genes in Arabidopsis T3 6-5 was investigated by POP-PCR analysis. The results
231
indicated that the selection marker genes were integrated into Arabidopsis chromosome 3 at
232
position 1,334,923 (Supplementary Figure S3). We chose the Arabidopsis BAC DNA
233
containing this region (BAC T9J14 derived chromosome 3) as a probe for the FISH
234
analysis. The other two BAC DNAs (BAC T1J8, T22P22 derived from chromosome 2, 5,
235
respectively) were also chosen based on the results of microarray analysis of expressed
236
genes from Arabidopsis as described below. The signals of each probe were detected in
237
different positions on PD chromosome-type T (Figure 4b). The results indicated that PD
238
chromosome had a complicated structure, consisting of the chromosomal regions from
239
Arabidopsis chromosome 2 (BAC T1J8), 3 (BAC T9J14), and 5 (BAC T22P22).
240 241
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
12
ACS Paragon Plus Environment
Page 12 of 37
Page 13 of 37
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
ACS Synthetic Biology
242
generated by a structural change in PD chromosome-type T. Arabidopsis genomic regions
243
were not detected in the human chromosome 15 in the hybrid cells (Supplementary Figure
244
S4). This result suggests that PD chromosome-type S and -type A are formed after
245
separation from the human chromosome 15 and also supports the idea that PD
246
chromosome-type S and -type A were converted from type T.
247 248
Expression of Arabidopsis genes in hybrid cells; the conservation of gene expression
249
system between humans and plants
250
The conservation of gene expression systems and gene functions between different
251
organisms has been investigated in several studies. Most of these studies are limited to
252
in-silico comparisons among plants19 and animals 20-23. As a few examples to investigate
253
the conservation of gene expression systems experimentally, Wilson et al. introduced a
254
human chromosome 21 into mouse cells and analyzed the expression of the human genes in
255
mice. They showed that, in equivalent mouse and human tissues, DNA sequences play a
256
primary role in directing transcriptional programs 5. However, experimental studies are
257
limited to the evolutionary close organisms. In order to show the feasibility of our hybrid
258
cell line for gene expression analysis, we examined whether endogenous genes on the
259
Arabidopsis chromosome were expressed in the hybrid cells using the Arabidopsis Oligo
260
microarray. As a result, 462 Arabidopsis genes were detected to be expressed in hybrid cells
261
only and not in human HT1080 cells, as shown in the heat map of Figure 5a (Yellow, red
262
colors in Hybrid cells). The expressed genes were mapped mainly on chromosome 2, 3, and
263
5 of Arabidopsis thaliana (Supplementary Figure S5). The genes whose expression was
13
ACS Paragon Plus Environment
ACS Synthetic Biology
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
264
detected only in human HT1080 cells could be the human genes which cross-hybridized
265
with the microarray (Blue color in Hybrid Cells).
266
The reverse-transcription (RT)-PCR analysis of three highly expressed genes
267
confirmed that several types of Arabidopsis genes were expressed in the hybrid cells
268
(Figure 5b). For example, NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NADHU7)
269
has homologues in human and Arabidopsis genomes. The expression of AtNADHU7 was
270
detected in the hybrid cells and Arabidopsis protoplasts but not in human HT1080 cells.
271
This demonstrated that AtNADHU7 was expressed from PD chromosomes in the human
272
cell background. The Arabidopsis Aquaporin TIP1-1 (GAMMA TIP) gene also has a human
273
homologue. However, for this gene, expression was detected only in the hybrid cells and
274
not in Arabidopsis protoplasts or human HT1080 cells. This indicated the cell-type specific
275
expression of AtGAMMA TIP from the Arabidopsis genomic region. The PATATIN-like
276
protein-6 (PLP-6) gene was also selected as a representative gene that is present
277
exclusively in the Arabidopsis genome; no homologue is present in the human genome.
