Genomic and Developmental Mechanisms of SIX2-related Craniofacial Syndrome

In this project, we study genomic disruptions associated with frontonasal dysplasia (FND), a congenital craniofacial disorder that profoundly affects the structure and function of the orofacial complex, as a model for understanding genome organization and molecular mechanisms underlying craniofacial development and malformations. Several independent studies have associated FND with partly overlapping heterozygous microdeletions at Chromosome 2p21 in which SIX2 is the only protein-coding gene. SIX2 is a member of the SIX- and homeo-domain containing DNA-binding transcription factors. In all vertebrate genomes, Six2 is physically linked to Six3 in a tail-to-tail configuration, with these two genes organized into separate topologically associating domains (TADs) flanking a conserved TAD boundary. Whereas Six2+/- mice are phenotypically normal and Six2-/- mice exhibit kidney hypoplasia with normal frontonasal structures, our preliminary study found that deleting Six2 together with part of the Six2/Six3 intergenic region, but not including the Six3 gene or Six2 distal enhancers, caused midline facial clefting in heterozygous mice. On the other hand, mice carrying a heterozygous deletion including Six2, Six3, and their intergenic region in between, could survive with no frontonasal defects. We hypothesize that SIX2-related FND is caused by gain of SIX3 expression in developing frontonasal mesenchyme due to TAD boundary disruption and enhancer adoption rather than by SIX2 haploinsufficiency as previously believed. This project will test this novel hypothesis and unravel the genomic and molecular developmental mechanisms underlying SIX2-related FND. Data from these studies will provide novel insights into mechanisms of craniofacial development, and lead to improvements in molecular diagnosis, medical assessment and interpretation of clinical genomics data, and treatment of developmental disorders associated with genomic structural variations.