Genetics: Advance Online Publication, published on October 31, 2012 as 10.1534/genetics.112.146126
Heritable Loss of Replication Control of a Minichromosome Derived From the B
Chromosome of Maize
Rick E. Masonbrink1, Shulan Fu2, Fangpu Han2, & James A. Birchler1*
1
Division of Biological Sciences, University of Missouri, Columbia, MO 65211,
USA;
2
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of
Genetics and Developmental Biology, Chinese National Academy of Sciences,
Beijing China
*Corresponding Author
Copyright 2012.
Running Title: Minichromosome Amplification
Keywords: replication, minichromosome, B chromosome
Corresponding Author: James Birchler, 311 Tucker Hall, University of Missouri,
Columbia, MO 65211, USA
Tel: (573) 882-4905; Fax: (573) 882-0123
E-mail address: [email protected]
2 ABSTRACT
During an accumulation regime of a small telomere-truncated B chromosome, a
derivative with large variations in size and multiple punctate centromere loci exhibiting
amplified copy numbers was discovered. Multiple centromere satellite loci or transgene
signals were documented in amplified chromosomes suggesting over-replication.
Immunolocalization studies revealed multiple foci of biochemical markers characteristic
of active centromeres such as CENP-C and phosphorylation of histones H3S10 and
H2AThr133. The amplified chromosomes exhibit an absence of chromosome
disjunction in meiosis I and an infrequent chromosome disjunction in meiosis II. Despite
their unusual structure and behavior these chromosomes were observed in the lineage
for seven generations during the course of this study. While severely truncated relative
to a normal B chromosome, the progenitor minichromosome is estimated to be at least
several megabases in size. Given that the centromere and transgene signals at
opposite ends of the chromosome generally match in copy number, the replication
control is apparently lost over several megabases.
3 Endoreduplication is essentially the replication of a genome without cell division,
and is typically found in terminally differentiated cells in specialized tissues. This
process is thought to increase the availability of DNA templates to amplify gene
expression, because the transcriptional and translational activities of the cell increase
proportionally with each genome doubling (D'AMATO 1984; LARKINS et al. 2001). Studies
in plants and animals show that endoreduplication is correlated with the lowering of Mphase cyclins, responsible for the G2 to M transition, and with a higher abundance of Sphase cyclins, responsible for the G1 to S transition (EVANS et al. 1983). The oscillation
of cyclins and a regulatory protein degraded by the anaphase promoting complex (Dbf4)
regulate the activity of cyclin dependent kinases (CDK) and Dbf4 dependent kinases
(DDK), respectively. They phosphorylate the mini-chromosome maintenance proteins 27 (MCM), which form the pre-replication complex necessary for replication licensing
(TUTEJA et al. 2011). While all previous descriptions of endoreduplication involve whole
genome duplication, (e.g. BAUER and BIRCHLER 2006), we describe a single,
supernumerary chromosome that routinely amplifies copy number without separation
while the remainder of the genome replicates normally.
The amplified chromosome originated from the B chromosome (B) of maize, a
supernumerary chromosome that persists in the genome with a selfish inheritance
resulting in higher-than-expected transmission (LONGLEY 1927). The B can accumulate
in the genome by surviving meiosis as a univalent, nondisjunction at the second pollen
mitosis, and preferential fertilization of the egg rather than the central cell by the B-
4 containing sperm, (CARLSON 1969; CARLSON and ROSEMAN 1992; ROMAN 1948).
Preferential fertilization is associated with a single gene in the normal karyotype (A
chromosomes), but nondisjunction and univalent survival are conditioned by the B
chromosome (CARLSON 1969; CARLSON and ROSEMAN 1992; CHIAVARINO et al. 2001).
Three regions of the B chromosome are essential for nondisjunction at the second
pollen mitosis; one cis-acting site in the proximal heterochromatin and two trans-acting
sites in the proximal and distal euchromatin (CARLSON 1973; CARLSON 1978; LIN 1978;
RHOADES and DEMPSEY 1972; RHOADES et al. 1967; ROMAN 1949; WARD 1973).
Nondisjunction of the B chromosome has also been observed in the tapetum,
endosperm, and roots, although these properties do not appear to contribute to its
selfish inheritance (ALFENITO and BIRCHLER 1990; CHIAVARINO et al. 2000; MASONBRINK
and BIRCHLER 2010).
