Multiple levels of epigenetic regulation are essential to maintain the pluripotent state of embryonic stem (ES) cells. Histone modification and DNA methylation have been shown to control the stemness of ES cells. Despite the importance of nucleosome positioning in epigenetic regulation, whether and how nucleosomes regulates stem cell functions remains poorly defined, in part due to technical obstacles to obtain high-resolution nucleosome maps in higher organisms. Recently we obtained a yeast nucleosome map at base-pair resolution by combining a novel chemical mapping approach and a Bayesian deconvolution algorithm. This current project seeks to extend this new approach to map nucleosomes for the mouse genome. Using ES cells expressing an engineered histone H4, our preliminary results have demonstrated the feasibility of constructing high-resolution nucleosome maps for higher organisms. The chemical mapping requires introducing a unique cysteine into histone H4 at position 47 to covalently attach a sulfhydryl-reactive copper-chelating label. This procedure is complicated by existence of multiple copies of histone H4 genes in the mouse genome. Thus our first aim is to develop a chemical mapping protocol, and carry out genome-wide chemical mapping in cultured mammalian cells. To define the center positions of nucleosomes based on chemical data requires deconvolution of cleavage signals from locally overlapping nucleosomes. The Bayesian algorithm we developed previously for yeast is computationally inefficient for the mouse data. Thus our second aim is to develop a more efficient computing algorithm and software tools. With above aims achieved, we will generate chemical maps of nucleosomes for both pluripotent ES cells and differentiated fibroblast cells, and quantify the gene expression in parallel by RNA-seq experiments. We will perform high-resolution analysis on global features of nucleosome positioning for the mouse genome, and investigate how nucleosomes regulate gene expression in coordination with other epi-regulators. Lastly, using chemical nucleosome maps we aim to determine the impact of the repressive chromatin mark H3K27me3 on nucleosome positioning throughout the genome. Taken together, the proposed work will delineate the nucleosome landscape of embryonic stem cells in unprecedented details and accuracy, providing insight into a new aspect of epigenetic regulation of the pluripotent cellular state.
Nucleosome positioning, a critical aspect of the epigenetic landscape of a cell, can dramatically influence gene expression patterns;however, genome-wide nucleosome map is not available at a desired resolution in higher organisms. In this study, we will employ a new computational biology approach to determine how the nucleosome landscapes differ in distinct epigenetic states of embryonic stem cells. Understanding the epigenetic changes associated with different cell states is an essential step to develop diagnostic and therapeutic strategies for a range of diseases.