- Email: [email protected]
Centromere Epigenetics in Plants
Accepted Manuscript Centromere Epigenetics in Plants James A. Birchler, Fangpu Han PII:
S1673-8527(13)00062-3
DOI:
10.1016/j.jgg.2013.03.008
Reference:
JGG 197
To appear in:
Journal of Genetics and Genomics
Received Date: 6 February 2013 Accepted Date: 6 March 2013
Please cite this article as: Birchler, J.A., Han, F., Centromere Epigenetics in Plants, Journal of Genetics and Genomics (2013), doi: 10.1016/j.jgg.2013.03.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Centromere Epigenetics in Plants
2 3
James A. Birchler1, Fangpu Han 2
4 5
1
6
MO, 65211-7400, US
7
2
8
Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
RI PT
Division of Biological Science, University of Missouri-Columbia, 311 Tucker Hall, Columbia,
SC
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and
9
Correspondence author: James A Birchler ([email protected])
11
Dr. James A. Birchler
12
Division of Biological Sciences
13
311 Tucker Hall
14
University of Missouri-Columbia
15
Columbia, MO. 65211-7400
16
USA
17
Email: [email protected]
18
Phone: +1 573-882-4905
19
Fax: +1 573-882-0123
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Abstract
22
The centromere is a chromosomal constriction that is essential for anaphase movement.
23
Eukaryotic centromeres contain complex DNA repeat sequences and transposable elements.
24
Paradoxically, the centromeric DNA sequences are rapidly evolving but the function is
25
conserved. In plants, the concept of centromere epigenetics was revealed from stable dicentric
26
chromosomes in which one centromere is inactive and can be inherited in this state for many
27
generations. The centromere repeats are present but a kinetochore is absent. In addition, new
28
centromeres have been found over unique sequences. The inactive centromeres can regain
29
function at a low frequency when released on their own. Collectively, these observations
30
illustrate DNA sequence alone does not dictate centromere function. Because of the ease of
31
manipulation, plant dicentric chromosomes comprise an excellent model for the study of
32
centromere epigenetics.
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1. INTRODUCTION The centromere is an essential chromosome site at which the kinetochore forms and loads
35
proteins needed for faithful segregation during the cell cycle and meiosis (Houben et al., 1999;
36
Cleveland et al., 2003; Ma et al., 2007; Birchler and Han, 2009). Centromere specific sequences
37
such as tandem repeats or transposable elements evolve quickly both within and between the
38
species but have conserved kinetochore proteins (Henikoff et al., 2001). The universal feature of
39
centromere function is a specific histone H3 variant CENH3 (Mendiburo et al., 2011).
40
RI PT
34
In eukaryotes, there are two kinds of centromeres classified as simple and complex. The first defined centromere was a simple one, which is also called a point centromere, and was found in
42
single-celled budding yeast (Furuyama and Biggins, 2007; Malik and Henikoff, 2009). It consists
43
of a single nucleosome, binds one spindle fiber per chromosome and strictly depends on a
44
specific DNA motif of 125 bp in length. The complex type, called regional centromeres, contain
45
tandem repeat satellite DNA and retrotransposon elements that are concentrated at all or most
46
primary constrictions. Their sizes can range into megabases.
M AN U
SC
41
Maize has a long history as a model for chromosome study that facilitate the study of
48
centromeres. It has 20 chromosomes called A chromosomes as well as a supernumerary or B
49
chromosome. Both A and B chromosomes contain CentC (156 bp, tandem repeat in maize) and
50
CRM (centromeric retrotransposon in maize) located in centromere regions (Jiang et al., 1996;
51
Ananiev et al., 1998; Kato et al., 2004; Lamb et al., 2005).