278
AtPLP-6 was expressed in the hybrid cells, suggesting that several kinds of
279
Arabidopsis-specific genes could be expressed in a human cell background.
280
The systematic humanization of yeast genes has also been reported recently and
281
indicated that critical ancestral functions of many essential genes were conserved in a
282
pathway-specific manner, despite differences in sequence, splicing, and protein interfaces24.
283
Integrated information on the conservation of gene expression systems and gene functions
284
would give valuable insight into understanding basic principles of living organisms. The
285
hybrid cell line established in this study will be a new experimental system to
14
ACS Paragon Plus Environment
Page 14 of 37
Page 15 of 37
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
ACS Synthetic Biology
286
systematically investigate the extent to which deeply divergent orthologues can stand in for
287
each other like the systematic humanization of yeast genes.
288 289
Conclusion
290
Here we succeeded in the first development of hybrid cells, although partial one, between
291
human HT1080 cells and plant Arabidopsis protoplasts. Using the hybrid cell line, we
292
showed that the essential chromosome functions including replication, segregation, gene
293
expression and inheritance are basically conserved between evolutionarily distinct
294
organisms such as humans and plants. Based on the results, the hybrid cell line is expected
295
to be a unique and powerful tool to examine functional conservation of chromosome
296
functions, gene regulation system, and chromosome evolution over long evolutionary
297
distances. The reunion of chromosomes derived from two evolutionarily far separated
298
organisms, plants and humans, has the potential to open a new avenue for the better
299
understanding of evolutionary alterations of chromosomes and will contribute to the
300
engineering of living organisms by synthetic biology approaches. In future, systematic
301
analyses of hybrid cells may lead to the production of a platform for the genome writing10.
302
Furthermore, PD chromosomes in the hybrid cells can be used for developing a
303
shuttle vector between plants and mammalians, if they replicate and segregate both in plant
304
and mammalian cells. Multinuclei formed from clusters of mis-segregated uncondensed
305
chromosome resulting from cytokinesis failure have been used for
306
Microcell-Mediated-Chromosome Transfer (MMCT)25. Since the efficient multinuclei
307
formation is not induced by the treatment of microtubule inhibitors such as colcemid in
15
ACS Paragon Plus Environment
ACS Synthetic Biology
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
308
HT1080 cells, the CHO cells would be a good material for this purpose. The transfer of PD
309
chromosomes into other types of animal cells is also expected to be useful to investigate
310
whether PD chromosomes are stable in other type of animal cells, and it will allow more
311
careful analysis of their structure and exclude the possibility that plant chromosomes gain
312
some “human DNA sequences”. In addition, it has been reported that the chicken DT40
313
cells have high homologous recombination efficiency and are useful for engineering of
314
HACs1. The transfer of PD chromosomes into chicken DT40 cells will give unique
315
opportunity to engineer the PD chromosomes by homologous recombination. Thus, the
316
shuttle vector will make it possible to engineer plant chromosomes in animal cells and
317
chicken DT40 cells in which chromosomes can be manipulated much more easily,
318
efficiently, and freely than in plant cells. The shuttle vector system will expand the
319
possibility of the production of useful plants in near future.
320 321
Methods
322
Plasmid construction
323
pCAMBIA1305.1 (Cambia, Canberra, Australia) was digested with Kpn I and Sac I. The
324
digested vector was ligated with a fragment containing both EGFP under the hCAG
325
promoter and Bsd under the pGK promoter. The resulting plasmid, pCAM-Bsd-EGFP, was
326
used to transform Arabidopsis plants. All restriction enzymes were purchased from New
327
England Biolab Inc., Ipswich, MA, USA.
