Abstract
Disruption of FOXF2, encoding a member of the Forkhead family transcription factors, has been associated with cleft palate in humans and mice. FOXF2 is located in a conserved gene cluster containing FOXQ1, FOXF2, and FOXC1. We found that expression of Foxq1 is dramatically upregulated in the embryonic palatal mesenchyme in Foxf2–/– mouse embryos. We show here that the Foxf2 promoter-deletion mutation caused dramatically increased expression of the cis-linked Foxq1 allele but had little effect on the Foxq1 allele in trans. We analyzed effects of the Foxf2 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 Foxf2 mutation resulted in significantly increased expression of the Foxq1 and Exoc2 genes located in the same topologically associated domain with Foxf2 but not the expression of the Foxc1 and Gmds genes located in the adjacent chromatin domain. We inactivated the Foxq1 gene in mice homozygous for a Foxf2 conditional allele using CRISPR genome editing and generated (Foxf2/Foxq1)+/– mice with loss-of-function mutations in Foxf2 and Foxq1 in cis. Whereas the (Foxf2/Foxq1)–/– mice exhibited cleft palate at birth similar as in the Foxf2–/– mice, systematic expression analyses of a large number of Foxf2-dependent genes revealed that the (Foxf2/Foxq1)–/– embryos exhibited distinct effects on the domain-specific expression of several important genes, including Foxf1, Shox2, and Spon1, in the developing palatal shelves compared with Foxf2–/– embryos. These results identify a novel cis-regulatory effect of the Foxf2 mutation and demonstrate that cis-regulation of Foxq1 contributed to alterations in palatal gene expression in Foxf2–/– 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 Foxf2 and Foxq1 genes in development and disease.
Highlights
The secondary palate separates the nasal cavity from the oral cavity and consists of the bony hard palate anteriorly and muscular soft palate posteriorly (Bush and Jiang, 2012)
In between Foxq1 and Foxf2, there is an uncharacterized long non-coding RNA gene, named 1700018A04Rik, which is transcribed from the opposite DNA strand with the most 5 transcription start site (TSS) located only about 300 bp from the TSS of Foxf2 (Figures 1A,B)
Our RNA sequencing (RNA-seq) and in vivo expression assays showed that expression of Foxq1 was the most dramatically upregulated gene in the embryonic palatal mesenchyme in Foxf2−/− mouse embryos
Summary
The secondary palate separates the nasal cavity from the oral cavity and consists of the bony hard palate anteriorly and muscular soft palate posteriorly (Bush and Jiang, 2012). The palatal shelves reorient to the horizontal position above the tongue, grow toward and fuse with each other at the midline to form the roof of the oral cavity. The Shh and Fgf signaling pathways have been shown to play a key role in regulating the palatal epithelial-mesenchymal interactions governing palatal shelf growth and patterning (Rice et al, 2004; Han et al, 2009; Lan and Jiang, 2009). Shh produced by the palatal epithelial cells signals to the palatal mesenchyme and forms a positive feedback loop with Fgf to coordinate cell proliferation in both the epithelium and mesenchyme during palate development (Rice et al, 2004; Lan and Jiang, 2009). We recently showed that a Shh-Foxf1/Foxf2-Fgf18-Shh molecular circuit regulates the proliferation of palatal mesenchymal cells during palatal shelf growth (Xu et al, 2016)
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