52
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47
Plant centromeric specific sequences cannot assemble a functional centromere when reintroduced into plant cells (Phan et al., 2007) unlike human satellite sequences that can be used
54
to create artificial chromosome in cell lines (Harrington et al., 2007). These results indicated that
55
plant centromeres have a strong epigenetic component making it unclear if centromere sequences
56
can organize a kinetochore in the absence of a pre-existing one (Birchler et al., 2011). However,
57
in the last few years, centromere inactivation and reactivation have been described in maize and
58
wheat (Birchler et al., 2011; Stimpson et al., 2012). Although plant centromeres are typically
59
composed of tandem repeat sequences and retrotransposons (Ma et al., 2007), neocentromeres
60
can form over unique sequences in barley and an oat-maize addition line (Nasuda et al., 2005;
61
Topp et al., 2009).
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62 63
2. Centromere inactivation 3
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Typically one chromosome has one centromere. Dicentric chromosomes were first described
65
in plants by McClintock who noted their unstable nature (McClintock, 1939, 1941). However, in
66
human, several cases of inactive centromeres and further analyses indicated that the silenced
67
centromere is accompanied by the loss of centromere chromatin (Kalitsis and Choo, 2012). In
68
fission yeast, artificial dicentric chromosomes have been developed, which revealed that a
69
centromere was inactivated epigenetically (Sato et al., 2012). In maize, stable dicentric
70
chromosomes have been found and their centromeric DNA sequences and histone modification
71
were studied in detail (Han et al., 2006, 2009; Gao et al., 2011). The inactive centromere shows
72
no constriction and no evidence of CENH3 association.
SC
73
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64
The types of dicentric chromosomes originally studied were minichromosomes that originated from a chromosome type breakage-fusion-bridge cycle from TB-9SbDp9 (Han et al.,
75
2006). The five dicentric minichromosomes examined were stable because only one centromere
76
was active (Han et al., 2006) (Fig. 1). It is interesting to note that centromere inactivation is not
77
related to centromere size because one of them, dicentric minichromosome #5, contained one
78
large and one small centromere, with the large one being inactive. The second type of dicentric
79
chromosome was recovered from a centromere tug-of-war (Han et al., 2009). In these cases, the
80
dicentric chromosome contained a 9S chromosome and formed a mirror image chromosome, but
81
with one large and one small B centromere located at the ends. The small version of the B
82
centromere was inactive based on the immunostaining for CENH3, CENP-C and
83
phosphorylation of H3 at Ser-10 (Han et al., 2009).
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The third type of dicentric chromosome was a maize A chromosome translocation involving
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chromosomes 1 and 5 (Gao et al., 2011). Both of the centromeres are large but one has lost
86
centromeric function. The chromosome originated from irradiation decades ago and appeared to
87
have captured a centromere between the breaks in chromosomes 1 and 5. This captured
88
centromere must have become inactive when the translocation was formed. In some other cases
89
we found a newly formed dicentric chromosome from the offspring of B9-Dp9, in which the new
90
dicentric chromosome contains two normal B centromeres with one of them being inactive (Fig.
91
2). We also found one chromosome that contains three centromere regions with only one active
92
and the other two inactive.
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3. Centromere reactivation 4
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The most common types of DNA sequences in centromere regions are tandem repeats and retrotransposon elements. However, neocentromeres can form anew at sites without these
97
repeats. This raises the question of whether the repeats can foster a centromere or whether they
98
accumulate at the sites of centromeric chromatin. In maize, an inactive centromere can be
99
reactivated through intrachromosomal recombination (Han et al 2009). A new dicentric
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chromosome was released from recombination in a fold back chromosome resulting from the
101
centromere tug-of-war described above. This forms a mirror image chromosome with only the
102
small inactive centromeres, but the core DNA sequences such as CentC, CRM and B repeats are
103
present. When the small inactive centromere separated from this dicentric condition, its
104
centromere function was regained in some cells and was stably transmitted to the next
105
generation. In this resurrection process, the centromeric core DNA sequences do not change
106
based on FISH (Fluorescence in situ hybridization) detection.
