Genome-wide identification of Foxf2 transcriptional target genes in palate development

Cleft palate is among the most common structural birth defects in humans. Previous studies have shown that mutations in FOXF2 are associated with cleft palate in humans and mice and that Foxf2 acts in a Shh-Foxf-Fgf18-Shh molecular network controlling palatal shelf growth. In this study, we generated mice carrying 3xFLAG epitope-tagged endogenous Foxf2 protein using the CRISPR/Cas9-mediated genome editing technology and characterized genome-wide Foxf2 binding sites in the developing palatal shelves using chromatin immunoprecipitation and genome sequencing (ChIP-seq). By combined analysis of ChIP-seq and RNA-seq datasets we identified a large list of Foxf2 target genes. Further analyses demonstrate that Foxf2 directly regulate expression of several genes encoding ECM or ECM modifiers during palate development. Moreover, our ChIP-seq and RNA-seq datasets provide an excellent resource for comprehensive understanding of the molecular network controlling palate development. We generated mice carrying 3xFLAG epitope-tagged endogenous Foxf2 protein using the CRISPR/Cas9-mediated genome editing technology. 50 pairs of palatal shelves were dissected from Foxf2FLAG/FLAG embryos at E13.5. ChIP was performed by using anti-FLAG antibodies (Sigma). Sequencing libraries were generated using Rubicon ThruPLEX DNA sequencing kit (Rubicon Genomics, Ann Arbor MI). Sequencing was performed on an Illumina HiSeq2500 (Illumina, San Diego CA). Raw FASTQ files were aligned to mm9 reference genome. Peak calling was performed using the HOMER (Hypergeometric Optimization of Motif EnRichment) software. The input samples were used as the control for calling peaks from the Foxf2-FLAG ChIP-seq data set.