The chromosomes of humans and other eukaryotes provide the seemingly contrasting functions of compacting the genomic DNA several thousand times its length while also allowing for efficient processes such as replicating the genome and expressing genes encoded within it. To accomplish this, the DNA is assembled into a complicated, multifaceted complex known as chromatin. The initial level of packaging DNA into chromatin involves wrapping short ~200 bp segments around protein spools comprised of the core histone proteins into structures known as nucleosomes. Immensely long, genome-sized strings of nucleosomes are assembled into a hierarchy of higher-order chromatin structures, to form chromosomes. Formation of levels of structure above the nucleosome involves essential inter-nucleosome interactions provided by the 'tail' domains of the core histone proteins, which protrude out from the main body of nucleosomes. Regulation of these domains is a key component of the regulation of gene expression. While nucleosome structure is fairly well understood, higher- order chromatin structures and inter-nucleosome interactions remain poorly defined. Moreover, how posttranslational modifications within the tail domains result in modulation of chromatin higher order structures to regulate gene expression is not well understood. Importantly, mutations that result in changes to core histone tail modifications have been linked to diseases including cancers in humans. In prior work we demonstrated that the core histone tail domains mediate inter-nucleosome interactions in higher-order chromatin structures. The primary goals of the work described in this proposal are to: 1) understand how the core histone tail domains mediate inter-nucleosome interactions in chromatin by characterizing short-range and long-range inter-nucleosomal interactions of the H3 and H4 tail domains in both reconstituted model complexes and in native chromatin, 2) to define the mechanisms by which linker histones bind to nucleosomes, and in particular how the H1 C-terminal domain stabilizes higher order structures and 3) understand how the mechanisms by which the HMGN 'architectural factors' and acetylation of specific lysines within the core histone tail domains alter chromatin structure to allow for gene expression. We will use several novel approaches including site-directed crosslinking of tail-DNA interactions, fluorescence-based methods (FRET) and other chemical probing approaches to investigate structures and interactions of the tail domains, H1, and the effect of HMGNs and acetylation on these domains. In addition, we will develop an in vivo FRET approach to assess core histone and linker histone structures in live cells. These results will provide a basis for understanding how transcription, replication, DNA repair, and other processes occur in a chromatin environment.
Within the eukaryotic cell nucleus, the human genome is associated with core histones and other proteins to form a multifaceted complex known as chromatin. This complex brings about the orderly packaging of the immense length of DNA within the tiny volume of the nucleus and is directly involved in critical functions such as regulation of gene expression. Defects in the regulation of chromatin structure result in cancer and other genetic diseases. This project aims to elucidate fundamental aspects of how chromatin structure is assembled and regulated, and impinges on process like gene expression.
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