Functional characterization of craniofacial enhancers at single cell and single base pair resolution

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Abstract Craniofacial abnormalities are some of the most commonly occurring human birth defects worldwide, with up to 200,000 children born every year with some type of craniofacial defect. These defects can occur as part of complex syndromes that involve multiple tissues and organs. The syndromic forms of these disorders have been successfully linked to nearly 500 genes including TWIST1 for craniosynostosis and IRF6 for orofacial clefting. However more frequently no other part of the body is directly involved (50% of craniosynostoses, 70% of orofacial clefts). Genome wide association studies indicate heritability for such defects, however the vast majority of associations fall outside of genes suggesting defective gene regulation is a major contributor to incidence of such defects. Gene regulatory elements can be located throughout the genome and typically have tissue-specific activity, making them difficult to identify and predict what gene they control. The overall objective of this application is to identify the cell types present in the developing human face and functionally characterize important regulatory elements that specify them as single base pair resolution. In Aim 1 we propose to systematically identify populations of cell types using single-cell based methods for measuring the transcriptome and active regulatory sites across the genome in human craniofacial tissue from 4 to 5 weeks of gestation and mouse from embryonic days 10.5 and 11.5. In Aim 2 we propose to identify physical interactions between regulatory sequences and target genes in these same tissue types. Finally, in Aim 3 we will identify regulatory elements from these tissues that can be tested in a cell culture model of cranial neural crest cells. These enhancers will be assessed for effects on gene expression when repressed or removed from the genome. Those with significant effects on gene expression will be tested for every variant to identify important locations within them. Our proposed studies will generate the most comprehensive view of the cell types active in the developing human face and reveal the contributions individual noncoding variants make on gene expression.