Genetics, Vol. 163, 1221-1225, March 2003, Copyright © 2003

Chromatin Immunoprecipitation Reveals That the 180-bp Satellite Repeat Is the Key Functional DNA Element of Arabidopsis thaliana Centromeres

Kiyotaka Nagakia, Paul B. Talbertb, Cathy Xiaoyan Zhongc, R. Kelly Dawec, Steven Henikoffb, and Jiming Jianga
a Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706,
b Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024
c Department of Plant Biology and Department of Genetics, University of Georgia, Athens, Georgia 30602

Corresponding author: Jiming Jiang, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706., jjiang1{at}facstaff.wisc.edu (E-mail)

Communicating editor: V. L. CHANDLER


*  ABSTRACT
*TOP
 *ABSTRACT
*LITERATURE CITED

The centromeres of Arabidopsis thaliana chromosomes contain megabases of complex DNA consisting of numerous types of repetitive DNA elements. We developed a chromatin immunoprecipitation (ChIP) technique using an antibody against the centromeric H3 histone, HTR12, in Arabidopsis. ChIP assays showed that the 180-bp centromeric satellite repeat was precipitated with the antibody, suggesting that this repeat is the key component of the centromere/kinetochore complex in Arabidopsis.

THE centromere is one of the most important domains of eukaryotic chromosomes. The centromere is responsible for sister-chromatid cohesion and serves as the site for spindle-fiber attachment during cell division. Thus, centromeres play a critical role in faithful chromosome segregation and transmission. Although the functions of centromeres are conserved among all eukaryotic species, the DNA sequences in centromeric regions often show little or no homology among related species. In most eukaryotic species, the centromeres are embedded in long tracks of highly repetitive DNA sequences. Satellite repeats are often the major DNA components of centromeres (CSINK and HENIKOFF 1998 Down).

Although the centromeric DNA sequences are significantly diverged among eukaryotic species, several proteins specific to the centromere/kinetochore complex are highly conserved (DOBIE et al. 1999 Down; YU et al. 2000 Down). The centromere-specific histone H3 variants (CenH3s) are well characterized and their role in centromere function have been demonstrated (see reviews by HENIKOFF et al. 2001 Down; SULLIVAN et al. 2001 Down). The first CenH3, CENP-A, was identified in humans as a histone-H3-related centromere protein (PALMER et al. 1987 Down, PALMER et al. 1991 Down). Since then CenH3s have been found in all model eukaryotes (reviews by HENIKOFF et al. 2001 Down; SULLIVAN et al. 2001 Down) and recently in plants (TALBERT et al. 2002 Down; ZHONG et al. 2002 Down). CenH3s replace the regular H3 histone in centromeric chromatin (YODA et al. 2000 Down; AHMAD and HENIKOFF 2001 Down; BLOWER et al. 2002 Down). Blocks of CenH3-associated nucleosomes and regular H3-associated nucleosomes are linearly interspersed in functional centromeres (BLOWER et al. 2002 Down). CENP-A is present only in the functional centromeres of dicentric chromosomes in humans (WARBURTON et al. 1997 Down). Thus, identification of DNA sequences that interact with CenH3 is an effective way to recognize specific DNA sequences involved in centromere function.

The centromeres of Arabidopsis thaliana have been genetically mapped (COPENHAVER et al. 1999 Down) and are cytologically located within distinctive centromeric heterochromatin (FRANSZ et al. 1998 Down, FRANSZ et al. 2000 Down). Various types of repetitive DNA elements, including retroelements, transposons, and telomere-like repeats, were identified in the centromeric regions (RICHARDS et al. 1991 Down; THOMPSON et al. 1996 Down; BRANDES et al. 1997 Down). The most abundant DNA element within the genetically mapped A. thaliana centromeres is the 180-bp satellite repeat (MARTINEZ-ZAPATER et al. 1986 Down; MALUSZYNSKA and HESLOP-HARRISON 1991 Down; MURATA et al. 1994 Down; ROUND et al. 1997 Down; HESLOP-HARRISON et al. 1999 Down). Each Arabidopsis centromere contains several megabases of the 180-bp repeat (KUMEKAWA et al. 2000 Down, KUMEKAWA et al. 2001 Down; HOSOUCHI et al. 2002 Down). The 180-bp repeat is organized into long tandem arrays (JACKSON et al. 1998 Down) that may be interrupted by the Athila retrotransposon (FRANSZ et al. 2000 Down; KUMEKAWA et al. 2000 Down, KUMEKAWA et al. 2001 Down). However, the complete sequences of individual Arabidopsis centromeres are impossible to determine using the currently available sequencing technologies (ARABIDOPSIS GENOME INITIATIVE 2000; HENIKOFF 2002 Down). It remains an open question whether we have identified all the DNA elements located in the centromeres of Arabidopsis chromosomes. It is not known which centromeric repeats, if any, are involved in centromere function.

