GABPα and CREB1 Binding to Double Nucleotide Polymorphisms of Their Consensus Motifs and Cooperative Binding to the Composite ETS ⇔ CRE Motif (ACCGGAAGTGACGTCA)

[Image: see text] Previously, cooperative binding of the bZIP domain of CREB1 and the ETS domain of GABPα was observed for the composite DNA ETS ⇔ CRE motif (A(0)C(1)C(2)G(3)G(4)A(5)A(6)G(7)T(8)G(9)A(10)C(11)G(12)T(13)C(14)A(15)). Single nucleotide polymorphisms (SNPs) at the beginning and end of the ETS motif (ACCGGAAGT) increased cooperative binding. Here, we use an Agilent microarray of 60-mers containing all double nucleotide polymorphisms (DNPs) of the ETS ⇔ CRE motif to explore GABPα and CREB1 binding to their individual motifs and their cooperative binding. For GABPα, all DNPs were bound as if each SNP acted independently. In contrast, CREB1 binding to some DNPs was stronger or weaker than expected, depending on the locations of each SNP. CREB1 binding to DNPs where both SNPs were in the same half site, T(8)G(9)A(10) or T(13)C(14)A(15), was greater than expected, indicating that an additional SNP cannot destroy binding as much as expected, suggesting that an individual SNP is enough to abolish sequence-specific DNA binding of a single bZIP monomer. If a DNP contains SNPs in each half site, binding is weaker than expected. Similar results were observed for additional ETS and bZIP family members. Cooperative binding between GABPα and CREB1 to the ETS ⇔ CRE motif was weaker than expected except for DNPs containing A(7) and SNPs at the beginning of the ETS motif.