The packaging of genomic DNA into repeating nucleosome units plays a pivotal role in the regulation of essential biological process including DNA transcription, DNA replication, and DNA repair. Cells must assemble and disassemble nucleosomes to read the information stored in the DNA sequence in a temporally and spatially regulated way. As such, cells have evolved several mechanisms that precisely regulate nucleosome dynamics and influence the local structure of chromatin. These mechanisms include: (1) the incorporation of histone variants into the nucleosome, (2) post-translational modification of histones, (3) histone chaperone activity, (4) ATP-dependent nucleosome remodeling, and (5) nucleosome-binding proteins. We currently do not know how these mechanisms act synergistically, nor how they influence intrinsic nucleosome dynamics. The research proposed in this MIRA application will focus on the quantification of nucleosome dynamics and the molecular basis of its regulation. We will develop a high-throughput assay for the quantification of nucleosome dynamics that will be applied to nucleosome assembly intermediates, mono-nucleosomes and multi- nucleosome arrays. This assay revolves around hydrogen-deuterium exchange analysis chromatin substrates in varied biologically-relevant states. We will also characterize the molecular mechanisms underpinning concerted activity of histone chaperones and histone acetyltransferases. Systems of interest at Rtt109, Vps75 and Asf1; as well as Nap1, CBP/p300 and linker histone H1. Varied approaches will be employed including biochemistry, high-resolution structural characterization, and in vivo complex analysis. The proposed work will impact translational studies motivated to develop anti-fungal therapeutics and identify new avenues for targeting of anti-cancer treatments.
Aberrant activity of the diverse factors that regulate local chromatin structure can lead to diseases associated with altered transcriptional signatures such as seen in human cancers. Our work to understand the molecular mechanisms underlying these regulatory factors may thus uncover new avenues for the development of anti- cancer drugs. Our work to understanding how these regulatory factors work in pathogenic species, such as yeast, may also provide targets for the development of novel anti-fungal therapeutics.