The CenH3 in Arabidopsis, HTR12, was characterized recently by TALBERT et al. 2002 Down. The antibody against HTR12 was localized at Arabidopsis centromeres in both mitotic and meiotic cells (TALBERT et al. 2002 Down). Using the anti-HTR12 antibody we developed a chromatin immunoprecipitation (ChIP) procedure to determine which centromeric repeats, if any, are incorporated into the centromere/kinetochore complex in Arabidopsis.

Approximately 20 g of young leaf tissues from the A. thaliana ecotype Columbia were ground to fine powder with liquid nitrogen, resuspended in 10 ml nuclei isolation buffer [60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 0.35 M sorbitol, protease inhibitor (Roche Applied Science, Indianapolis), and 0.1 mM phenylmethylsulfonyl fluoride, pH 6.7] containing 0.1% cellulase and 0.05% pectinase. The suspension was incubated at 37° for 30 min and filtered with cheesecloth of 120-, 45-, and 30-µm meshes. Nuclei were pelleted by centrifuge at 2000 x g for 10 min at 4°. The nuclei were washed twice using 10 ml nuclei isolation buffer and suspended in 1.2 ml of micrococcal nuclease digestion buffer (10% sucrose, 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2, and 1 mM CaCl2).

The ChIP procedure was based on protocols developed by LO et al. 2001 Down with only minor modifications. Nuclei were digested with 10 units of micrococcal nuclease (Sigma, St. Louis) to liberate nucleosomes. The nucleosome samples were first incubated with preimmune rabbit serum (1:100 dilution), then 4% protein A Sepharose (Amersham Biosciences, Piscataway, NJ) for 4 hr, and centrifuged. The supernatant was incubated with the anti-HTR12 antibody (1:400 dilution) overnight and 25% protein A Sepharose for 4 hr. After centrifugation, the samples were separated into Sup (unbound) and Pel (bound) fractions. The bound fraction was sequentially washed in 1.2 ml washing buffer (20 mM Tris-HCl pH 7.5, 5 mM EDTA) containing 50, 100, and 150 mM NaCl. Bound immune complex was eluted with 1 ml elution buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% SDS). Nucleic acids were extracted from both the supernatant and wash-dissociated bound fractions and resuspended in 100 µl of TE buffer (pH 8.0).

Equal amounts (10 µl each) of the Sup and Pel fractions were blotted on membranes. The membranes were sequentially probed with 32P-labeled centromeric DNA probes (Table 1). The amount of hybridization was quantified using a phosphorimager. Mock experiments using preimmunized rabbit serum served as nonspecific binding controls for each ChIP assay. The percentage of immunoprecipitation (IP) [defined as Pel/(Pel+ Sup)] of the mock experiments was subtracted in each case from the percentage of IP of the anti-HTR12 treatments. Each experiment was replicated in three independent tubes. We used the 18S·26S ribosomal RNA genes (rDNA) as negative controls. The rDNAs in Arabidopsis are located at the terminal regions of chromosome 2 and 4 and are distant from the centromeres (FRANSZ et al. 1998 Down).


 
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  		Table 1. DNA probes and their represented centromeric repeats used in ChIP assays

The ChIP experiment was repeated three times and the results are summarized in Fig 1. We used polymerase chain reaction (PCR)-amplified sequences of the 180-bp satellite repeat as a probe in the slot blot hybridization. The PCR-derived probe may include more variants of the 180-bp repeat family than included in the specific plasmid probes. On average, 15.5% [standard error (SE) = ±2.0%, n = 3] of the 180-bp repeat was found in the pellet, whereas only 0.7% (SE = ±0.57%, n = 3) of the DNA was detected in the pellet when the same blots were reprobed with the A. thaliana rDNA sequences. The 180-bp repeat was immunoprecipitated at a significantly higher level compared to the rDNA (t-test, P = 0.002).



