Analysis of Centromere Structure and Function:
A part of the NSF Plant Genome Initiative

      Centromere morphology has been well-characterized in higher eukaryotes, and a number of specific proteins that localize to distinct centromere domains have been identified. Although significant effort has been invested in determining how kinetochore proteins interact with mitotic spindles, far less is known of the mechanisms that regulate the assembly of those proteins onto precise sites on the DNA. At present, it is not clear if the primary sequence carries specific cues, or if, instead, secondary structures or epigenetic modifications are inherited through the cell cycle. An understanding of the basis of chromosome inheritance in higher organisms requires the coupling of the cell biology of meiosis with the genetic consequences of chromosome segregation and exchange — Arabidopsis is currently the only model system which presents such an opportunity.

      Using the quartet mutation, we performed a genome-wide analysis of recombination, demonstrating that higher cells carefully control the distribution and number of sites of genetic exchange. This analysis provided the first demonstration that higher centromere functions could be precisely mapped in the context of a natural chromosome (Copenhaver, Browne and Preuss, 1998, PNAS 95: 247-252). Using genetic markers as anchors, we have now assembled physical maps covering the unique DNA within each centromere — defining intervals ranging from 500 to 1700 kb per centromere (Copenhaver et al., Science 286: 2468). The efforts of sequencing teams at TIGR, Cold Spring Harbor, and Washington University yielded DNA sequences that nearly span two centromeric intervals. Within the next year, complete sequence characterization of all five Arabidopsis centromeres will be obtained. This work will make it possible to examine DNA within the centromeres and compare it to the surrounding regions in order to identify key genetic or epigenetic features that specify function.

      The centromeric regions that we defined contain a surprisingly large number of predicted genes, both in the areas immediately flanking the centromere, as well as within the centromere itself. To date, we have identified 27 genes within genetically-defined centromeres that are, surprisingly, expressed at measurable levels. Each of these genes is represented only once within the Arabidopsis genome. One of the centromeric genes, CUE1, is necessary for Arabidopsis growth, and three have known metabolic functions (Copenhaver, Science 286: 2468). In the past, genetic analyses revealed only a few examples of genes that can be expressed near centromeres; centromere-linked genes are more often subject to silencing or exhibit variable expression. It is possible that the expression of centromere-linked genes in Arabidopsis is controlled by unusual regulatory elements that ensure stable expression, a model which we are currently testing.

Centromere mapping in Chlamydomonas

Future objectives:

      Centromeres undergo dramatic changes in morphology through the cell cycle, alternating between an extended conformation during cell growth and a condensed form incorporating millions of base pairs during mitosis and meiosis. We plan to expand significantly our analysis of centromeres, determining the relationship between the primary DNA sequence and secondary and tertiary structure. In parallel, we will determine if the folding of centromere regions into ordered structures is an intrinsic feature of the DNA sequence, or instead depends on chromosomal context. The functions of individual centromeric sequences can be analyzed by addressing the following critical issues: Which regions are responsible for high-fidelity inheritance? Which sequences are necessary for the formation of characteristic centromere structures during cell division? Do different DNA regions differentially affect recombination frequencies?

      As the genomes of other higher eukaryotes are sequenced, we will explore the diversity and evolution of centromeric sequences. First, we are performing a systematic PCR analysis of centromeric DNA in twenty different varieties of Arabidopsis, obtaining an overview of centromere conservation within the species. In addition, we are collaborating with groups that have mapped centromeres in other organisms in order to determine if syntenic regions, common to higher genomes, can be identified..