Previously, maize embryos with B chromosomes were bombarded with telomere
repeats resulting in chromosomal truncations of A and B chromosomes (YU et al. 2007).
One of these truncations (86-B23) had most of the B chromosome long arm removed,
resulting in a chromosome slightly larger than a B chromosome centromere. This
chromosome was accumulated to multiple numbers when a normal B chromosome was
in the genome. During this program of accumulation, we fortuitously discovered a
lineage in which this chromosome replicated multiple times, but remained a single
entity.
MATERIALS AND METHODS
5 Mitotic in-situ Hybridization: Root tip FISH was performed as described with a
slight modification (MASONBRINK and BIRCHLER 2010). To make cell wall digestion times
more predictable, distilled water was substituted in washes for ethanol. Fluorescently
labeled oligonucleotide probes consisted of a telomere repeat that strongly cross
hybridizes to the B repeat; and CentC, which hybridizes to the centromere of all
chromosomes and in the B chromosome long arm (ALFENITO and BIRCHLER 1993;
ANANIEV et al. 1998; LAMB et al. 2005). The telomere oligonucleotide will label the B
chromosome centromere because of the partial homology of the B centromere specific
repeat (ALFENITO and BIRCHLER 1993) and produces such a strong signal that the
exposure time for the A chromosome telomeres is seldom long enough for visualization.
Nick translated PCR products consisted of the Stark repeat (LAMB et al. 2007), the
centromeric retrotransposon of maize (CRM) (NAGAKI et al. 2003), the 180bp knob
repeat (PEACOCK et al. 1981), and the cassette from the telomere-truncation
experiments without the telomere array (WY96) (YU et al. 2006).
Meiotic Fluorescence in-situ Hybridization: Meiotic FISH was performed as
described previously (MASONBRINK et al. 2012). FISH probes consisted of fluorescein
labeled telomere oligonucleotide (green) and a Texas Red labeled CentC
oligonucleotide.
Immunocytochemistry: Immunolocalization for mitosis and meiosis were
performed as described (HAN et al. 2009). The polyclonal antibody against maize
CENP-C was raised in rabbit [GL Biochem (Shanghai) Ltd.]. A monoclonal rabbit
antibody (04-817) raised against histone H3 phosphorylated at Ser-10 was obtained
from Upstate. The phosphorylated H2a antibody was previously described (DONG and
6 HAN 2012). The images were taken as a confocal z-stack (Zeiss LSM 710 NLO), and a
flat projection of the three-dimensional image was created with the ZEN 2009 Light
Edition (Zeiss), and processed with Photoshop CS 3.0.
RESULTS
A small B chromosome derived-minichromosome (86-B23) (YU et al. 2007)
(Figure 1a) was accumulated to multiple copies using the maize B chromosome
accumulation mechanism. In two generations this minichromosome was accumulated to
five copies before finding an unusually large chromosome with multiple distinct
centromere loci. In the next generation we observed many of these minichromosomes
changing in size and quantity between cells of the same root. Smaller sized
chromosomes typical of the progenitor were usually found in every cell, while the larger
sized chromosomes were restricted to fewer cells as if they were present only in certain
developmental lineages. These chromosomes had multiple distinct CentC signals per
minichromosome (Figure 1b). In addition, we probed the chromosome with CRM, the
centromeric retrotransposon of maize (Figure 1c); Stark repeat (LAMB et al. 2007), which
is found in the centromere and the distal heterochromatin of the B chromosome (Figure
1d); WY96, the truncation transgene cassette without the telomere array (Figure 1e);
and the 180 bp knob repeat, a constituent of maize heterochromatin (Figure 1f)
(PEACOCK et al. 1981). Each probe hybridized to multiple sites on the endoreduplicated
chromosomes and the quantity of these signals was correlated with the size of the
chromosome. For the medium sized chromosome in Figure 1e, the transgene, which
exhibits the most discrete signals, hybridized to between 16-20 sites. In a single cell of
one individual, the chromosome deviated from the typical spherical appearance and
7 was observed to form a large circle (Supplemental Figure 1). While we attempted to
increase the copy number of the amplifying chromosome, we were not able to observe
a cell with more than eight. As a sample of the frequency of their occurrence, the
numbers were recorded from three different root tips. 29 of 48 cells (60%) of the cells
showed a large chromosome, whereas the normal appearing minichromosome was
observed in the remaining cells.