107 108 109
4. Nondisjunction of the B centromere
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B chromosomes (Bs), also called supernumerary chromosomes, are dispensable components in the genomes of plants, fungi and animals. B chromosomes do not confer any advantages on
111
the organism and have a non-Mendelian transmission. They are considered selfish elements that
112
maintain a high transmission in populations (Jones and Houben, 2003; Banaei-Moghaddam et al.,
113
2012). In plants, the B chromosomes undergo nondisjunction constituting a post meiotic drive. It
114
occurs at the first pollen mitosis in rye and second pollen mitosis in maize. The availability of an inactive B centromere on chromosome arm 9S provided an
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opportunity to examine whether centromere function is necessary for nondisjunction. The normal
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nondisjunction property of the B centromere involved the centromere and trans-acting sites—
118
one of which was at the very distal tip of the long arm. The stock with the inactive B centromere
119
on 9S was missing in this region. Thus, when normal B chromosomes were added to the
120
genotype with the inactive B centromere on 9S, this combination revealed if the inactive
121
centromere underwent nondisjunction. Indeed, chromosome 9 was induced to undergo
122
nondisjunction but more frequently 9S was broken in the process, which was a byproduct of
123
attempted nondisjunction (Han et al., 2007) (Fig. 3). These results reveal that centromere
124
function is independent of nondisjunction. This inactive B centromere in the dicentric
125
chromosome serves as a good model for the study of nondisjunction.
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5. Concluding remarks and future challenges The centromere has a complex structure and conserved function, which is not surprising
128
given its important function. The H3 histone variant CENH3 and other proteins are critical to
129
centromere function whereas the DNA sequence is not necessarily determinant. In general,
130
dicentric chromosomes are not stable under most circumstances. However, they have led to the
131
recognition of centromere inactivation and their subsequent reactivation. These processes must
132
occur within the timeframe of a single cell cycle but can be perpetuated in the changed state over
133
generations. Further, study of centromere epigenetics will aid in an understanding of their normal
134
functions and their changes over evolutionary time (Wang and Bennetzen, 2012).
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References
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Ananiev, E.V., Phillips, R.L., and Rines, H.W. (1998). Chromosome-specific molecular organization of maize (Zea mays L.) centromeric regions. Proc. Natl. Acad. Sci. USA 95,
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Banaei-Moghaaam, A.M., Schubert, V., Kumke, K., Weib, O., Klemme, S., Nagaki, K., Macas, J., Gonzalez-Sanchez, M., Heredia, V., Gomez-Revilla, D., Gonzalez-Garcla, M.,
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Vega, J.M., Puertas, M.J., and Houben, A. (2012). Nondisjunction in favor of a
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chromosome: the mechanism of rye B chromosome drive during pollen mitosis. Plant Cell,
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Birchler, J.A., and Han, F. (2009). Maize Centromeres: Structure, Function, Epigenetics. Ann.
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Birchler, J.A., Gao, Z., Sharma, A., Presting, G.G., and Han, F. (2011). Epigenetic aspects of centromere function in plants. Curr. Opin. Plant. Biol. 14, 217-222. Choo, K.H.A. (2001). Domain organization at the centromere and neocentromere. Dev. Cell 1, 165-177.
Cleveland, D.W., Mao, Y.H., and Sullivan, K.F. (2003). Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 112, 407-421.
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Furuyama, S., and Biggins, S. (2007). Centromere identity is specified by a single centromeric nucleosome in budding yeast. Proc. Natl. Acad. Sci. USA 104, 14706-14711. Gao, Z., Fu, S., Dong, Q., Han, F., and Birchler, J.A. (2011). Inactivation of a centromere during the formation of a translocation in maize. Chromosome Res. 19, 755-761.
