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. To accomplish this, the DNA is assembled into a complicated, multifaceted complex known as chromatin. The packaging of genome DNA into chromatin first involves wrapping short (~200 bp) segments around protein spools comprised of histone proteins into structures known as nucleosomes. Immensely long, genome-sized strings of nucleosomes are folded and assembled into a hierarchy of structures of complex structures, to form chromosomes. Formation of these large condensed structures involves essential inter-nucleosome interactions provided by the `tail' domains of the core histone proteins, which protrude out from the main body of nucleosomes as well as binding of an additional `linker' histone, which stabilizes higher order structures. Regulation of inter-nucleosome interactions by posttranslational modifications within the core histone tail domains and linker histones is a key component of the regulation of gene expression. While much is know about the structure of individual nucleosomes, higher-order chromatin structures and inter-nucleosome interactions remain poorly defined. Moreover, how posttranslational modifications within the histones modulate chromatin higher order structures to regulate gene expression is not well understood. Importantly, mutations that alter histone posttranslational modifications have been linked to diseases including cancers in humans. In prior work we documented inter-nucleosome interactions in higher-order chromatin structures and defined critical aspects of how linker histones bind to nucleosomes in chromatin. The primary goals of the work described in this proposal are to: 1) define impact of modifications that transition inactive chromatin structures to those hospitable to active genes in a model chromatin system; 2) define aspects of how linker histones bind to nucleosomes and oligonucleosome arrays, and; 3) investigate newly discovered communication between the core histone tail domains, the architectural proteins HMGN1 and HMGN2, and linker histones. In addition we will determine the molecular basis for our newly discovered effect of H1 phosphorylation on H1 CTD structure and determine if there is a corresponding effect on chromatin folding/compaction. We will use several novel approaches including site-directed crosslinking of tail-DNA interactions, fluorescence and FRET based methods, and novel chemical probing approaches to investigate structures and interactions in chromatin. 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 chromatin structure, and how chromatin modifiers regulate access to DNA within chromatin and contribute to control of gene expression.
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