Immunocytochemistry was used to characterize the chromatin of the amplified
chromosomes, and to determine if multiple centromere loci on a single chromosome
could condition multiple active centromeres. CENPC is an inner kinetochore protein that
is a biochemical marker of centromere activity. An antibody against CENPC was found
to localize to multiple loci on the large amplified chromosomes (Figure 2). The larger of
such chromosomes were found to exhibit up to three or more detectable foci. In
contrast, the smaller versions and the A chromosomes never had more than one
binding site for each chromosome. Sister chromatid cohesion is indicated by
phosphorylation of serine 10 of histone H3 (KASZAS and CANDE 2000). The amplified
chromosomes had multiple sites of sister chromatid cohesion indicated by bound
phosphorylated H3S10 antibody (Figure 3). An antibody to phosphorylated H2AThr133,
a histone modification associated with centromere function (DONG and HAN 2012), also
had multiple foci (Figure 4).
Throughout meiosis the amplified chromosome continued to vary in size and
frequency between cells of the same tassel and anther. In one anther nearly all the
stages of meiosis were observed, excluding pachynema, from the same plant that had
multiple amplified chromosomes. In pachynema these chromosomes appeared small
8 and compact, a deviation from the typical string-like morphology of pachytene
chromosomes (Figure 5a). In diplonema two spherical copies of the chromosome had
"sticky" behavior, a characteristic of B chromosomes (Figure 5b) (MCCLINTOCK 1933). In
diakinesis we observed three smaller sized chromosomes of various sizes, and a
noticeable division of B repeat to opposite ends of a chromosome (Figure 5c). At
metaphase I, the amplified chromosomes appeared to nondisjoin as demonstrated by
being closer to one pole than all other chromosomes (Figure 5d). This was also seen in
somatic cells of the anther (Supplemental Figure 2). All amplified chromosomes that did
not nondisjoin lagged at the metaphase plate. They apparently eventually moved to one
pole as evidenced by the lack of micronuclei in subsequent stages. Chromosome
disjunction was never observed with this chromosome at meiosis I (Figure 5e). Unequal
numbers of amplified chromosomes were observed in prometaphase II, probably the
result of precocious monopolar movement at metaphase I and nondisjunction at
anaphase I (Figure 5f). At anaphase II some sister separation was apparent, but many
times the chromosome nondisjoined to one pole (Figure 5g). In telophase II, the large
spherical shaped chromosomes were still present, as well as a higher quantity of small
versions (Figure 5h).
While screening the amplifying chromosome line, a small version was found that
resembled the progenitor of the amplifying chromosome with two CentC FISH signals
and a characteristic small size. It retained these characteristics and remained at one
copy in every cell examined in the root. Because this minichromosome was stable, we
self-pollinated and outcrossed the plant to see if amplification would reinitiate. In
screening the next generation we observed enlarged chromosomes in root tips from ten
9 of eighteen seedlings, demonstrating that the amplifying phenotype could reinitiate. We
also screened forty seeds by FISH from the original transformant seed stocks to
determine if amplification was a property of this chromosome but were unable to find an
amplifying minichromosome. While the earliest seed stocks did not contain an amplified
chromosome, we did find one plant in which the minichromosome frequently
nondisjoined between cells of the same root resulting in quantity shifts from zero to four
minis and increased chromosome size (Supplemental Figure 3), but the amplifying
phenotype was not recapitulated.
DISCUSSION
Here we describe a chromosome that changes size and structure among cells of
the same plant. Its behavior is similar to a univalent chromosome during meiosis and
mitosis, frequently nondisjoining even in the absence of a normal B chromosome. The
fact that the normal karyotype replicates and separates normally indicates the presence
of a cis-acting modification in this amplifying chromosome.
A potential explanation for this behavior is that there is an enhancement of
nondisjunction without the subsequent sister separation. The sister chromatids of the
normal B chromosome eventually separate, but the replicated sisters may remain
adhered through successive replication cycles, thus doubling the size of the
chromosome. For this hypothesis to explain the observed structure, the chromosome's
sister chromatids would have to remain adhered through multiple mitoses and thus
would drastically lower its presence among monitored cells. This possibility would halve
the number of cells in the lineage receiving such a chromosome for each instance of
10 nondisjunction. Considering that we found an instance of eight such chromosomes in
one cell with an approximate ten to sixteen centromere sites (Figure 1b), the odds of
finding this would be extremely low. In this scenario, nondisjunction could explain the
number of centromere loci present, but the observed frequency of amplified
chromosomes would be much lower than that observed. Moreover, nondisjunction of
the B chromosome has never been observed to produce such a conglomerate
(MASONBRINK and BIRCHLER 2010).
