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The shape of the face is one of the most distinctive features among humans, and differences in facial morphology have substantial implications in areas such as social interaction, psychology, forensics, and clinical genetics. Craniofacial shape is highly heritable, including the normal spectrum of morphological variation as well as susceptibility to major craniofacial birth defects. In this study, we explored the role of transcriptional enhancers in the development of the craniofacial complex. Our study is based on the rationale that such enhancers, which can be hundreds of kilobases away from their target genes, regulate the spatial patterns, levels, and timing of gene expression in normal development.
To identify distant-acting enhancers active during craniofacial development, we used chromatin immunoprecipitation on embryonic mouse face tissue followed by sequencing to identify noncoding genome regions bound by the enhancer-associated p300 protein. We used LacZ reporter assays in transgenic mice and optical projection tomography (OPT) to determine three-dimensional expression patterns of a subset of these candidate enhancers. Last, we deleted three of the craniofacial enhancers from the mouse genome to assess their effect on gene expression and craniofacial morphology during development.
We identified more than 4000 candidate enhancer sequences predicted to be active in the developing craniofacial complex. The majority of these sequences are at least partially conserved between humans and mice, and many are located in chromosomal regions associated with normal facial morphology or craniofacial birth defects. Characterization of more than 200 candidate enhancer sequences in transgenic mice revealed a remarkable spatial complexity of in vivo expression patterns. Targeted deletions of three craniofacial enhancers near genes with known roles in craniofacial development resulted in changes of expression of those genes as well as quantitatively subtle but definable alterations of craniofacial shape.
Our analysis identifies enhancers that fine tune expression of genes during craniofacial development in mice. These results support that variation in the sequence or copy number of craniofacial enhancers may contribute to the spectrum of facial variation we find in human populations. Because many craniofacial enhancers are located in genome regions associated with craniofacial birth defects, such as clefts of the lip and palate, our results also offer a starting point for exploring the contribution of noncoding sequences to these disorders.
No Two Faces Are Alike
Gene disruptions can cause severe dysmorphologies like cleft palate, but what causes the subtle shifts in facial morphology that make each face unique? Studying mice, Attanasio et al. (1241006) identified over 4000 candidate genetic enhancers around genes driving craniofacial development. To avoid the challenge of recognizing individual mouse faces, optical projection tomography was used to link changes in facial morphology with alterations in the function of specific enhancers.
The shape of the human face and skull is largely genetically determined. However, the genomic basis of craniofacial morphology is incompletely understood and hypothesized to involve protein-coding genes, as well as gene regulatory sequences. We used a combination of epigenomic profiling, in vivo characterization of candidate enhancer sequences in transgenic mice, and targeted deletion experiments to examine the role of distant-acting enhancers in craniofacial development. We identified complex regulatory landscapes consisting of enhancers that drive spatially complex developmental expression patterns. Analysis of mouse lines in which individual craniofacial enhancers had been deleted revealed significant alterations of craniofacial shape, demonstrating the functional importance of enhancers in defining face and skull morphology. These results demonstrate that enhancers are involved in craniofacial development and suggest that enhancer sequence variation contributes to the diversity of human facial morphology.
↵* Present address: EMD Millipore, 28820 Single Oak Drive, Temecula, CA 92590, USA.