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  		Figure 1. ChIP analysis of Arabidopsis centromeric repeats. The nucleosome samples were prepared from leaf tissues of A. thaliana ecotype Columbia. Unbound (Sup) and bound (Pel) fractions precipitated with preimmune blood (Pre) and anti-HTR12 antibody (HTR12) were blotted to membranes and probed with the centromeric repeats. Three samples were blotted in each of the three independent ChIP experiments. Note that the Pel fraction hybridization signals derived from the 180-bp repeat are significantly darker in samples precipitated with anti-HTR12 antibody than in samples precipitated with Pre.

A number of repetitive DNA elements previously identified in the centromeric regions were tested in the ChIP assays. A 620-kb mitochondrial DNA (mtDNA) in chromosome 2 (STUPAR et al. 2001 Down) was located within the genetically mapped centromere (COPENHAVER et al. 1999 Down). This mtDNA locus is only ~100 kb away from the 180-bp repeat array (ARABIDOPSIS GENOME INITIATIVE 2000). Bacterial artificial chromosome clone T17H1, which contains 76 kb of mtDNA (STUPAR et al. 2001 Down), was used as a probe in the slot blot hybridization. The IP percentage of mtDNA was 1.2% on average (Fig 2). Similarly, the 5S ribosomal RNA genes were located in the centromeric regions of Arabidopsis chromosomes 2, 3, and 4 (ARABIDOPSIS GENOME INITIATIVE 2000). The IP percentage of 5S rDNA is 2.6% on average (Fig 2).



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  		Figure 2. The IP percentage derived from different Arabidopsis centromeric repeats. The columns represent the average IP percentage from three independent experiments. Only the IP percentage from the 180-bp repeat is significantly higher than that of the control (rDNA).

Several medium repetitive DNA sequences, including 106B, 163A, 164A, 278A, and mi167, were reported in the pericentromeric regions of Arabidopsis chromosomes (THOMPSON et al. 1996 Down; BRANDES et al. 1997 Down). The 106B repeat shows homology with the long-terminal-repeat region of the Athila retrotransposon, but other repeats are not homologous to any known sequences (THOMPSON et al. 1996 Down). The IP percentage of these repeats ranged from 2.0 to 3.9%.

The majority of the transposable elements in the Arabidopsis genome, including Athila, Tat, Tim, Copia, and another Ty3/gypsy element with homology to the centromere-specific retrotransposons in cereals (referred to as the CR homolog hereafter; see also LANGDON et al. 2000 Down), are concentrated in the centromeric regions (ARABIDOPSIS GENOME INITIATIVE 2000). Members of all of these transposable elements can be found within the genetically mapped Arabidopsis centromeres (KUMEKAWA et al. 2000 Down, KUMEKAWA et al. 2001 Down). Athila elements are highly concentrated in pericentromeric regions. Sequencing analysis has revealed insertions of the Athila elements into the 180-bp satellite arrays (KUMEKAWA et al. 2000 Down, KUMEKAWA et al. 2001 Down). Athila had an IP percentage of 4.5%, and the IP percentage of other transposable elements ranged from -0.6 to 3.9% (Fig 2).

In summary, the 180-bp repeat was significantly increased in the precipitated fractions (P = 0.002) in ChIP assays. The levels of precipitation of other centromeric repeats were not significantly higher than that of the rDNA control (P > 0.05). The ChIP data suggest that the 180-bp repeat is the main DNA element incorporated into the centromere/kinetochore complex. We cannot rule out the possibility that other centromeric repeats are also incorporated into the centromere/kinetochore complex. However, the copy numbers of these repeats in the functional centromeric chromatin domains would be low and beyond the detection sensitivity of our current ChIP technique.