328 329
Agrobacterium-mediated transformation of Arabidopsis by the floral dip method
16
ACS Paragon Plus Environment
Page 16 of 37
Page 17 of 37
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
ACS Synthetic Biology
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
ACS Paragon Plus Environment
ACS Synthetic Biology
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
ACS Paragon Plus Environment
Page 18 of 37
Page 19 of 37
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
ACS Synthetic Biology
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
ACS Paragon Plus Environment
ACS Synthetic Biology
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
ACS Paragon Plus Environment
Page 20 of 37
Page 21 of 37
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
ACS Synthetic Biology
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
ACS Paragon Plus Environment
ACS Synthetic Biology
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
ACS Paragon Plus Environment
Page 22 of 37
Page 23 of 37
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
ACS Synthetic Biology
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
ACS Paragon Plus Environment
ACS Synthetic Biology
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
24
ACS Paragon Plus Environment
Page 24 of 37
Page 25 of 37
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
ACS Synthetic Biology
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
References
527
[1] Oshimura, M., Uno, N., Kazuki, Y., Katoh, M., and Inoue, T. (2015) A pathway from
25
ACS Paragon Plus Environment
ACS Synthetic Biology
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
528
chromosome transfer to engineering resulting in human and mouse artificial chromosomes
529
for a variety of applications to bio-medical challenges. Chromosome Res. 23, 111-133.
530
[2] Tomizuka, K., Yoshida, H., Uejima, H., Kugoh, H., Sato, K., Ohguma, A., Hayasaka, M.,
531
Hanaoka, K., Oshimura, M., and Ishida, I. (1997) Functional expression and germline
532
transmission of a human chromosome fragment in chimaeric mice. Nat. Genet. 16,
533
133-143.
534
[3] Ramulu, K. S., Dijkhuis, P., Rutgers, E., Blaas, J., Krens, F. A., Dons, J. J. M.,
535
ColijnHooymans, C. M., and Verhoeven, H. A. (1996) Microprotoplast mediated transfer of
536
single specific chromosomes between sexually incompatible plants. Genome 39, 921-933.
537
[4] Ananiev, E. V., RieraLizarazu, O., Rines, H. W., and Phillips, R. L. (1997) Oat-maize
538
chromosome addition lines: A new system for mapping the maize genome. Proc. Natl. Acad.
539
Sci. USA 94, 3524-3529.
540
[5] Wilson, M. D., Barbosa-Morais, N. L., Schmidt, D., Conboy, C. M., Vanes, L.,
541
Tybulewicz, V. L., Fisher, E. M., Tavare, S., and Odom, D. T. (2008) Species-specific
542
transcription in mice carrying human chromosome 21. Science 322, 434-438.
543
[6] Wang, D. Y., Kumar, S., Hedges, S. B. (1999) Divergence time estimates for the early
544
history of animal phyla and the origin of plants, animals and fungi. Proc. R. Soc. Lond. 266,
545
163-171.
546
[7] Jones, C. W., Mastrangelo, I. A., Smith, H. H., Liu, H. Z., and Meck, R. A. (1976)
547
Interkingdom fusion between human (HeLa) cells and tobacco hybrid (GGLL) protoplasts.
548
Science 193, 401-403.
549
[8] Lermontova, I., Fuchs, J., Schubert, V., and Schubert, I. (2007) Loading time of the
26
ACS Paragon Plus Environment
Page 26 of 37
Page 27 of 37
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
ACS Synthetic Biology
550
centromeric histone H3 variant differs between plants and animals. Chromosoma 116,
551
507-510.
552
[9] Cocking, E. C. (1984) Plant animal-cell fusions. Ciba Foundation Symposia 103,
553
119-123.
554
[10] Boeke, J. D., Church, G., Hessel, A., Kelley, N. J., Arkin, A., Cai, Y., Carlson, R.,
555
Chakravarti, A., Cornish, V. W., Holt, L., Isaacs, F. J., Kuiken, T., Lajoie, M., Lessor, T.,
556
Lunshof, J., Maurano, M. T., Mitchell, L. A., Rine, J., Rosser, S., Sanjana, N. E., Silver, P.
557
A., Valle, D., Wang, H., Way, J. C., and Yang, L. (2016) The Genome Project-Write.
