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 among the most common human structural birth defects, affecting 1 in 800 newborns, and 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. Our 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. In this project, integrated genome-wide methylation and bulk and single-cell transcriptome approaches will be applied to define molecular and cellular mechanisms of OFC pathogenesis resulting from disrupted DNA methylation in the cranial neural crest. The role of DNA methylation in multifactorial OFC susceptibility will then be defined by integrating multiple environmental and dietary modulators of DNA methylation to genetic (Wnt9b KO) and chemical (Shh antagonist) mouse models of incompletely penetrant OFCs. Finally, epigenome editing will be applied to evaluate the functional impact of OFC-associated methylation changes on gene expression and cranial neural crest biology. Completion of the proposed studies will bring fundamental insight into how DNA methylation regulates cranial neural crest biology and orofacial morphogenesis. By defining environmental- and dietary-mediated methylome- transcriptome responses that alter OFC susceptibility, these studies will also provide a necessary foundation for identification of environmental influences that modulate DNA methylation and contribute to OFC risk. Pursuing 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.