Orofacial clefting, including cleft lip and cleft palate, is the second most common birth defect in the world, present in about 1 in 700 newborns. According to data from the Center for Disease Control (CDC), every year in the United States, about 2650 babies are born with cleft palate, and 4440 babies are born with cleft lip with or without a cleft palate. The causes of orofacial clefting are mostly unknown but are thought to be a combination of genes and other environmental factors. One such gene that has been linked to this disease is transcription factor p63.
The transcription factor p63 is a protein almost exclusively found in epithelial cells that plays a key role in the establishment and differentiation of epithelial tissues during development. Mice lacking this protein do not develop proper epidermal layers, lack hair follicles and eyelids, show craniofacial and limb malformations, and ultimately die at birth because their skin is permeable.
Point mutations in p63 in human patients similarly lead to developmental defects, including orofacial clefting. Despite the overwhelming evidence showing p63’s importance in development, knowledge is limited about how p63 engages with chromatin during development and how disease-specific point-mutations act at the molecular level. A key reason for this is the lack of appropriate models to study the de novo expression of p63 and its role at developmentally regulated genes in humans.
To overcome this limitation, we developed an inducible trans-differentiation model in which we convert human fibroblasts into keratinocyte-like cells, a type of epithelial cells. We show that co-expression of p63 and a reprogramming factor KLF4 for 72 hours is enough to turn on a transcriptional and chromatin profile that is similar to that observed in human basal keratinocytes. Specifically, we observed that p63 establishes enhancers at critical craniofacial genes, such as IRF6 and GRHL3, genes that when mutated have been shown to lead to clefting of the lip or the palate.
Enhancers are regions of non-coding DNA that regulate transcription of primarily neighboring genes in a cell type-specific manner; they are thought to loop on to the promoter region of genes — places where genes start — to enhance expression of a specific gene. Enhancers are further characterized by being regions where chromatin is more accessible and flanking histones are marked by acetylation, a covalent modification that also leads to more openness of chromatin. Importantly, enhancers are active, open, and marked differently depending on the cell type, playing a key role in maintaining cell identity. For this reason, there is great interest in understanding how these elements are established and maintained and how they might be involved in the disease.
In this study, we firstly showed that p63 plays a crucial role in establishing epithelial specific enhancers at critical craniofacial genes. We also demonstrated that mutations in p63 found in human patients with limb and craniofacial malformations lead to aberrant enhancer establishment, which might explain the severity of phenotypes observed in these patients.
In the final part of our project, we partnered with the Ludwig laboratory at the University of Bonn. This lab has analyzed genome-wide association studies (GWAS) of patients with cleft lip and cleft palate and identified specific genetic variants — differences in their DNA sequence — compared to healthy controls that seem to be enriched in cleft lip and cleft palate (CL/P) patients, suggesting they might be causal for the disease. Combining the data from our study with their findings, we observed that many of these genetic variants that are enriched in patients with cleft lip and cleft palate colocalize with enhancers newly established by p63. This indicates a novel and exciting molecular link between p63 enhancer function and CL/P, in which genetic variants enriched in CL/P patients disrupt recruitment of p63 and lead to defects in enhancer establishment and dysregulation of gene expression during lip and palate fusion. Further, our system identifies over 100 new candidate genes, upregulated by p63 and enriched for CL/P associated genetic variants.
Taken together, our findings address how p63 binds and remodels chromatin with its first encounter, identifies and prioritizes CL/P candidate genes and the vital regulatory elements that regulate them, and provides new explanations for how p63 mutations and genetic variants associated with CL/P underlie disease.
These findings are described in the article entitled p63 establishes epithelial enhancers at critical craniofacial development genes, recently published in the journal Science Advances.