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Han, F., Gao, Z., and Birchler, J.A. (2009). Reactivation of an inactive centromere reveals epigenetic and structural components for centromere specification in maize. Plant Cell 21,
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Han, F., Lamb, J.C., Yu, W., Gao, Z., and Birchler, J.A. (2007). Centromere function and
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Han, F.P., Lamb, J.C., and Birchler, J.A. (2006). High frequency of centromere inactivation
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and Fukui, K. (1999). The cell cycle dependent phosphorylation of histone H3 is correlated
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Jones, N., and Houben, A. (2003). B chromosomes in plants: escapees from A chromosome genome? Trends Plant Sci. 8, 417-423.
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Jiang, J.M., Nasuda, S., Dong, F.G., Scherrer, C.W., Woo, S.S., Wing, R.A., Gill, B.S., and
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cereal chromosomes. Proc. Natl. Acad. Sci. USA 93, 14210-14213.
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Kalitsis, P., and Choo, K.H.A. (2012). The evolutionary life cycle of the resilient centromere. Chromosoma 121, 327-340.
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a dynamic structure with conserved functions. Trends Genet. 23, 134-139. Malik, H.S., and Henikoff, S. (2009). Major evolutionary trnasitions in centromere complexity.
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McClintock, B. (1939). The behavior in successive nuclear divisions of a chromosome broken at meiosis. Proc. Natl. Acad. Sci. USA 25, 405-416. McClintock, B. (1941). The stability of broken ends of chromosomes in Zea mays. Genetics 26, 234-282. Mendiburo, M.J., Padeken, J., Fulop, S., Schepers, A., and Heun P. (2011). Drosophila CENH3 is sufficeint for centromere formation. Science 334, 686-690.
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Nasuda, S., Hudakova, S., Schubert, I., Houben, A., and Endo, T.R. (2005). Stable barley chromosomes without centromeric repeats. Proc. Natl. Acad. Sci. USA 102, 9842-9847. Phan, B.H., Jin, W., Topp, C.N., Zhong, C.X., Jiang, J., Dawe, R.K., Parrott, W.A. (2007). Transformation of rice with long DNA-segments consisting of random genomic DNA or
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centromere-specific DNA. Transgenic Res. 16, 341-351.
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Sato, H., Masuda, F., Takayama, Y., Takahashi, K., and Saitoh, S. (2012). Epigenetic
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inactivation and subsequent heterochromatinization of a centromere stabilize dicentric
202
chromosomes. Curr. Biol. 22, 658-667.
205
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Stimpson, K.M., and Sullivan, B.A. (2010). Epigenomics of centromere assembly and function. Curr. Opin. Cell. Biol. 22, 772-780.
Topp, C.N., Okagaki, R.J., Melo, J.R., Kynast, R.G., Phillips, R.L., and Dawe, R.K. (2009).
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Identification of a maize neocentromere in an oat-maize addition line. Cytogenet. Genome
207
Res. 124, 228-238.
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maize from a tetraploid ancestor. Proc. Natl. Acad. Sci., USA 109, 21004-21009.
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Wang, H., and Bennetzen, J.L. (2012). Centromere retention and loss during the descent of
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Figure legends
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Figure 1. Immunostaining of CENH3 in dicentric minichromosome at diakinesis of meiosis.
213
Red represents the signal of CENH3, which is a marker for centromere activity; DAPI-stained
214
chromosomes are blue; green is B specific centromere repeat signal. The arrows denote the
215
dicentric chromosome with one site of CENH3 binding. Bar =10 µm.
216
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Figure 2. FISH pattern of a newly formed dicentric chromosome.
218
Arrow indicates a newly formed dicentric chromosome, which contained two similar sized
219
centromere regions. Red is B specific centromere repeat signals; green is knob and DAPI-stained
220
chromosomes are blue. Bars =10 µm.
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Figure 3. Progeny of an inactive B centromere when it undergoes nondisjunction.
