Centromeric Chromatin
Accurate chromosome segregation requires that kinetochores assemble exclusively at centromeres. However, centromeric DNA sequences are not conserved, so unique epigenetic and genetic features of the underlying centromeric and pericentromeric chromatin propagate centromere identity. A hallmark of all centromeric chromatin is an essential histone H3 variant (CENP-A) that is required for kinetochore assembly and is likely to be the major epigenetic mark that specifies centromere identity. In addition, the CENP-T protein has a histone fold domain and is also important for kinetochore assembly and function. There are also a number of post-translational modifications to histones within the centromeric and pericentromeric chromatin. To fully understand the mechanisms that ensure faithful chromosome segregation, it is essential to elucidate the underlying features of centromeric and pericentromeric chromatin that contribute to the diverse functions of kinetochores. Surprisingly, the yeast centromere is a poor nucleosome positioning sequence so there are mechanisms required to stabilize the yeast centromeric nucleosome we are studying as well.
Projects
How does centromeric chromatin faciliate kinetochore assembly?
We are using a single molecule TIRF microscopy assay to assemble kinetochores de novo to identify the underlying chromatin requirements. This method allows us to easily manipulate centromeric chromatin and determine the effects on kinetochore function. We also collaborate with Andrew Stergachis’ lab to use Fiber-seq techniques to understand the structure of centromeric chromatin.
Fiber-seq technique to map centromeric chromatin structure in yeast
What are the key properties of centromeric DNA required for centromeric chromatin formation?
We are collaborating with the Asbury and TJ Ha’s labs to explore the properties of centromeric DNA and their role in CENP-A assembly. We are also identifying mechanisms that stabilize the centromeric nucleosome.
What is the composition of pericentromeric chromatin?
We are purifying pericentromeric chromatin to identify the proteins and post-translational modifications associated with this unique and important region of the chromosome. We then use in vivo and in vitro approaches to determine the precise functions of the relevant modifications.
How does proteolysis regulate Cenp-A?
We previously found that ubiquitin-mediated proteolysis keeps Cenp-A from mislocalizing to euchromatin. We are now studying the regulation of the ligase and identifying additional mechanisms that ensure Cenp-A levels are properly regulated to prevent ectopic kinetochore formation.
How does centromeric chromatin facilitate kinetochore assembly?
We are using a method to assemble kinetochores de novo to identify the underlying chromatin requirements. This method allows us to easily manipulate centromeric chromatin and determine the effects on kinetochore function. We are also using this method to identify the RNAs associated with the centromere and their corresponding functions.
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