Nucleosomes are the basic units of eukaryotic chromatin structure. They consist of approx. 150 bp DNA wrapped around a protein core formed by eight histone proteins.
In this highly stable complex the histone subunits hinder binding of other proteins to nucleosomal DNA. Thereby they play an important role in the regulation of all processes that require access to DNA, e.g. transcription and replication. The stability of nucleosomes suggests that elaborate mechanisms must have evolved to guide nucleosome dis- and reassembly and further structural changes. A detailed analysis of nucleosome dynamics is essential for a profound understanding of these processes. We analyse the dynamics of nucleosomes by measuring distances between different points within the nulceosome with Fluorescence Resonance Energy Transfer, a method to measure intramolecular distances. We use nucleosomes that are labeled with fluorescent dyes at specific positions. As a dynamic system nucleosomes adopt different conformations. To avoid averaging over different conformations and to unravel heterogeneities we also analyze these processes on a single-molecule level.
The nucleosome as the basic repeating unit of chromatin regulates DNA accessibility and has significant influence on genetic function. Two copies of each histone protein H2A, H2B, H3, H4 build up the protein octamer where short fragments of approximately 150 bp of DNA are wrapped around. This complex of DNA and histone octamer is called nucleosome. The N-terminal tails of the histone proteins are not part of the core structure and protrude from the nucleosome. It is known that they play an important role for inter- and intranucleosomal interactions. This project focuses mainly on the role of histone tails. One goal is to analyse the contribution of the tails to the overall stability of the nucleosome. To answer this question I will use truncated versions of H3 and H4 in which parts of the N-terminal tail regions are removed. With the help of this constructs I will experimentally control previously obtained results of MD simulations. Furthermore I will design site-specific mutated recombinant H2A proteins (R81A and R88A) with a single amino acid exchange at two important postitions. Until now it is not clear if these amino acids are crucial for the reconstitution of the nucleosome or if they only contribute to the stability of the complexe. To analyse the above mentioned issues I will perform an in vitro assay which is already established in the work group. This assay comprises fluorescently labeled nucleosome which are labeled on different positions within the histone core and along the DNA. The most important techniques for the analysis are flurorescence correlation sprectroscopy (FCS) and single pair Förster resonance energy transfer (spFRET). FCS measures the average diffusion coefficients of a fluorescently labeled sample by analyzing fluctuations in fluorescence emission. The diffusion coefficient provides informations about the molecule size and shape and can thereby be used to analyse potential interactions. The second technique spFRET is based on the non radiative energy transfer of two fluorophores which depends on the distance between them. This enables us to distinguish between particular subpopulations of a heterogenous sample. Both techniques will be used to investigate differences in the stability and dynamic of different compositions of in vitro reconstituted nucleosomes. The outcome of this study will increase and strengthen the current knowledge of nucleosome dynamics and thereby lead to a further understanding of the basic regulation of DNA accessibility and the associated regulation of gene function.