Identification of FOXL3-binding motif by iDamIDseq-iDEAR

FOXL3 is a member of Forkhead family proteins, which have a highly conserved Forkhead/winged-helix-domain. We discovered the FOXL3-binding motif by a iDamIDseq-iDEAR method (J. A. Gutierrez-Triana, et al., 2016). mRNA of DNA adenine methyltransferase fused with foxl3 (Dam-f-foxl3) was injected into one-cell-stage medaka embryos. Dam-f-foxl3 allows a restricted adenime-methylation of genomic GATC near the FOXL3-binding sites. As a negative control, we injected Dam fused with GFP (Dam-f-GFP). Genomic DNA at 2-day-post-fertilization was used for NGS-library preparation. GATC sites with adenine-methylation were specifically digested with DpnI restriction enzyme, and the DpnI-digested fragments were used for ligation- and PCR-based enrichement. NGS reads were mapped on medaka genome and the resultant contigs of DpnI-derived fragments) were used for iDEAR, an analysis pipeline for establishing profiles of transcription factor-binding sites. Dam-f-FOXL3-enriched contigs were further used as the input of de novo sequence discovery tool, DREME.