Endodermal maternal transcription factors establish super enhancers during zygotic genome activation

We examined the combinatorial function of the Xenopus tropicalis maternal transcription factors Foxh1, Vegt and Otx1. The study involved combinatorial ectopic expression of vegt and otx1 RNA in the putative ectoderm; and combinatorial knock-down of vegt and otx1 in the putative endoderm both followed by transcriptomic analysis (RNA-seq). We performed chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq) to assess the chromatin association of Vegt and Otx1 prior to zygotic gene activation. In addition, we performed ChIP-seq on H3K4me1 on endoderm tissue to identify active regulatory regions in an unbiased manner. Overall design: Screen for animally and vegetally expressed transcription factors: In biological triplicates, the animal and vegetal blastomeres of 8-cell stage embryos were separated. Subsequently, RNA was harvested from both set of blastomeres and whole embryos at the same stage and prepared for RNA sequencing. Combinatorial ectopic expression experiment: In biological duplicates, titrating levels of vegt and otx1 RNA were combinatorially microinjected into 1-cell stage embryos. Animal caps (putative ectoderm) were microdissected at stage 9 (6 hpf) from uninjected and injected samples, and RNA was harvested at stage 10.5 (7 hpf) for RNA sequencing. Double morpholino experiment: In biological duplicates, Vegt and Otx1 morpholinos were injected in 1-cell stage embryos independently or together. Vegetal masses (putative endoderm) were microdissected at stage 9 (6 hpf) and RNA was harvested from uninjected and injected samples at stage 10.5 (7 hpf) for RNA sequencing. Vegt and Otx1 ChIP-seq analysis: Developmentally synchronized embryos were harvested at stage 8 (prior to zygotic gene activation) and fixed with formaldehyde in biological duplicates. Chromatin immunoprecipitation as performed using anti-Vegt or anti-Otx1 antibody and DNA was purified for sequencing. H3K4me1 ChIP-seq analysis: Developmentally synchronized embryos were harvested at stage 10.5 and fixed with formaldehyde in biological duplicates. The vegetal tissue was disssected from the fixed embryos, and ChIP-seq was performed using anti-H3K4me1 antibody. Previously published Foxh1 ChIP-seq from GSE85273 was re-analyzed along with this study. GSM2263590 and GSM2263598 used to generate: GSE118024_foxh1_IDR_optimal_peaks.bed.gz GSM2263598 used to generate: GSE118024_otx1_IDR_optimal_peaks.bed.gz, GSE118024_vegt_IDR_optimal_peaks.bed.gz