Integrating chromatin structure and global chromosome dynamics

The role of DNA is to store an individual's genetic information such that it can be used during normal growth and development and be accurately copied during the different divisions of the cell. Human cells contain DNA totalling about 2 m in length that has to be packed within the cell nucleus which is only 0.01 mm in diameter. Importantly, the DNA must be organised in such a way that it is readily accessible for a variety of crucial processes. The information it contains must be easily read (transcription) so that the cell can rapidly produce proteins. It must be readily duplicated (DNA replication) and accurately separated during cell division (mitosis) and sexual reproduction (meiosis). Also, it is essential that any break, knot or tangle that might occur can be repaired (DNA repair). DNA associates with different proteins forming a nucleo-protein complex called chromatin. This enables the compaction necessary to fit the naked DNA inside the cell nucleus whilst maintaining access to the genetic information. The chromatin is divided into individual structures constituting chromosomes. During the process of cell division when the individual chromosomes have been duplicated chromosome condensation is necessary to ensure their accurate distribution. Miss-regulation of chromosome condensation can lead to cell death, cancer and improper chromosome segregation during cell cycle or during the production of gametes. There are different levels of compaction involved in packaging DNA into chromosomes. The basic structure is the nucleosome, formed by wrapping naked DNA around a core of proteins known as histones. The nucleosomes are arranged along the DNA forming a 10nm diameter fibre, likened to beads on a string. Despite the old impression that nucleosomes were static structures, nowadays, a nucleosome is considered as a highly dynamic assemblage. Changes to this organised structure are facilitated through histone modifications, modelling factors and exchange of histone proteins. The nucleosomal fibre is further compacted by winding it into a 30 nm fibre whose structure remains controversial. This fibre is additionally arranged into loops that are attached to a multi-protein axis called the chromosome scaffold. Although the biochemistry of histones and other chromosome-associated proteins has been studied intensively, their interactions to achieve chromosome condensation are still poorly understood. My research project aims to unravel the biological significance that the different levels of DNA compaction structures and components have on chromosome condensation in the nucleus. The correct chromosome condensation is essential for the stability of the genetic information. This project will contribute to the understanding of different important and interesting subjects like cell division, cancer, stem cells, chromosome alterations, fertility and plant breeding. The key proteins involved in chromosome condensation are conserved throughout eukaryotic evolution indicating that they are likely to have fundamental roles that are species-independent. I will be using Arabidopsis thaliana, a plant model organism for basic research in genetics and molecular biology and a good experimental system without any of the ethical issues related to working with animals. Furthermore, I have developed a range of molecular cytogenetic techniques that have contributed to the study of chromosome dynamics in Arabidopsis. I have recently found exciting evidence that some histone and chromosome scaffold mutants are affected in chromosome condensation at different levels. Thus, I would like to conduct a thorough analysis of these and other related proteins. I propose to use a multidisciplinary approach combining new high-resolution cytogenetic techniques, mutant characterisation, proteomic analysis, and mathematical models to resolve the complicated interactions of individual chromatin components that result in accurate chromosome condensation.

Eukaryotic DNA is packaged into chromatin and chromosomes to enable the entire genome to fit into the nucleus. This compaction must be organized in order to preserve the vital DNA processes necessary for transcription, replication, repair, segregation and recombination. The chromatin structure classical model has been used to explain all aspects of chromosome condensation. However, recent studies have led to reconsider the role of the components coordinating chromosome condensation. This proposal aims to investigate the key protein components and interactions involved in providing the accurate chromosome condensation in the model organism Arabidopsis thaliana. The key proteins and mechanisms involved in chromatin structure and chromosome condensation are conserved throughout evolution in eukaryotes, thus this investigation will be reciprocally informative to apply in plants and animals. I propose to use a multidisciplinary approach using functional analysis of mutants, genomics and proteomics, different imaging technologies and dynamic studies in combination with mathematical and bioinformatics to integrate the complexity of the biological data obtained. This study will investigate the role of histone proteins in chromosome condensation by analysis of knockdown and knockout mutant lines. A detailed characterisation of chromatin defects of the identified mutants will be achieved by different imaging techniques: fluorescence, confocal, electron and atomic force microscopy. Furthermore, the protein interactions between the histone proteins involved in chromosome condensation will be inferred by a proteomic approach. Additionally, this proposal aims to explore the role of chromosome scaffold proteins in chromosome condensation. An integrative and systems biology approach is proposed to study the wide range of protein components and interactions involved in chromatin compaction to predict new interactions and produce a computational model for chromosome condensation.