ABSTRACT The neural crest is a migratory stem cell population that is essential for the development of various tissues and organs during embryogenesis. Disruptions in neural crest development contribute to the pathogenesis of craniofacial defects and other congenital malformations, imposing a significant burden on individuals and society. Understanding the molecular mechanisms underlying neural crest formation is crucial for proper diagnosis of these conditions. While previous research has focused on protein-DNA interactions in activating neural crest genes, the role of chromatin conformation changes in this process remains poorly understood. Previous studies characterizing the architecture of the neural crest genome revealed that these cells display a complex enhancer-promoter interactome characterized by a reliance on long-range interactions. This proposal aims to investigate the contribution of architectural proteins, specifically CTCF and YY1, in establishing this unique chromatin organization. We hypothesize that architectural proteins interact with pioneer transcription factors to assemble the neural crest interactome and promote activation of gene regulatory circuits. We will test this hypothesis in three specific aims. Aim 1 will define the function of architectural proteins in neural crest formation by identifying the chromatin loops mediated by CTCF and YY1 through chromatin conformation capture. We will also target architectural proteins in loss-of-function studies to uncover their relative contributions to the establishment of the neural crest enhancer interactome. Aim 2 seeks to determine how tissue-specific enhancer-promoter loops are established during neural crest specification. To accomplish this, we will investigate the functional and physical interactions between architectural proteins and the pioneer transcription factors that define the neural crest lineage. Aim 3 will define how disease-linked mutations in architectural proteins affect neural crest chromatin organization. We will employ genome engineering to reproduce CTCF/YY1 mutations in embryonic stem cells and drive these cells into adopting a cranial neural crest fate. By elucidating the function of architectural proteins in neural crest development and their role in the genesis of craniofacial malformations, this research has the potential to pave the way for future therapeutic strategies aimed at preventing or treating these debilitating conditions.