Scientists have discovered that two proteins, previously known for helping to construct "silent" regions of chromosomes, also play an important but unforeseen role in building special structures that cells need to ensure accurate chromosome copying during cell division.
The work was performed by researchers in Lawrence Berkeley National Laboratory's Life Sciences Division.
"When cells divide, they must make sure that both daughter cells receive exactly one copy of each chromosome," says Paul Kaufman, a staff scientist at Berkeley Lab and assistant adjunct professor of biochemistry and molecular biology at the University of California at Berkeley. "This process is known as chromosome segregation, and if it goes awry, cells can lose chromosomes or acquire more than one chromosome copy." In humans, lack of a chromosome can cause blood disorders including leukemia; an extra chromosome 21 causes Down Syndrome.
As cell division begins, spindles form that will eventually pull the original chromosomes and their copies apart into two daughter cells. These spindles attach to constricted regions of chromosomes called centromeres: complexes of proteins called kinetochores fasten the centromeres to the spindles.
To elucidate this process, Kaufman and his graduate students, Judith Sharp and Alexa Franco, focused their research on Saccharomyces cerevisiae, the familiar single-celled organism used for centuries to ferment beer and wine and to cause bread dough to rise.
"We use yeast as a model system in which to investigate the fundamental building blocks of chromosomes," Kaufman says. "The structures we're studying are evolutionarily conserved and are much the same in many organisms, including humans."
In particular, the researchers looked at two kinds of proteins known to be important for depositing proteins onto chromosomes. One, CAF-I (for Chromatin Assembly Factor I), puts together nucleosomes, the fundamental building blocks of chromosomes. Nucleosomes consist of DNA wrapped around groups of structural proteins called histones.
Kaufman and his coworkers had previously demonstrated that CAF-I and another set of proteins, called Hir (for histone regulatory), are important for the formation of so-called "silenced" regions of chromosomes, where large stretches of DNA are enveloped in protein structures that repress gene expression.
Silencing is vital to chromosome stability and accurate segregation. In higher organisms, loss of silencing can lead to cancer; even in yeast it can lead to developmental abnormalities and premature aging.
"We knew that CAF-I could assemble nucleosomes in a test tube, but it wasn't until we applied genetic tools that we discovered how much more there was to the picture," Kaufman says. "This is the advantage of working with yeast. It's easy to get rid of a specific gene and find out what happens when the protein it codes for is missing."
When the researchers removed the genes that code for both CAF-I and Hir proteins, the growth rate of the yeast slowed markedly. Moreover, yeast lacking these genes lost chromosomes or gained extra ones hundreds of times more often than ordinary wild yeast. Yeast that lacked only one of the two genes was not similarly affected, however.
The delay in cell division that occurred when both genes were missing seemed due to the activation of something called the "spindle assembly checkpoint," a mechanism that monitors the proper attachment of chromosomes to spindles before separation begins. This clue pointed to the involvement of kinetochores.
Kaufman and his colleagues performed a series of tests indicating that both CAF-I and Hir proteins are highly localized on centromeres and therefore act directly to affect structures at these locations. Their functions seem to overlap; thus they can partially substitute for each other if one is missing. But when both are missing, defects in centromere structures occur.
"This the first demonstration that proteins that control histone deposition contribute to the formation of functional kinetochores," Kaufman says. "Kinetochores are essential to proper chromosome segregation during the cell division process."
The Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California. - By Paul Preuss
(Reference: "Chromatin Assembly Factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae," by Judith A. Sharp, Alexa A. Franco, Mary Ann Osley, and Paul D. Kaufman, appears in the 1 January 2002 edition of Genes & Development. As of today, the text is accessible online at this URL.)
[Contact: Paul Preuss]