In a similar ChIP study in maize, ZHONG et al. 2002 Down showed that a centromeric satellite repeat (CentC) and a centromeric retrotransposon (CRM) were precipitated using an antibody against the maize CenH3. CRM belongs to a special retrotransposon family that is highly specific to the centromeric regions of grass chromosomes (MILLER et al. 1998 Down; PRESTING et al. 1998 Down; LANGDON et al. 2000 Down). Frequent insertions of this centromeric retrotransposon into the centromeric satellites were recently demonstrated in rice (CHENG et al. 2002 Down) and maize (NAGAKI et al. 2003 Down). The ChIP assays in maize suggested that ~33% of the CRM elements are located within the functional domains of maize centromeres (ZHONG et al. 2002 Down). In contrast, the Athila elements were not detected in our ChIP assays, suggesting that the Athila elements may rarely insert into the functional domains of Arabidopsis centromeres.

It is not surprising that the 180-bp repeat appears to be a functional component of Arabidopsis centromeres. Satellite repeats are often the main DNA components of eukaryotic centromeres (CSINK and HENIKOFF 1998 Down). The centromeres of maize and rice chromosomes have also been well studied. Both maize and rice centromeres contain satellite repeats (ALFENITO and BIRCHLER 1993 Down; ANANIEV et al. 1998 Down; DONG et al. 1998 Down). The functional roles of the centromeric satellite repeats in maize and rice have been suggested using ChIP and centromere misdivision studies (KASZAS and BIRCHLER 1996 Down, KASZAS and BIRCHLER 1998 Down; CHENG et al. 2002 Down; ZHONG et al. 2002 Down).

Our ChIP results showed that ~15% of the 180-bp repeat was incorporated into the centromere/kinetochore complex. This ChIP value may be an underestimation because some of the 180-bp repeat bound by HTR12 may not be recovered in ChIP assays. However, the ChIP data suggest that only subsets of the 180-bp satellite arrays are involved in centromere function. Similar phenomena have also been reported in maize and humans. ZHONG et al. 2002 Down showed that ~38% of the centromeric satellite repeat CentC in maize was immunoprecipitated using the anti-CenH3 antibody. Human centromeres contain up to several megabases of the 171-bp {alpha}-satellite repeat. However, only subsets of the {alpha}-satellite repeats are found in association with CENP-A (WARBURTON et al. 1997 Down; BLOWER et al. 2002 Down) and show centromeric function when used in artificial chromosome construction (IKENO et al. 1998 Down; SCHUELER et al. 2001 Down).


*  ACKNOWLEDGMENTS

We thank Dr. N. Kumekawa for sharing plasmid clones derived from transposable elements in Arabidopsis. This research was supported by grants DE-FG02-01ER15266 from DOE to J.J. and partially supported by grant 9975827 from the National Science Foundation to R.K.D. and J.J.

Manuscript received November 4, 2002; Accepted for publication December 16, 2002.


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W. Jin, J. C. Lamb, J. M. Vega, R. K. Dawe, J. A. Birchler, and J. Jiang
Molecular and Functional Dissection of the Maize B Chromosome Centromere
PLANT CELL, May 1, 2005; 17(5): 1412 - 1423.
[Abstract] [Full Text] [PDF]

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 Mol. Biol. CellHome page
N. S. Edwards and A. W. Murray
Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat
Mol. Biol. Cell, April 1, 2005; 16(4): 1800 - 1810.
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 J. Cell Sci.Home page
F. Shibata and M. Murata
Differential localization of the centromere-specific proteins in the major centromeric satellite of Arabidopsis thaliana
J. Cell Sci., June 15, 2004; 117(14): 2963 - 2970.
[Abstract] [Full Text] [PDF]

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 Nucleic Acids ResHome page
Y. Zhang, Y. Huang, L. Zhang, Y. Li, T. Lu, Y. Lu, Q. Feng, Q. Zhao, Z. Cheng, Y. Xue, et al.
Structural features of the rice chromosome 4 centromere
Nucleic Acids Res., April 2, 2004; 32(6): 2023 - 2030.
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 Plant CellHome page
W. Jin, J. R. Melo, K. Nagaki, P. B. Talbert, S. Henikoff, R. K. Dawe, and J. Jiang
Maize Centromeres: Organization and Functional Adaptation in the Genetic Background of Oat
PLANT CELL, March 1, 2004; 16(3): 571 - 581.
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 GeneticsHome page
R. J. Mroczek and R. K. Dawe
Distribution of Retroelements in Centromeres and Neocentromeres of Maize
Genetics, October 1, 2003; 165(2): 809 - 819.
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