223
Left, the inactive B centromere on the tip of 9S (9-Bic-1) together with a normal B chromosome
224
that supplies the trans-acting function for nondisjunction. A: the inactive centromere separates
225
to two cells at the second pollen mitosis; B: the two inactive centromeres enter the same cell at
226
the end of the second pollen mitosis. These cells eventually become the two sperm; C:
227
chromosome breakage. The inactive centromere is held together when the normal centromeres
228
move to different poles. The two sperm produced will fertilize the egg or the polar nuclei in the
229
process of double fertilization. If the broken chromosome contributes to the endosperm, a mosaic
230
kernel for the C1 pigment gene will be produced.
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S1673-8527(13)00062-3
DOI:
10.1016/j.jgg.2013.03.008
Reference:
JGG 197
To appear in:
Journal of Genetics and Genomics
Received Date: 6 February 2013 Accepted Date: 6 March 2013
Please cite this article as: Birchler, J.A., Han, F., Centromere Epigenetics in Plants, Journal of Genetics and Genomics (2013), doi: 10.1016/j.jgg.2013.03.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Centromere Epigenetics in Plants
2 3
James A. Birchler1, Fangpu Han 2
4 5
1
6
MO, 65211-7400, US
7
2
8
Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
RI PT
Division of Biological Science, University of Missouri-Columbia, 311 Tucker Hall, Columbia,
SC
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and
9
Correspondence author: James A Birchler ([email protected])
11
Dr. James A. Birchler
12
Division of Biological Sciences
13
311 Tucker Hall
14
University of Missouri-Columbia
15
Columbia, MO. 65211-7400
16
USA
17
Email: [email protected]
18
Phone: +1 573-882-4905
19
Fax: +1 573-882-0123
TE D
EP AC C
20
M AN U
10
ACCEPTED MANUSCRIPT
Abstract
22
The centromere is a chromosomal constriction that is essential for anaphase movement.
23
Eukaryotic centromeres contain complex DNA repeat sequences and transposable elements.
24
Paradoxically, the centromeric DNA sequences are rapidly evolving but the function is
25
conserved. In plants, the concept of centromere epigenetics was revealed from stable dicentric
26
chromosomes in which one centromere is inactive and can be inherited in this state for many
27
generations. The centromere repeats are present but a kinetochore is absent. In addition, new
28
centromeres have been found over unique sequences. The inactive centromeres can regain
29
function at a low frequency when released on their own. Collectively, these observations
30
illustrate DNA sequence alone does not dictate centromere function. Because of the ease of
31
manipulation, plant dicentric chromosomes comprise an excellent model for the study of
32
centromere epigenetics.
AC C
EP
TE D
M AN U
SC
RI PT
21
2
ACCEPTED MANUSCRIPT
33
1. INTRODUCTION The centromere is an essential chromosome site at which the kinetochore forms and loads
35
proteins needed for faithful segregation during the cell cycle and meiosis (Houben et al., 1999;
36
Cleveland et al., 2003; Ma et al., 2007; Birchler and Han, 2009). Centromere specific sequences
37
such as tandem repeats or transposable elements evolve quickly both within and between the
38
species but have conserved kinetochore proteins (Henikoff et al., 2001). The universal feature of
39
centromere function is a specific histone H3 variant CENH3 (Mendiburo et al., 2011).
40
RI PT
34
In eukaryotes, there are two kinds of centromeres classified as simple and complex. The first defined centromere was a simple one, which is also called a point centromere, and was found in
42
single-celled budding yeast (Furuyama and Biggins, 2007; Malik and Henikoff, 2009). It consists
43
of a single nucleosome, binds one spindle fiber per chromosome and strictly depends on a
44
specific DNA motif of 125 bp in length. The complex type, called regional centromeres, contain
45
tandem repeat satellite DNA and retrotransposon elements that are concentrated at all or most
46
primary constrictions. Their sizes can range into megabases.
