Chromatin Structure and DNA Processing in Euplotes Crassus

Northwestern University, Evanston IL

Investigators

Abstract

0078182 Jahn The ciliated protozoan Euplotes crassus is being studied as a model system in order to uncover the mechanisms producing rearrangements of DNA. Ultimately, investigating this system should help answer questions about the structure of chromosomes and the nucleus, and how cells manipulate these structures such that the genome remains functionally stable while providing genome plasticity, which can be evolutionarily and developmentally advantageous. By studying a controlled rearrangement process such as that occurring in the E. crassus during the formation of a macronucleus, it can be determined what chromosomal proteins, DNA sequences and enzymes are involved and how the process is controlled. E. crassus, like other ciliated protozoa, possesses two types of nuclei, macronuclei and micronuclei, that differ in their properties. Most notably, chromosome segregation is absent from the macronucleus and transcription is absent from the micronucleus. Chromatin and chromosome structure and genomic organization differ in these two types of nuclei. In addition, during the developmental process of forming macronuclei from micronuclei, extensive DNA elimination occurs that includes site-specific deletions and chromosome breakage with telomere addition. The abundance of these rearrangements, their developmental programming, and their occurrence in single-celled organisms that can be developmentally synchronized en masse, provide a unique model system for the analysis of chromosome structure and the mechanisms of genome rearrangements. The principal investigator has identified an unusual chromatin structure of the highly abundant Tec element transposons that are undergoing elimination during macronuclear development. In addition, her laboratory has identified a lysine/arginine-rich, histone H1 or HMG-like,'chromosome scaffold' protein (p85) that has the following properties: a) it is only found in the developing macronucleus, b) it is associated with eliminated DNA (including the Tec elements), c) it colocalizes with topoisomerase II, and d) it affects topoisomerase II activities in vitro. This points to a role for topoisomerase II-mediated, chromosome condensation processes that are conserved in all organisms in ciliate genomic rearrangements. This project aims to determine the sequence of p85 to allow identification of any similar proteins in other organisms. In addition, the role of p85 in DNA elimination will be determined by defining what DNA sequences it binds to and what sequence specificity it has, and by determining how this protein affects chromatin structure. Finally, the interaction of p85 with topoisomerase II will be further analyzed.