Another potential consideration might be that the minichromosome formed a ring
structure. Then with sister chromatid exchange within the ring, multiple centromeres
would be present. This scenario also seems unlikely because the sister centromeres of
rings typically separate at anaphase and rupture the chromosome. On this hypothesis
an atypical behavior of rings would need to occur to form larger and larger structures
due to repeated failure of sister chromatid separation. Also, this scenario would not
predict the finding of rare cells with many different sized chromosomes spanning ten to
twenty copies (Figure 1) nor is it consistent with the observation that when they are not
present, the normal minichromosome was observed. Lastly, McClintock studied small
ring chromosomes in maize (MCCLINTOCK 1938) and their structure and behavior are
not concordant with the chromosomes described here.
A more likely scenario might be that an initial modification occurred that
circumvents replication licensing, thus initiating multiple rounds of replication per cell
cycle in select cells (NISHITANI and LYGEROU 2002). The minichromosome could then
increase to various sizes between cells and permit the accumulation of multiple copies.
This scenario would also group the larger amplified chromosomes in selected cells,
11 while the smaller versions would be present in most cells. A combination of overreplication and nondisjunction could also allow multiple chromosome copies of varying
sizes to accumulate, while also limiting the number of cells having large variants.
The latter mechanism would double the number of centromere signals at each
replication cycle, which would create chromosomes with 2, 4, 8, 16, 32, etc. centromere
signals. The number of centromere signals is directly proportional to the number of
single copy transgenes, which have distinct condensed FISH signals (Fig 1e inset). In
counting the number of transgene signals, we noticed that the number deviates from the
strict doubling predicted. This deviation might result from the splitting of some of the
larger conglomerates in some mitoses, not all copies of the chromosome continuing to
replicate in synchrony, or limitations on the detection of all copies.
The number of centromeric FISH signals (Figure 1) and antibody binding sites
associated with sister chromatid cohesion and centromeric activity (Figures 2-4) were
often at unequal frequencies. Inactivated centromeres, i. e., centromeres that do not
recruit a kinetochore (HAN et al. 2006), within the amplified structure may account for
this signal discrepancy, possibly explaining the relative stability of these chromosomes.
The precocious monopolar movement and nondisjunction observed in metaphase I and
II and anaphase I and II were more frequent with larger versions of the amplified
chromosome. In fact, the only sister separation observed in meiosis occurred with small
versions of the chromosome in anaphase II. Early monopolar movement is frequent with
univalent B chromosomes (CARLSON and ROSEMAN 1992), but is at much lower levels
than what was seen with the amplified chromosome.
12 This unusual chromosome was found in a lineage derived from a truncated B
chromosome. This amplifying behavior acts in cis, namely only the minichromosome is
affected, thus suggesting some change, be that mutational or epigenetic, that was
perpetuated over the seven generations that the chromosome was under scrutiny.
Nevertheless, a progenitor- sized chromosome was still observed in this lineage
suggesting that single copies were possible and that the amplification could repeatedly
occur from a single copy. Many small B chromosome derivatives have been studied in
our laboratory over many years (HAN et al. 2007) without any similar cases being
observed.
This minichromosome contains the knob heterochromatin that is adjacent to the
B centromere indicating that the knob is proximal to the site of truncation and that the B
centromere is intact. The core of the B centromere is approximately 700 kilobases and
the B repeat cluster spans a minimum estimate of 3.7 megabases (JIN et al. 2005).
Thus, this example provides insight into the size range of replication control. Rereplication of this chromosome is regularly unrestricted and apparently spans
megabases given that the transgene signals at the chromosome terminus generally
match the number of centromere signals. The discovery of this chromosome will provide
a model system for future analyses of replication control.
ACKNOWLEDGEMENTS
This work was supported by National Science Foundation grant DBI0701297 and DBI
0922703.
