A major goal of regenerative medicine is to understand the regulation of cell fate, and then to use that understanding to develop cell-based and drug-based therapies. Cell fate is determined by the immediate history and the environment of a cell. History and environment are in some senses erased at the formation of the zygote and the restoration of totipotency. This change in developmental potential is associated with genome-wide changes in chromatin, which enable recapitulation of the normal patterns of gene expression that emerge during development. Our methods for purifying and characterizing intact chromatin from genes that regulate self-renewal provide the first opportunity to identify all of the proteins that control these fate determinants. We will focus our study on a gene, Nanog, which is specifically required for ES cell self-renewal and on a second gene, MIR145, which is required for differentiation. In ES cells, Oct4 is an activator of Nanog but a repressor of MIR145. MIR145 encodes the microRNAs, miR143 and miR145; they suppress accumulation of the Oct4, Sox2, and Klf4 proteins. Our goal is to understand how these genes are regulated and, therefore, we must examine them in both their active and inactive states. Accordingly, we will identify all of the proteins that bind these genes in self-renewing hES cells and we will describe the changes in bound proteins that occur when the cells differentiate into extra-embryonic endoderm (XEN). The most promising of these changes will be tested genetically to determine whether they play a causal role in either the maintenance or the repression of stem cell self-renewal. These experiments will provide a deeper understanding of how stem cells maintain and then exit their self-renewing state. The work will also provide a new general method for studying the regulation of gene expression in any stem cell or differentiated cell of interest, accelerating the rate of discovery broadly across the entire field of regenerative medicine.
Statement of Benefit to California:
The benefits of this research will include (1) an acceleration in our understanding of the key determinants of cell fate, (2) new technology to understand gene regulation in stem cells at its most mechanistic level, and (3) the potential to start new companies that exploit this new technology and bring its benefits directly to the public.