Centromere and Kinetochore Assembly
The key site for attachment of the chromosome to the mitotic spindle is the kinetochore. Kinetochores demonstrate amazing structural diversity: budding yeast employ a minimalist kinetochore that binds a single microtubule, human kinetochores bind approximately 20 microtubules, and nematode worms expand their kinetochores so that they occupy the entire length of the chromosome. Despite organizational differences, many of the core proteins of the kinetochore are conserved throughout eukaryotes. We are attempting to understand the underlying principles that give rise to this diversity of kinetochore structures but still allow the common function of microtubule binding during mitosis.
The kinetochore is a transient structure that only exists during mitosis and is disassembled after cells segregate their chromosomes and rebuild the nuclear envelope. However, the underlying foundation for the kinetochore, the centromere, persists throughout the cell cycle. The centromere is comprised of a uniquely specialized region of chromatin and a core complex of approximately 20 constitutively associated proteins. Centromeric chromatin is distinguished by the replacement of histone H3 in the nucleosome with the histone H3 variant centromere protein A (CENP-A) that is thought to epigenetically specify centromere function. We are particularly interested in how centromeric chromatin is assembled and how that chromatin directs the assembly of the centromere and the mitotic kinetochore. We are studying how the specialized centromeric nucleosomes are deposited in centromeric chromatin, how this chromatin is stably propagated through many cell divisions, and how CENP-A chromatin is recognized to build the centromere and mitotic kinetochore.
Terry Winters 2000
We are studying the process of chromosome segregation in eukaryotic cells. Accurate chromosome segregation is critical to ensure that cells are duplicated without genome loss or damage. Chromosome segregation mechanisms are conserved from yeast cells to humans. Chromosomes are replicated during S-phase and are then segregated by a microtubule spindle during mitosis.We are interested in understanding how the chromosomes attach to the microtubule spindle during mitosis so that each daughter cell receives one and only one of each sister chromatid. We study how the forces for chromosome segregation are generated within the mitotic spindle and along the length of the chromosome and the mechanisms cells use to detect and correct errors during mitosis.
RNA Directed Chromatin Modification
Noncoding RNAs play essential roles in regulating the organization of chromatin domains. Two of the most well studied examples are the Xist RNA in humans that is required silencing transcription of one X chromosome in females so that the proper gene dosage is maintained between male and female cells and the short RNAs used by the RNA interference machinery in fission yeast to form heterochromatin at centromeres and ensure accurate chromosome segregation. In both of these examples, RNA molecules direct the modification of chromosomes resulting in changes in chromatin organization, transcriptional output and the functional properties of the chromosome. Although the importance of RNA in the process of chromosome specialization is widely appreciated, the mechanisms through which RNAs guide the modification and reorganization of chromatin domains are only beginning to be understood.Recent efforts in our laboratory have focused on RNAs that are associated with vertebrate mitotic chromosomes. Our goal is to decode the identities and functions of RNAs that regulate mitotic chromosome structure and to understand the mechanisms through which RNA molecules are targeted to chromosomal domains and used alter chromatin. Of particular interest are RNAs transcribed from human centromeres that direct the post-transcriptional modifications of histonesto ensure faithful chromosome segregation during vertebrate mitosis.
Chromosomes undergo dynamic structural changes during the cell cycle and during cell differentiation that dictate the functional properties of the chromosome. One of the most dramatic examples of this is the mitotic condensation of chromosomes that is required for chromosome segregation during cell division. We currently have a very limited understanding of the higher order folding of the chromosome. The X-ray crystal structure of the nucleosome has provided a detailed understanding of the primary level of organization of the chromosome (Luger et al, Nature 1997). However, the organization of the chromosome beyond the wrapping of DNA around the nucleosome is unknown and the mechanisms that control chromosome structural rearrangement are only beginning to be deciphered.We are developing techniques to map the higher order folding of chromosomes from the nucleosome level to the level of the condensed mitotic chromosome. These methods should allow us to interrogate the changes in chromosome structure that occur as cells divide and differentiate and as cells silence some regions of their genome while mantaining transcriptional activity in others. We are also developing methods to analyze the contribution of non-coding RNAs to the structural rearrangements in chromosomes that occur during mitosis and gene silencing. Through these approaches we hope to gain insight into the higher order organization of chromosomes and the mechanisms that control chromosome structural changes.