Functional Genomics of Maize Centromeres
R. Kelly Dawe (1), Jiming Jiang (2), James A. Birchler (3), Wayne A. Parrott (1) , Gernot Presting (4)
(1) University of Georgia, (2) University of Wisconsin, (3) University of Missouri, (4) University of Hawaii
Objectives and results from first period
1) Sequence 300 kb of maize centromeric DNA from the A centromeres, and develop a database of repetitive DNA elements associated with maize centromeres. Results: The sequencing is complete and provide further support for the idea that centromeres are composed of satellite repeats and retrotransposons, many of which are centromere-specific.
2) Fully sequence the centromeres from two derivatives of the maize B chromosome that have suffered a series of centromere deletions. Results: We carried out about half of the sequencing, but ran into contiging problems. The problems were partially solved by generating a complete Fiber-FISH map, complete with at least four deficiency breakpoints.
3) Identify putative functional repeats by chromatin immunoprecipitation using antibodies to maize CENH3 (Centromeric Histone 3). Results: We used ChIP to demonstrate that CentC and the centromeric retrotransposon CRM interact strongly with CENH3, providing the first evidence in plants that centromere-localized DNAs are functional components of the centromere/kinetochore complex. A phosphorylated form of CENH3 was also identified.
4) Transform maize with promising centromeric BACs to determine whether they organize a kinetochore and generate chromosome movement. Results: Transformation efforts with maize have been slow. We have turned to rice transformation to speed the process, and have successfully incorporated rice and maize centromeric DNA into rice with very encouraging results.
Objectives for renewal
1) Identification of centromeric contigs using the public BAC library and database. Public BAC end sequence will be examined for the presence of known centromere repeats. Low pass sequence will be obtained from roughly 10 Mb of centromeric contigs. Low-copy markers and variants of CentC (a tandem repeat occupying the bulk of maize centromeres) will be cataloged and mapped to chromosomes.
2) Sequencing a maize centromere. On maize centromere 4 the longest continuous CentC array is 50 kb, much smaller than an average BAC clone. A BAC library from an oat-maize addition line containing chromosome 4 will be prepared and maize centromeric BACs identified by colony hybridization. These will be fingerprinted and high-pass sequenced.
3) ChIP using anti-kinetochore antisera. Antibodies to phospho-CENH3, CENPC (Centromere Protein C), Mis12 (Minichromosome instability 12) will be mapped to centromeres using chromatin immunoprecipitation (ChIP) and newly identified single copy genetic markers.
4) Competition assays for centromere function. To explain the rapid evolution of centromeres, it has been proposed that homologous centromeres compete for access to female reproductive cells by a meiotic drive-like mechanism. We will test this hypothesis by pairing and test-crossing chromosomes containing different maize B centromeres (differing in size and sequence complexity). Genetic tests will allow us to determine if preferential recovery is caused by meiotic drive or random chromosome loss.
5) Making artificial centromeres in maize and rice. Preliminary results indicate that introduced centromeric DNA does not always display centromere (kinetochore) activity. New maize and rice centromeric BAC constructs will be introduced biolistically, and cell lines subjected to treatments that alter the epigenetic environment of trans-centromeres, and (in principle) promote the recruitment of kinetochore proteins. Once appropriate conditions are established, we will select for free-replicating artificial chromosomes by adding telomere repeats to the centromere constructs
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Figure 1. Organization of maize centromeres. As illustrated by a centromeric BAC sequence (A), maize centromeres are composed of CentC tandem repeat arrays, centromeric retrotransposons (CRs) and a variety of other repeats. One particularly well-conserved CR element, CRM (B) occurs primarily as complete and uninterrupted elements, and is thoroughly interspersed with CentC at the level of the light microscope. C) Oat-maize chromosome addition lines were used to study the DNA organization of individual maize centromeres. FISH analysis using CentC and CRM probes confirmed the presence of maize chromosomes in the seven oat-maize addition lines used. The distributions of CentC and CRM sequences in the centromeres of maize chromosomes 1, 2, 3, 4, 6, 7, and 9 were analyzed using fiber-FISH. The lengths of the fiber-FISH signals (in micrometers) were converted to kilobases using a 3.21 kb/μm conversion rate (Table 1).
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Figure 2. ChIP analysis of maize centromeres. A) CENH3, a highly conserved protein that replaces histone H3 in centromeres, is thought to recruit many of the proteins required for chromosome movement. CENH3 is present throughout the cell cycle, and localizes to the inner kinetochore (beneath MAD2). B) ChIP Results. The data were quantified by determining the percentage of probe sequence in the pellet, and subtracting the percentage from the negative control (preimmune). ~38% of the CentC satellite array, and 33% of the CRM elements are immunoprecipitated by anti-CenH3 antibodies (probes used on the blots are indicated below, on a map of the CRM element). There was no significant immunoprecipitation of the major knob (180bp) repeat or a retrotransposon related to CRM, Tekay. C) Immunocytochemical association of CENH3 and CentC/CRM. Chromatin from purified nuclei was gently stretched, fixed, and treated with the maize antibody to CENH3. Two fibers are shown: for each fiber the upper signal is derived from CENH3 and the lower signals are derived from CentC (green) and CRM (red). We frequently observed fiber-FISH signals that spanned regions not covered by anti-CENH3 signals. However, chromatin fibers stained by CENH3 antibodies nearly always coincided with CentC/CRM sequences.
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Figure 3. CENH3, ph(Ser50)CENH3, and ph(Ser28)H3 localization in meiosis and mitosis. A) The locations of peptides used to prepare antisera. The top line shows a portion of histone H3, ranging from amino acids 24 to 46. Ser28 is indicated. The second line shows the corresponding region of maize CENH3 with Ser50 indicated. The two peptides used for antibody production are shown below. B) Immunolocalization of CENH3, ph(Ser50)CENH3, and ph(Ser28)H3 at meiosis and mitosis. Histones are shown in red, chromosomes in blue, and tubulin in green. The sole exception is anaphase II, where CENPC instead of tubulin is shown in green. The data illustrate that CENH3 is phosphorylated at diakinesis-anaphase, with a peak at metaphase. Pericentromeric histone H3 is phosphorylated at serine 28 at roughly the same time. Bars (=5µm) indicate the scale for all images in a row.
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Figure 4. FISH localization of BAC-derived trans-centromeres. A) Rice centromeric BAC inserted into rice, as detected using the rice CentO satellite (arrow). B) Maize centromeric BAC 16H10 inserted into rice, as detected using CentC. C) Fiber-FISH of the insert shown in B. The trans-centromere is roughly 120 kb long.
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