M AN U
SC
41
Maize has a long history as a model for chromosome study that facilitate the study of
48
centromeres. It has 20 chromosomes called A chromosomes as well as a supernumerary or B
49
chromosome. Both A and B chromosomes contain CentC (156 bp, tandem repeat in maize) and
50
CRM (centromeric retrotransposon in maize) located in centromere regions (Jiang et al., 1996;
51
Ananiev et al., 1998; Kato et al., 2004; Lamb et al., 2005).
52
TE D
47
Plant centromeric specific sequences cannot assemble a functional centromere when reintroduced into plant cells (Phan et al., 2007) unlike human satellite sequences that can be used
54
to create artificial chromosome in cell lines (Harrington et al., 2007). These results indicated that
55
plant centromeres have a strong epigenetic component making it unclear if centromere sequences
56
can organize a kinetochore in the absence of a pre-existing one (Birchler et al., 2011). However,
57
in the last few years, centromere inactivation and reactivation have been described in maize and
58
wheat (Birchler et al., 2011; Stimpson et al., 2012). Although plant centromeres are typically
59
composed of tandem repeat sequences and retrotransposons (Ma et al., 2007), neocentromeres
60
can form over unique sequences in barley and an oat-maize addition line (Nasuda et al., 2005;
61
Topp et al., 2009).
AC C
EP
53
62 63
2. Centromere inactivation 3
ACCEPTED MANUSCRIPT
Typically one chromosome has one centromere. Dicentric chromosomes were first described
65
in plants by McClintock who noted their unstable nature (McClintock, 1939, 1941). However, in
66
human, several cases of inactive centromeres and further analyses indicated that the silenced
67
centromere is accompanied by the loss of centromere chromatin (Kalitsis and Choo, 2012). In
68
fission yeast, artificial dicentric chromosomes have been developed, which revealed that a
69
centromere was inactivated epigenetically (Sato et al., 2012). In maize, stable dicentric
70
chromosomes have been found and their centromeric DNA sequences and histone modification
71
were studied in detail (Han et al., 2006, 2009; Gao et al., 2011). The inactive centromere shows
72
no constriction and no evidence of CENH3 association.
SC
73
RI PT
64
The types of dicentric chromosomes originally studied were minichromosomes that originated from a chromosome type breakage-fusion-bridge cycle from TB-9SbDp9 (Han et al.,
75
2006). The five dicentric minichromosomes examined were stable because only one centromere
76
was active (Han et al., 2006) (Fig. 1). It is interesting to note that centromere inactivation is not
77
related to centromere size because one of them, dicentric minichromosome #5, contained one
78
large and one small centromere, with the large one being inactive. The second type of dicentric
79
chromosome was recovered from a centromere tug-of-war (Han et al., 2009). In these cases, the
80
dicentric chromosome contained a 9S chromosome and formed a mirror image chromosome, but
81
with one large and one small B centromere located at the ends. The small version of the B
82
centromere was inactive based on the immunostaining for CENH3, CENP-C and
83
phosphorylation of H3 at Ser-10 (Han et al., 2009).
TE D
The third type of dicentric chromosome was a maize A chromosome translocation involving
EP
84
M AN U
74
chromosomes 1 and 5 (Gao et al., 2011). Both of the centromeres are large but one has lost
86
centromeric function. The chromosome originated from irradiation decades ago and appeared to
87
have captured a centromere between the breaks in chromosomes 1 and 5. This captured
88
centromere must have become inactive when the translocation was formed. In some other cases
89
we found a newly formed dicentric chromosome from the offspring of B9-Dp9, in which the new
90
dicentric chromosome contains two normal B centromeres with one of them being inactive (Fig.
91
2). We also found one chromosome that contains three centromere regions with only one active
92
and the other two inactive.
