Chan:Research

Chan Lab

Department of Plant Biology at UC Davis

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Centromeres: Controllers of Inheritance We study basic mechanisms of genetic inheritance. Centromeres control chromosome segregation during cell division, because they are the loci at which chromosomes attach to spindle microtubules via the kinetochore protein complex. Centromere DNA in most plants and animals consists of megabases of simple tandem repeats. These sequences can be dispensable for centromere function. Instead, the centromere is epigenetically specified by CENH3, a centromere-specific histone H3 variant that replaces conventional H3 in centromeric nucleosomes, and is essential for recruiting other kinetochore proteins. The plant Arabidopsis thaliana is ideal for studying chromosome segregation, because it combines facile genetics and cytology with large centromere structure that is similar to vertebrate cells (by contrast, laboratory yeasts have very small centromeres). We have three major research directions: 1) Quantitative characterization of large tandem repeat centromeres The repeated nature of centromere DNA and the large size of plant and animal kinetochores make it difficult to analyze centromere structure in vivo. We have developed a method to count the absolute number of proteins in individual Arabidopsis kinetochores. This will allow us to address what subset of the centromere tandem repeats is bound by CENH3, and to determine the stoichiometric relationship between CENH3 and other kinetochore proteins. Despite the essential nature of centromeres, their DNA sequence and the CENH3 protein evolve rapidly. In collaboration with Ian Korf, we are using comparative genomics to study how centromere DNA evolution has been constrained by functional demands. 2) Regulation of centromere function during meiosis Meiosis creates gametes with half the chromosome number of the preceding cell. In meiosis I, sister chromatids segregate together instead of separating as they do in mitosis. Chromosome segregation errors in meiosis I are the major cause of miscarriages and birth defects in humans, so understanding this process is important for public health. We have identified a mutant form of CENH3 that causes a specific defect in meiotic chromosome segregation. This mutant may help us to understand how chromosome behavior in meiosis is specialized. 3) Engineering centromeres to produce haploid plants Haploid plants that are converted back into diploids can greatly accelerate plant breeding. Such “doubled haploids” produce instant homozygous lines from heterozygous F1s, a process that normally takes 8-10 generations of inbreeding. We have discovered a simple method for producing haploid plants through seed by manipulating CENH3. When Arabidopsis plants expressing altered CENH3 proteins are crossed to wild type, chromosomes from the mutant parent are eliminated, yielding haploid progeny. Haploids are easily converted to diploids, so Arabidopsis geneticists can produce large populations of plants with chromosomes from only one parent. We have written a detailed protocol that describes how to produce Arabidopsis haploid plants, available by clicking this link File:Haploid Arabidopsis protocol.pdf Our method has key advantages over current procedures that often require tissue culture and are limited to specific species or genotypes. As CENH3 is found in all eukaryotes, the procedure should theoretically work in any plant species. To learn more about our technology, please see this website. Chromosome spread from metaphase II of meiosis photographed by Ravi. DNA is stained with DAPI, and five pairs of sister chromatids can be seen on either side of the meiocyte (cytokinesis in Arabidopsis is delayed until after meiosis II).