Comprehensive analysis of CRMs with RNA-seq data revealed that Lim1/Otx2-bound CRMs co-localizing with TLE rather than p300 are strongly associated with region/tissue-specific genes.

In vertebrates, homeodomain proteins Otx2 and Lim1/Lhx1 are required for head formation, but the regulatory principles underlying their functions in the head organizer remain unsolved. Here we show using ChIP-seq analysis that Otx2, Lim1, the coactivator p300 and the corepressor TLE/Groucho colocalize on cis-regulatory modules (CRMs) of thousands of genes including almost all ‘head-organizer’ genes in the Xenopus tropicalis gastrula. Comprehensive analysis of CRMs with RNA-seq data revealed that Lim1/Otx2-bound CRMs co-localizing with TLE rather than p300 are strongly associated with region/tissue-specific genes. Together with reporter analyses, our data suggest that Otx2 activates head-organizer genes with Lim1 and represses non-head-organizer genes with transcriptional repressors such as Goosecoid. Thus, it is likely that each of thousands of genes interprets Otx2 as a ‘positional tag’ to determine its expression in the head organizer, corroborating the idea that positional information directly contributes “massively parallel (distributive) gene regulation” rather than “hierarchal top-down gene control.” This general principle of gene regulation by region/tissue specific transcription factors may underlie the extraordinary diversities of morphologies in animals.