Foxi2 and Sox3 are master regulators controlling ectoderm germ layer specification in Xenopus [ChIP-Seq]

In vertebrates, germ layer specification represents a critical transition where pluripotent cells acquire lineage-specific identities. We identify the maternal transcription factors Foxi2 and Sox3 to be pivotal master regulators of ectodermal germ layer specification in Xenopus. Ectopic coexpression of Foxi2 and Sox3 in prospective endodermal tissue induces the expression of ectodermal markers while suppressing mesendodermal markers. Transcriptomics analyses reveal that Foxi2 and Sox3 jointly and independently regulate hundreds of ectodermal target genes. During early cleavage stages, Foxi2 and Sox3 pre-bind to key cis-regulatory modules (CRMs), marking sites that later recruit Ep300 and facilitate H3K27ac deposition, thereby shaping the epigenetic landscape of the ectodermal genome. These CRMs are highly enriched within ectoderm-specific super-enhancers (SEs). Our findings highlight the pivotal role of ectodermal SE-associated CRMs in precise and robust ectodermal gene activation, establishing Foxi2 and Sox3 as central architects of ectodermal lineage specification. Overall design: Foxi2 ChIP-seq: Developmentally synchronized embryos were harvested at stage 8, 9 and 10.5, then fixed with formaldehyde in biological duplicates. Chromatin immunoprecipitation was performed using anti-Foxi2 or anti-Sox3 antibodies and DNA was purified for sequencing. P300, H3K27ac, H3K27me3 ChIP-seq analysis: Developmentally synchronized embryos were harvested at stage 10.5 and fixed with formaldehyde in biological duplicates. The presumptive ectoderm (animal) and endoderm (vegetal) tissues were dissected from the fixed embryos, and ChIP-seq was performed using anti-P300, anti-H3K27ac, and anti-H3K27me3 antibodies. Previously published wild-type blastula and gastrula whole-embryo ChIP-seq of H3K27ac, H3K4me1 were ren-analyzed from GSE56000 and H3K27me3 from GSE67974.