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

The tongue is a specialized muscular organ that performs multiple essential functions including mastication, deglutition, oral sensation, oral cleansing, airway maintenance and vocalization. In this study, we show Foxf1/Foxf2 serves as key mediators of hedgehog signaling in regulating myoblast migration, differentiation, and intrinsic tongue muscle organization. We took advantage of the Foxf2FLAG mice which carries 3xFLAG epitope-tagged endogenous Foxf2 protein and characterized genome-wide Foxf2 binding sites in the developing tongues using chromatin immunoprecipitation and genome sequencing (ChIP-seq). Further analyses demonstrate that Foxf1/2 transcription factors directly control the expression of Hgf, Tgfb2, and Tgfb3, to regulate tongue myogenesis. Overall design: We generated mice carrying 3xFLAG epitope-tagged endogenous Foxf2 protein using the CRISPR/Cas9-mediated genome editing technology. 75 tongue tissues were dissected from Foxf2FLAG/FLAG embryos at E12.5. ChIP was performed by using anti-FLAG antibodies (Sigma). Sequencing libraries were generated using ThruPLEX DNA sequencing kit (Takara). Sequencing was performed on an Illumina Nextseq500 (Illumina, San Diego CA). For ChIP-Seq data analysis, raw FASTQ files were aligned to mm10 reference genome using Bowtie2. Peak calling and de novo motif identification were 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 sets.