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Centromere Structure and Chromosome Dynamics

K.F. Sullivan, R.D. Shelby, O. Vafa, T. Kanda,* G. Wahl*

* The Salk Institute for Biological Sciences, La Jolla, CA

The key event in cellular replication is transmission of the genome into daughter cells during mitosis. This transmission is accomplished through the interaction of microtubules of the mitotic spindle apparatus with the centromere, a specialized domain present in a single copy on each chromosome. Centromeres direct the assembly of a motile center at the surface of the chromosome: the kinetochore. Work in this laboratory focuses on understanding how centromere function is specified on human chromosomes.

Analysis of centromere-specific proteins revealed a group of specialized chromatin proteins that are important for establishing centromere function. One of these is centromere protein-A (CENP-A), a homolog of the core nucleosomal protein histone H3. CENP-A is located in a narrow zone at the surface of the centromere known as the inner kinetochore plate and forms a distinctive chromatin substrate upon which additional components of the kinetochore are assembled. We used a biochemical approach to isolate native CENP-A chromatin and its associated DNA and showed, for the first time, that centromeric satellite DNA (alphoid DNA in human chromosomes) is a major component of the inner kinetochore plate. These experiments were extended to isolate kinetochore-associated DNA from a nonprimate species, the Indian muntjac. The results revealed that the distinctive centromere satellite DNA of this species assembles into the kinetochore.

Centromeric satellite DNA sequences are not conserved evolutionarily, and so the detection of mammalian kinetochore DNA as centromeric satellite DNA poses a puzzle: how is the conserved structure of the kinetochore determined by the underlying DNA? One emerging model is that in higher eukaryotes, centromeric DNA does not in fact specify centromere function in the same way that it does in simpler unicellular eukaryotes. Rather, centromeres may be maintained as epigenetic elements of the chromosome, with the molecular information for centromere function residing at the level of DNA modification, protein modification, or protein assembly within the chromatin fiber.

We are focusing on the assembly of kinetochore chromatin. Previously, we discovered that the timing of expression of CENP-A is a critical factor for the assembly of CENP-A at kinetochores. Now, we are determining when kinetochore DNA is replicated within S phase and when newly synthesized CENP-A is deposited onto the chromosome. By elucidating the pathway by which kinetochore assembly takes place, we hope to uncover potential mechanisms for propagating centromere identity through successive cell cycles.

Aberrant segregation of chromosomes is a hallmark of cancer cells and is thought to play a central role in establishing the transformed state. One feature common to many types of tumors is the presence of acentric chromosome fragments known as double minute chromosomes. Double minute chromosomes contribute to the development of tumors by providing amplified oncogenes or drug-resistance genes that confer a growth advantage to cells that harbor the genes.

In collaboration with T. Kanda and G. Wahl at the Salk Institute for Biological Studies, La Jolla, California, we have developed a highly sensitive labeling method that allows visualization of chromosomes and chromosome fragments in living cells during cell division. In this method, the nucleosomal protein histone H2B is modified by linking it to green fluorescent protein. The modified histone is efficiently incorporated into chromosomes, resulting in intensely labeled chromatin that can easily be visualized.

By introducing this probe into a human tumor cell line that contains double minute chromosomes, we discovered that these acentric chromosomal fragments are efficiently distributed into daughter cells during mitosis through a "hitchhiking" mechanism. Double minute chromosomes appear to be sticky; they adhere to each other to form large clusters that in turn adhere to normal chromosomes. As the normal chromosomes are separated through the directed motility of the centromere, clusters of double minute chromosomes are passively dragged into daughter cells. We have also observed double minute chromosomes adhering to and preventing the segregation of normal chromosomes during mitosis by forming bridges between the segregating chromosomes at anaphase. Thus, double minute chromosomes may contribute to the genetic instability of tumor cells at a second level by disrupting normal chromosomal segregation.

PUBLICATIONS

Kanda, T., Sullivan, K.F., Wahl, G.M. Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8:377, 1998.

Sullivan, K.F. A moveable feast: The centromere/kinetochore complex in cell division. In: The Dynamics of Cell Division. Endow, S.A., Glover, D.M. (Eds.). Oxford University Press, New York, in press.

Vafa, O., Sullivan, K.F. Chromatin containing CENP-A and -satellite DNA is a major component of the inner kinetochore plate. Curr. Biol. 7:897, 1997.