PROJECT SUMMARY/ABSTRACT Stem cells have the unique property to self-renew or differentiate into any of the 200+ different cell types of the body. Understanding the mechanisms that contribute to stem cell identity and specific cellular differentiation is essential to direct them to specialized cell states which can then be utilized for targeted therapy for countless diseases. There has been increased interest in understanding the role of chromatin regulatory factors in stem cell identity due to their essential roles in cellular processes and during development. During development, lineage specification is accompanied by genome-wide chromatin reorganization and gene expression pattern changes. Therefore, it is not surprising that epigenetic factors represent one of the largest protein classes that are mutated in disease states, including developmental disorders. Our research interests focus on the similarities and differences in chromatin structure among different cell types and how chromatin regulatory factors that modulate these differences regulate cell fate. The long-term goals of our laboratory are to comprehensively understand the functions, targets, regulation, and mechanisms of action of non-coding RNAs (ncRNAs) and chromatin regulatory factors with critical functions in gene regulatory networks. Here, we propose three related but distinct research directives. We will determine the mechanism through which FACT, an essential histone chaperone, acts to maintain embryonic stem (ES) cell identity. We propose FACT is regulating cis regulatory elements (CREs) by modulating chromatin structure and transcription factor (TF) binding to regulate transcription of genes required to maintain stem cell identity. In parallel, we will capitalize on our system biology approach of understanding how nucleosome remodeling complexes contribute to cell identity through ncRNA regulation. To that end, we will examine the functional interactions among understudied nucleosome remodeling complexes, including how some remodelers may compensate for loss of another or work redundantly. Finally, we will uncover the molecular contribution of an essential epigenetic factor, BAF, in neural differentiation focusing, in part, on the complexes role in regulating non-coding transcription. Over the next five years, our laboratory will define the mechanism of FACT function in ES cells, determine the interplay between a subset of nucleosome remodelers in gene regulatory networks, and understand the contribution of one remodeler in regulating differentiation. The proposed research is significant because it will uncover fundamental mechanisms that choreograph the interplay of chromatin dynamics with ncRNA function, providing a crucial step in understanding cell fate decisions.