558
Science.
559
[11] Ohzeki, J., Bergmann, J. H., Kouprina, N., Noskov, V. N., Nakano, M., Kimura, H.,
560
Earnshaw, W. C., Larionov, V., and Masumoto, H. (2012) Breaking the HAC Barrier:
561
Histone H3K9 acetyl/methyl balance regulates CENP-A assembly. EMBO J. 31,
562
2391-2402.
563
[12] Hansen, D., and Stadler, J. (1977) INCREASED POLYETHYLENE
564
GLYCOL-MEDIATED FUSION COMPETENCE IN MITOTIC CELLS OF A MOUSE
565
LYMPHOID-CELL LINE. Somatic Cell Genetics 3, 471-482.
566
[13] Saffery, R., Irvine, D. V., Griffiths, B., Kalitsis, P., Wordeman, L., and Choo, K. H. A.
567
(2000) Human centromeres and neocentromeres show identical distribution patterns of >20
568
functionally important kinetochore-associated proteins. Hum. Mol. Gene. 9, 175-185.
569
[14] Gieni, R. S., Chan, G. K. T., and Hendzel, M. J. (2008) Epigenetics regulate
570
centromere formation and kinetochore function. J. Cell. Biochem. 104, 2027-2039.
571
[15] Barnhart, M. C., Kuich, P., Stellfox, M. E., Ward, J. A., Bassett, E. A., Black, B. E.,
27
ACS Paragon Plus Environment
ACS Synthetic Biology
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
572
and Foltz, D. R. (2011) HJURP is a CENP-A chromatin assembly factor sufficient to form a
573
functional de novo kinetochore. J. Cell Biol. 194, 229-243.
574
[16] Hori, T., Shang, W. H., Takeuchi, K., and Fukagawa, T. (2013) The CCAN recruits
575
CENP-A to the centromere and forms the structural core for kinetochore assembly. J. Cell
576
Biol. 200, 45-60.
577
[17] Scott, K. C., and Sullivan, B. A. (2014) Neocentromeres: a place for everything and
578
everything in its place. Trends Genet. 30, 66-74.
579
[18] Ohzeki, J., Larionov, V., Earnshaw, W. C., and Masumoto, H. (2015) Genetic and
580
epigenetic regulation of centromeres: a look at HAC formation. Chromosome Res. 23,
581
87-103.
582
[19] Davidson R.M., Gowda M., Moghe G., Lin H., Vaillancourt B., Shiu S.H., Jiang N.,
583
and C., R. B. (2012) Comparative transcriptomics of three Poaceae species reveals patterns
584
of gene expression evolution. Plant J. 71, 492-502.
585
[20] Liao, B. Y., and Zhang, J. Z. (2006) Evolutionary conservation of expression profiles
586
between human and mouse orthologous genes. Mol. Biol. Evol. 23, 530-540.
587
[21] He, Z., Eichel, K., and Ruvinsky, I. (2011) Functional Conservation of Cis-Regulatory
588
Elements of Heat-Shock Genes over Long Evolutionary Distances. PLos One 6.
589
[22] Villar, D., Berthelot, C., Aldridge, S., Rayner, T. F., Lukk, M., Pignatelli, M., Park, T.
590
J., Deaville, R., Erichsen, J. T., Jasinska, A. J., Turner, J. M., Bertelsen, M. F., Murchison, E.
591
P., Flicek, P., and Odom, D. T. (2015) Enhancer Evolution across 20 Mammalian Species.
592
Cell 160, 554-566.
593
[23] Nitta, K. R., Jolma, A., Yin, Y., Morgunova, E., Kivioja, T., Akhtar, J., Hens, K.,
28
ACS Paragon Plus Environment
Page 28 of 37
Page 29 of 37
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
ACS Synthetic Biology
594
Toivonen, J., Deplancke, B., Furlong, E. E. M., and Taipale, J. (2015) Conservation of
595
transcription factor binding specificities across 600 million years of bilateria evolution.
