Overlapping ETS and CRE Motifs (G/CCGGAAGTGACGTCA) Preferentially Bound by GABPα and CREB Proteins

Previously, we identified 8-bps long DNA sequences (8-mers) that localize in human proximal promoters and grouped them into known transcription factor binding sites (TFBS). We now examine split 8-mers consisting of two 4-mers separated by 1-bp to 30-bps (X-4-N1-30-X-4) to identify pairs of TFBS that localize in proximal promoters at a precise distance. These include two overlapping TFBS: the ETS double left right arrow ETS motif ((C)/(G)CCGGAAGCGGAA) and the ETS double left right arrow CRE motif ((C)/(G)CGGAAGTGACGTCAC). The nucleotides in bold are part of both TFBS. Molecular modeling shows that the ETS double left right arrow CRE motif can be bound simultaneously by both the ETS and the B-ZIP domains without protein-protein clashes. The electrophoretic mobility shift assay (EMSA) shows that the ETS protein GABP alpha and the B-ZIP protein CREB preferentially bind to the ETS double left right arrow CRE motif only when the two TFBS overlap precisely. In contrast, the ETS domain of ETV5 and CREB interfere with each other for binding the ETS double left right arrow CRE. The 11-mer (CGGAAGTGACG), the conserved part of the ETS double left right arrow CRE motif, occurs 226 times in the human genome and 83% are in known regulatory regions. In vivo GABP alpha and CREB ChIP-seq peaks identified the ETS double left right arrow CRE as the most enriched motif occurring in promoters of genes involved in mRNA processing, cellular catabolic processes, and stress response, suggesting that a specific class of genes is regulated by this composite motif.