Notch signaling pathway maintains stem cells through transcriptional activation of HES/HEY family members to repress tissue-specific transcription factors. Here, comparative integromic analyses on HES/HEY family members were carried out. HES3 gene encodes two isoforms due to alternative promoters. Complete coding sequence of HES3 variant 2 was determined by curating CX755241.1 EST. Refined phylogenetic analysis using HES3 variant 2 instead of variant 1 revealed that mammalian bHLH transcription factors with Orange domain were grouped into HES subfamily (HES1, HES2, HES3, HES4, HES5, HES6, HES7) and HEY subfamily (HEY1, HEY2, HEYL, HESL/HELT, DEC1/BHLHB2, DEC2/BHLHB3). Eight amino-acid residues were added to the C-terminal WRPW motif in human HES3 due to lineage specific T to G nucleotide change at stop codon of chimpanzee, rat, and mouse HES3 orthologs. HES1 and HES3 were expressed in undifferentiated embryonic stem (ES) cells. HES1 was also expressed in fetal tissues, and regenerating liver. HES1, HEY1 and HEY2 were expressed in endothelial cells. HES1, HES4 and HES6 were expressed in gastric cancer, HES1 and DEC1 in pancreatic cancer, HES1, HES2, HES4, HES6 and DEC2 in colorectal cancer. HES6 was also expressed in other tumors, such as brain tumors, melanoma, small cell lung cancer, retinoblastoma, ovarian cancer, and breast cancer. Double NANOG-binding sites, CSL/RBPSUH-binding site and TATA-box in HES1 promoter, NANOG-, SOX2-, POU5F1/OCT3/OCT4-binding sites and TATA-box in HES3 promoter, double CSL-binding sites in HES5 promoter, SOX2-, POU-binding sites and TATA-box in HES6 promoter, and CSL-binding site in HEY1, HEY2 and HEYL promoters were evolutionarily conserved. However, double CSL-binding sites in mouse Hes7 promoter were not conserved in human HES7 promoter. Together these facts indicate that HES1 and HES3 were target genes of the ES cell-specific network of transcription factors, and that HES1, HES5, HEY1, HEY2 and HEYL were target genes of Notch signaling pathway.

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