596
Elife 4.
597
[24] Kachroo, A. H., Laurent, J. M., Yellman, C. M., Meyer, A. G., Wilke, C. O., Marcotte,
598
E. M. (2015) Evolution. Systematic humanization of yeast genes reveals conserved
599
functions and genetic modularity. Science 348, 921-925.
600
[25] Nakayama, Y., Uno, N., Uno, K., Mizoguchi, Y., Komoto, S., Kazuki, Y., Nanba, E.,
601
Inoue, T., and Oshimura, M. (2015) Recurrent micronucleation through cell cycle
602
progression in the presence of microtubule inhibitors. Cell Struct. Funct. 40, 51-59.
603
[26] Yoo, S. D., Cho, Y. H., and Sheen, J. (2007) Arabidopsis mesophyll protoplasts: a
604
versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565-1572.
605
[27] Li, H. X., Ding, D. Q., Cao, Y. S., Yu, B., Guo, L., and Liu, X. H. (2015) Partially
606
Overlapping Primer-Based PCR for Genome Walking. Plos One 10 (3): e0120139.
607
doi:10.1371/journal.pone.0120139.
608
[28] Takiguchi, M., Kazuki, Y., Hirarnatsu, K., Abe, S., Iida, Y., Takehara, S., Nishida, T.,
609
Ohbayashi, T., Wakayama, T., and Oshimura, M. (2014) A Novel and Stable Mouse
610
Artificial Chromosome Vector. Acs Synth. Biol. 3, 903-914.
611
[29] Uchiyama, S., Kobayashi, S., Takata, H., Ishihara, T., Hori, N., Higashi, T.,
612
Hayashihara, K., Sone, T., Higo, D., Nirasawa, T., Takao, T., Matsunaga, S., and Fukui, K.
613
(2005) Proteome analysis of human metaphase chromosomes. J. Biol. Chem. 280,
614
16994-17004.
615
[30] Kubiura, M., Okano, M., Kimura, H., Kawamura, F., and Tada, M. (2012)
29
ACS Paragon Plus Environment
ACS Synthetic Biology
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
616
Chromosome-wide regulation of euchromatin-specific 5mC to 5hmC conversion in mouse
617
ES cells and female human somatic cells. Chromosome Res. 20, 837-848.
618
[31] O'Keefe, C. L., and Matera, A. G. (2000) Alpha satellite DNA variant-specific
619
oligoprobes differing by a single base can distinguish chromosome 15 homologs. Genome
620
Res. 10, 1342-1350.
621
[32] Ijdo, J. W., Wells, R. A., Baldini, A., and Reeders, S. T. (1991) Improved telomere
622
detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acids Res.
623
19, 4780.
624
[33] Shibata, F., and Murata, M. (2004) Differential localization of the centromere-specific
625
proteins in the major centromeric satellite of Arabidopsis thaliana. J. Cell Sci. 117,
626
2963-2970.
627
30
ACS Paragon Plus Environment
Page 30 of 37
Page 31 of 37
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
ACS Synthetic Biology
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
ACS Paragon Plus Environment
ACS Synthetic Biology
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
32
ACS Paragon Plus Environment
Page 32 of 37
Page 33 of 37
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
671
672 673 674
ACS Synthetic Biology
Figures
Figure 1
33
ACS Paragon Plus Environment
ACS Synthetic Biology
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
675
676 677 678 679 680
Figure 2
34
ACS Paragon Plus Environment
Page 34 of 37
Page 35 of 37
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
681 682 683
ACS Synthetic Biology
Figure 3
35
ACS Paragon Plus Environment
ACS Synthetic Biology
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
684 685 686 687 688
Figure 4
36
ACS Paragon Plus Environment
Page 36 of 37
Page 37 of 37
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
ACS Synthetic Biology
689 690 691
692 693 694 695
Figure 5
37
ACS Paragon Plus Environment