PROJECT SUMMARY This application proposes to investigate the newly discovered function of histone H3 as an oxidoreductase enzyme, catalyzing the reduction of cupric (Cu+2) ions to the biousable cuprous (Cu+1) form. The eukaryotic histone H3-H4 tetramer contains a putative Cu2+ binding site at the interface of the apposing H3 proteins with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We have extensive evidence that histones are required for efficient use of copper inside cells, which depend on availability of copper ions in their reduced, +1 oxidation state. It is the Cu+1 ions that are trafficked intracellularly by protein chaperones to destination target proteins. We show that the H3-H4 tetramer, assembled from recombinant histones, binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ levels and copper-dependent activities such as mitochondrial respiration and superoxide dismutase 1 (Sod1) function in S. cerevisiae. Our data have uncovered a function of the histone H3-H4 tetramer with little precedence in literature, revealing that the eukaryotic genome is wrapped around an enzyme. We now propose to develop a mechanistic understanding of this new function of histones and how it is regulated and linked to cellular copper homeostasis. In Aim 1, we seek to understand the mechanism of catalysis by determining the structure of copper-bound H3-H4 tetramer and the contributions of the residues in and around the active site. In Aim 2, we will discern how the enzyme activity is regulated, especially through post-translational modifications of histones and certain histone variants. The enzymatic activity of histones indicates that there must be a previously undiscovered biological network that shuttles Cu2+ to histones and then distributes the reaction product (Cu1+) to different parts of the cell for use by proteins in the nucleus, cytoplasm and mitochondria. In Aim 3, we plan to systematically identify the protein effectors involved in this novel copper biological network in yeast by utilizing a high-throughput CRISPR-interference (CRISPRi) technology. We aim to identify the genes and pathways that integrate the enzymatic activity of histones with other cellular functions. Our proposal will begin to build the scientific foundation for understanding chromatin structure and function as an enzyme and its impact on eukaryotic biology with instructive consequences for the evolution of the eukaryotic cell as well as a range of human pathologies such as cancer and neurodegeneration in which copper homeostasis is altered.

PROJECT NARRATIVE Histones are the main protein constituent of chromatin, the physiological relevant form of the genome, and function to compact the large eukaryotic genomes and regulate DNA-based processes such as gene expression. However, we have discovered that histones also functions as an enzyme to generate copper ions that can be used by the cell for many important processes and functions. We propose to investigate this novel role of histones in regulation of copper biology inside the cell that has important consequences for basic understanding of eukaryotic biology as well as human diseases such as cancer.