The DNA of a single eukaryotic cell is over two meters in length, but compacts in the cell nucleus approximately 100,000 fold by a hierarchical scheme of packaging into nucleoprotein assemblies (""""""""chromatin"""""""") of increasing complexity and largely unknown architecture. Compaction beyond the nucleosome, the basic repeating unit of chromatin, is promoted by linker histone H1, with profound implications for the transcriptional competence of the underlying DNA. The location and mode of interaction of linker histones with the nucleosome is a subject of intense debate, as is the question of how sequence-specific DNA binding proteins recognize their binding site in the context of a nucleosome. Structural models are lacking for both types of interaction. ? ? The objective of this proposal is to determine the structure of two transcriptional regulators (the global regulator H1 and the specific transcription activator FoxA) in complex with the nucleosome, using x-ray crystallography and biophysical methods. It is proposed to investigate the role of linker histone tails in the organization of linker DNA, and to test the hypothesis that histone variants have evolved to modulate interaction with linker histones. These studies will culminate in a long-overdue structure of the nucleosome-H1 complex, which will reveal the location and mode of interaction of this key organizer of chromatin structure and function (specific aim 1).
In specific aim 2, the interaction of the transcription activator FoxA with highly defined nucleosomes will be studied. The hypotheses that the histone octamer contributes to FoxA binding, and that the histone tails modulate accessibility of nucleosomal DNA will be tested. Structural studies will illuminate the mechanism by which FoxA engages and opens chromatin, and the results will represent a paradigm for the mechanism by which the pervasive Fox (Forkhead) factors function in diverse biological regulatory contexts. Together, the proposed studies will provide insight into chromatin higher order structure DNA recognition in a natural chromatin context, and are thus essential to understand how the information stored in the genome is organized and accessed. In particular, the following specific aims are proposed: ? ? 1) Test the hypothesis that linker histone interacts with core histones and organizes the path of linker DNA. ? ? 1.A. Biophysical investigation of linker histone binding to nucleosomes; test the hypothesis that histone variants have evolved to modulate the interaction with linker histones. ? ? 1.B. Determine the crystal structure of H1-bound nucleosomes. ? ? 2) Test the hypothesis that FoxA binding results in an altered nucleosome structure. ? ? 2.A. Biophysical analysis of the binding of FoxA to mono-nucleosomes with defined binding sites. ? ? 2.B. Determine the crystal structure of the FoxA-nucleosome complex. ? ?
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