Our research objective is to measure and interpret the conformational stability of nucleosomes. This is important if we are to understand the functioning of nucleosomes in vivo during cellular transcription and replication. We want to know what conformational changes are possible, and how modification of chromatin structure can affect those changes. Most of our work to date has involved studies of nucleosome core particles, and we now propose to develop a number of new chromatin model systems. These will include nucleosomes with more than 146 bp of DNA, and oligomers of nucleosomes. We particularly want to examine oligomer, because they will give us a means for measuring conformational transitions related to the higher-order folding of chromatin. The oligomeric structures will be isolated from natural chromatin preparations, or generated synthetically on genetically-engineered tandem repeats of DNA. We will examine the solution-induced conformational changes of these chromatin systems, before and after altering them by the binding of various proteins, such as H1 and HMGs 14 and 17. H1 is involved in the higher order folding in chromatin, while the HMG proteins are believed to be associated with transcriptionally-active chromatin. In addition, we will generate the synthetic model systems using different combinations of acetylated histones, to better understand the functions of acetylation, another important chromatin modification. Although we use a number of different biophysical methods in our studies, most of our work makes use of fluorescence measurements. These include measurements of steady-state fluorescence anisotropy, and measurements of the time-resolved decay of the fluorescence anisotropy using a picosecond laser-based fluorometer. We have shown that the intrinsic tyrosine fluorescence of nucleosomes is very sensitive to conformational changes. We will continue to use the tyrosine fluorescence in our studies, but will develop applications of extrinsic fluorescent proves as well. We plan to use extrinsic protein probes to learn about protein-DNA interactions. Other extrinsic probes, with long lifetimes, will be used to observe shape changes of nucleosomes and other chromatin models. This latter goal will be accomplished by rotational diffusion measurements using the decay of the fluorescence anisotropy of the probes. Finally, the fluorescence of DNA- intercalating probes will enable us to examine the torsional flexibility of the DNA in the various chromatin preparations.
Showing the most recent 10 out of 15 publications
