Accurate 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.
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.
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.
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.
We are working on understanding the role that centromere transcription plays in regulating centromeric chromatin and kinetochore function. We are identifying regulators of transcription and identifying the RNAs associated with the centromere and their corresponding functions.