Abstract Embryonic stem cells (ESCs) exhibit two unique features that make them potential tools for the development of therapies for degenerative diseases. First, ESCs have the capacity to differentiate into any cell type, a property termed pluripotency. Second, ESCs have the ability to proliferate indefinitely in culture in an undifferentiated state without accumulating genetic or epigenetic alterations, a process called self- renewal. The decision of ESCs to self-renew or differentiate is ultimately controlled by several transcription factors called ESC master regulators, along with regulators of chromatin structure. In recent years, the gene targets and functions of the ESC master regulators have become better understood. In contrast, the targets and gene regulatory functions of most chromatin regulators required for ESC self-renewal or pluripotency are unknown. This project aims to understand the functions and mechanisms of action of three chromatin regulatory complexes with crucial functions in ESC self-renewal and pluripotency. The Tip60- p400 complex has lysine acetyltransferase (KAT) and nucleosome remodeling activities, and functions to silence differentiation-induced genes in self-renewing ESCs. This finding was unexpected, given the fact that most KATs function primarily in activation of transcription, and the documented roles of Tip60-p400 complex in gene-activation in somatic cells. We will examine the mechanism by which the Tip60-p400 complex silences differentiation genes in murine ESCs, and determine how this activity is regulated during differentiation. In addition, we recently found that two additional chromatin regulatory complexes, NURD and BAF, oppositely regulate an overlapping set of target genes, which are expressed at moderate levels as a result of this opposition. We will examine the mechanisms underlying this opposition, its importance in the maintenance of the pluripotent state, and its function in ESC differentiation. PUBLIC HEALTH RELEVANCE: Project Narrative While ESCs have tremendous potential for the development of new therapies for degenerative diseases, it is currently impossible to robustly differentiate ESCs into many cell types in quantities sufficient for therapies. Therefore, an increased understanding of the factors regulating ESC self-renewal and differentiation should lead to more efficient protocols for their therapeutic differentiation, and should enhance the safety of such protocols. We are examining the functions of such regulatory factors using murine ESCs as a model system, due to their amenability to genetic manipulation. This study should enhance our understanding of the gene regulatory network controlling ESC self-renewal and differentiation.