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17 Figure Legends
FIGURE 1. Centromeric sequences in the amplified chromosome. Telomere probes,
which cross-hybridize to the B repeat and were used for detection of the B repeat, are
green. The inset pictures are the red channel of an amplified chromosome. Red arrows
denote amplified chromosomes and white arrows denote unmodified full-sized maize B
chromosomes. (a) CentC (red), a centromeric satellite repeat, localizes to a single locus
on the progenitor chromosome of the amplified chromosome. (b) CentC (red) had
multiple distinct signals on the amplified chromosome. (c) Multiple CRM (red) signals
are on the larger amplified chromosome, while the smaller versions have only two. (d)
Stark repeat (red), which is found in the distal heterochromatin of the long arm and in
the centromere of the B chromosome, hybridized throughout the amplified chromosome.
(e) WY96 (red), the single copy truncation cassette probe, hybridized to multiple distinct
loci on the amplified chromosome. (f) Knob (red) is found in the proximal
heterochromatin of the B chromosome, and also in the amplified chromosome. Scale
bars are 10 microns.
FIGURE 2. Multiple sites of bound CENPC on larger amplified chromosomes. The
yellow arrows denote amplified chromosomes with multiple sites of bound CENPC,
which is a marker for centromere activity. The red arrows denote small amplified
chromosomes with one site of CENPC binding. The top inset is the red CENPC
antibody channel showing multiple signals on each large amplified chromosome. The
top left box within the inset shows multiple CENPC signals on the amplified
18 chromosome. The middle inset is B repeat, which is found on the B chromosome and
throughout the amplified chromosomes. The bottom inset is the DAPI channel. The
white arrows denote three B chromosomes. B repeat is green and CENPC is red. Note
that A chromosomes have one distinct signal per sister chromatid. Scale bar is 10
microns.
FIGURE 3. Active sites of sister chromatid cohesion (H3S10) in the amplified
chromosome. The yellow arrow denotes an amplified chromosome with multiple sites of
bound H3S10 antibody, which is a marker for sister chromatid cohesion. The red arrows
denote smaller amplified chromosomes. The white arrows denote B chromosomes.
Notice all A chromosomes have one distinct signal per sister chromatid. The red
channel is the H3S10 antibody. Scale bar is 10 microns.
FIGURE 4. Sites of functional centromeres (phosphorylated H2A) in the amplified
chromosome. The yellow arrow denotes an amplified chromosome with multiple sites of
bound phosphorylated H2AThr133 antibody, which is a marker for centromere function.
The red arrow denotes a small amplified chromosome. The top inset is the red H2A
antibody channel showing multiple signals per large amplified chromosome. The middle
inset is the B repeat channel, which is found on both B chromosome arms at distal
positions and throughout the amplified chromosome. The bottom inset is the DAPI
channel. The white arrow denotes a B chromosome. B-repeat probes are green. Note
that A chromosomes have one distinct H2A signal per sister chromatid. Scale bar is 10
microns.
19 FIGURE 5. The meiotic behavior of an amplified chromosome. All meiocytes were
obtained from a single anther, except the pachytene cell. Yellow arrows denote
amplified chromosomes, and white arrows denote B chromosomes. CentC probes are
red, and telomere probes, which strongly cross-hybridized with the B repeat, are green.
(a) The amplified chromosome appears small at pachynema with diffuse B repeat
chromatin. (b) Two amplified chromosomes in diplonema. (c) Three amplified
chromosomes in diakinesis. (d) Two amplified chromosomes, both with early monopolar
movement typical of univalent B chromosomes at metaphase I. (e) Two amplified
chromosomes, the larger with persistent early monopolar movement and the smaller
with late monopolar movement at anaphase I. A univalent B chromosome lagging and
separating sister chromatids is present (white arrow). (f) Prometaphase II with the top
cell having two amplified chromosomes, while the bottom cell has one larger amplified
chromosome and a univalent B chromosome. (g) Anaphase II with the left cell
segregating sister chromatids of three amplified chromosomes and one large amplified
chromosome nondisjoining to one pole without sister chromatid separation. The right
cell has one amplified chromosome lagging at the metaphase plate while a larger
amplified chromosome has nondisjoined to one pole. (h) Telophase II with seven
amplified chromosomes. The larger amplified chromosomes are in distinct spherical
shapes. Scale bars are 10 microns.
20