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The most common types of DNA sequences in centromere regions are tandem repeats and retrotransposon elements. However, neocentromeres can form anew at sites without these
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repeats. This raises the question of whether the repeats can foster a centromere or whether they
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accumulate at the sites of centromeric chromatin. In maize, an inactive centromere can be
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reactivated through intrachromosomal recombination (Han et al 2009). A new dicentric
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chromosome was released from recombination in a fold back chromosome resulting from the
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centromere tug-of-war described above. This forms a mirror image chromosome with only the
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small inactive centromeres, but the core DNA sequences such as CentC, CRM and B repeats are
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present. When the small inactive centromere separated from this dicentric condition, its
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centromere function was regained in some cells and was stably transmitted to the next
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generation. In this resurrection process, the centromeric core DNA sequences do not change
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based on FISH (Fluorescence in situ hybridization) detection.
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4. Nondisjunction of the B centromere
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B chromosomes (Bs), also called supernumerary chromosomes, are dispensable components in the genomes of plants, fungi and animals. B chromosomes do not confer any advantages on
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the organism and have a non-Mendelian transmission. They are considered selfish elements that
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maintain a high transmission in populations (Jones and Houben, 2003; Banaei-Moghaddam et al.,
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2012). In plants, the B chromosomes undergo nondisjunction constituting a post meiotic drive. It
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occurs at the first pollen mitosis in rye and second pollen mitosis in maize. The availability of an inactive B centromere on chromosome arm 9S provided an
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opportunity to examine whether centromere function is necessary for nondisjunction. The normal
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nondisjunction property of the B centromere involved the centromere and trans-acting sites—
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one of which was at the very distal tip of the long arm. The stock with the inactive B centromere
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on 9S was missing in this region. Thus, when normal B chromosomes were added to the
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genotype with the inactive B centromere on 9S, this combination revealed if the inactive
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centromere underwent nondisjunction. Indeed, chromosome 9 was induced to undergo
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nondisjunction but more frequently 9S was broken in the process, which was a byproduct of
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attempted nondisjunction (Han et al., 2007) (Fig. 3). These results reveal that centromere
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function is independent of nondisjunction. This inactive B centromere in the dicentric
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chromosome serves as a good model for the study of nondisjunction.
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5. Concluding remarks and future challenges The centromere has a complex structure and conserved function, which is not surprising
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given its important function. The H3 histone variant CENH3 and other proteins are critical to
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centromere function whereas the DNA sequence is not necessarily determinant. In general,
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dicentric chromosomes are not stable under most circumstances. However, they have led to the
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recognition of centromere inactivation and their subsequent reactivation. These processes must
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occur within the timeframe of a single cell cycle but can be perpetuated in the changed state over
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generations. Further, study of centromere epigenetics will aid in an understanding of their normal
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functions and their changes over evolutionary time (Wang and Bennetzen, 2012).
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Figure legends
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Figure 1. Immunostaining of CENH3 in dicentric minichromosome at diakinesis of meiosis.
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Red represents the signal of CENH3, which is a marker for centromere activity; DAPI-stained
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chromosomes are blue; green is B specific centromere repeat signal. The arrows denote the
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dicentric chromosome with one site of CENH3 binding. Bar =10 µm.
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Figure 2. FISH pattern of a newly formed dicentric chromosome.
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Arrow indicates a newly formed dicentric chromosome, which contained two similar sized
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centromere regions. Red is B specific centromere repeat signals; green is knob and DAPI-stained
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chromosomes are blue. Bars =10 µm.
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Figure 3. Progeny of an inactive B centromere when it undergoes nondisjunction.
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Left, the inactive B centromere on the tip of 9S (9-Bic-1) together with a normal B chromosome
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that supplies the trans-acting function for nondisjunction. A: the inactive centromere separates
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to two cells at the second pollen mitosis; B: the two inactive centromeres enter the same cell at
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the end of the second pollen mitosis. These cells eventually become the two sperm; C:
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chromosome breakage. The inactive centromere is held together when the normal centromeres
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move to different poles. The two sperm produced will fertilize the egg or the polar nuclei in the
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process of double fertilization. If the broken chromosome contributes to the endosperm, a mosaic
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kernel for the C1 pigment gene will be produced.
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