Understanding the role of malleable epigenetic mechanisms in birth defects is a direct path to prevention strategies. Orofacial clefts (OFCs) of the lip and palate are common human structural birth defects, affecting 1 in 800 newborns, that pose serious individual, familial, and societal burdens. Prevention strategies for OFCs are elusive because our current understanding of causative factors is inadequate. Epidemiologic and traditional genetic studies have shown that OFCs are etiologically complex outcomes that result from multifactorial genetic and environmental influences. Epigenetic mechanisms are an exciting new focus in understanding the genesis of OFCs because they mediate the effect of environmental influences on the genome during sensitive embryonic periods. This proposal specifically focuses on DNA methylation because this epigenetic mechanism is environmentally sensitive and a practical target of prevention and therapeutic strategies. While implicated by multiple lines of evidence, the biological role of DNA methylation in orofacial development is unclear. We have established novel models and generated key proofs of concepts that poise us to uncover how DNA methylation regulates orofacial morphogenesis and to define the role that DNA methylation plays in modulating OFC susceptibility. We will apply integrated genome-wide methylation and bulk and single-cell transcriptome approaches to a novel mouse model in which OFCs result from disruption of DNA methylation in the cranial neural crest. We will also define the role of DNA methylation in multifactorial OFC susceptibility by integrating multiple environmental and dietary modulators of DNA methylation to genetic (Wnt9b KO) and chemical (Shh antagonist) mouse models of incompletely penetrant OFCs. Pursuit of the proposed studies will bring fundamental insight into how DNA methylation regulates cranial neural crest biology and orofacial morphogenesis and provide a critical foundation for future work interrogating the role of specific methylation events. Completion of these studies will also define environmental- and dietary-mediated methylome-transcriptome responses that alter OFC susceptibility and set the stage for definition of additional environmental influences that modulate DNA methylation and contribute to OFC risk. Pursing this line of investigation will advance our long-term goal of developing prevention strategies for etiologically complex birth defects by identifying culpable environmental influences and defining their mechanisms of action.