GABPα and CREB1 binding to double nucleotide polymorphisms of their consensus motifs and cooperative binding to the ETS-CRE motif (ACCGGAAGTGACGTCA).

Previously, cooperative binding to DNA between the bZIP domain of CREB1 and the ETS domain of GABPα was observed for the composite ETS-CRE motif (A0C1C2G3G4A5A6G7T8G9A10C11G12T13C14A15). Single nucleotide polymorphisms (SNPs) at the beginning and end of the ETS motif (ACCGGAAGT) increased cooperative binding. Here, we use a microarray containing all double nucleotide polymorphisms (DNPs) of the ETS-CRE motif to explore GABPα and CREB1 binding to their canonical motifs and their cooperative binding to the ETS-CRE motif. For GABPα, binding to SNPs predicted binding the DNPs. In contrast, CREB1 binding to DNPs showed cooperativity. Similar results were observed for other ETS and bZIP family members. Cooperative binding between GABPα and CREB1 was typically weaker than expected except for DNPs containing A7 and SNPs at the beginning of the ETS motif. Protein binding microarray (PBM) experiments were performed for the DNA binding domain of GABPa and CREB1. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to double-stranded custom Agilent microarrays (He et al., G3 2015) in order to determine their cooperative binding preferences to composite DNA motifs.