-Repression of Expression Affects -Mediated Gene Expression in Palate Development.

Disruption of , encoding a member of the Forkhead family transcription factors, has been associated with cleft palate in humans and mice. is located in a conserved gene cluster containing , , and . We found that expression of is dramatically upregulated in the embryonic palatal mesenchyme in mouse embryos. We show here that the promoter-deletion mutation caused dramatically increased expression of the -linked allele but had little effect on the allele in . We analyzed effects of the mutation on the expression of other neighboring genes and compared those effects with the chromatin domain structure and recently identified enhancer-promoter associations as well as H3K27ac ChIP-seq data. We show that the mutation resulted in significantly increased expression of the and genes located in the same topologically associated domain with but not the expression of the and genes located in the adjacent chromatin domain. We inactivated the gene in mice homozygous for a conditional allele using CRISPR genome editing and generated mice with loss-of-function mutations in and in . Whereas the mice exhibited cleft palate at birth similar as in the mice, systematic expression analyses of a large number of Foxf2-dependent genes revealed that the (/ embryos exhibited distinct effects on the domain-specific expression of several important genes, including , , and , in the developing palatal shelves compared with embryos. These results identify a novel -regulatory effect of the mutation and demonstrate that regulation of contributed to alterations in palatal gene expression in embryos. These results have important implications for interpretation of results and mechanisms from studies of promoter- or gene-deletion alleles. In addition, the unique mouse lines generated in this study provide a valuable resource for understanding the cross-regulation and combinatorial functions of the and genes in development and disease.