The expression profile of a gene is controlled by DNA sequences called cis-regulatory modules (CRMs). CRMs can function over large genomic distances and can be located many kilobases away from their target promoters. hes1 is a key developmental gene that is overexpressed in certain cancers and is a primary target of NOTCH signaling. Despite this, analysis of hes1 transcriptional control has been limited solely to its promoter. Here, we identify seven conserved DNA sequence blocks, representing the hes1 promoter and six novel CRMs, within 57 kb upstream of the mouse hes1 gene. We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions. We validate RBP-Jκ and TEAD family occupancy in cells in culture and test the response of each of these CRMs to active NOTCH. We show that two regions, CRM5 and CRM7, function as enhancers, and four can repress transcription. A pair of RBP-Jκ motifs arranged in a tail-tail configuration in CRM5 and the M-CAT motif in CRM7 are necessary for enhancer function. Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus. This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.
J Biol Chem
5687 - 5697
Animals, Basic Helix-Loop-Helix Transcription Factors, Cell Line, Computational Biology, Conserved Sequence, Enhancer Elements, Genetic, Gene Expression Regulation, Homeodomain Proteins, Immunoglobulin J Recombination Signal Sequence-Binding Protein, Mice, Myoblasts, Nucleotide Motifs, Promoter Regions, Genetic, Receptors, Notch, Regulatory Sequences, Nucleic Acid, Signal Transduction, Transcription Factor HES-1, Transcription Initiation Site