α-Enolase Is the c-myc Promoter-Binding Protein.

Quadruplex-forming sequences occur disproportionately in the promoters of growth regulatory genes, including c-myc. These sequences appear to play important roles in the regulation of gene expression. The c-myc quadruplex-forming sequence occurs in the c-myc P1 promoter region which is responsible for more than 80% of c-myc expression. We have used proteomic techniques used to identify quadruplex-binding proteins. Two dimensional electrophoretic gels of nuclear extracts were blotted and screened with G-rich (quadruplex forming) and C-rich (control) oligonucleotides. We were surprised to observe that α-enolase binds the G-rich, but not C-rich oligonucleotide. Using electrophoretic mobility shift assays (EMSA), we confirmed that both α-enolase and its alternative translation product, MBP-1, bind quadruplex-forming oligonucleotides. We then tested the hypothesis that both α-enolase and MBP-1 may play important physiologic roles by binding the c-myc quadruplex-forming sequence. Electrophoretic mobility shift assays were used to characterize the binding of α-enolase and MBP-1 to the c-myc quadruplex-forming sequence. Binding of AS1411, a quadruplex forming oligonucleotide, was used as a control. Both α-enolase and MBP-1 demonstrated strong binding to the c-myc P2 promoter sequence (containing the quadruplex-forming sequence). A “supershift” was seen when an anti-α-enolase antibody was included in the incubation although control antibodies did not alter the EMSA pattern. The quadruplex oligonucleotide aptamer, AS1411, competed for α-enolase and MBP-1 binding to the c-myc promoter sequence. However, a nonquadruplex-forming oligonucleotide did not compete. A mutation in the c-myc quadruplex sequence abrogated binding of both α-enolase and MBP-1. In order to test the biological effect of the α-enolase/MBP-1 interaction with the c-myc promoter, we characterized the effect of constitutive overexpression of each α-enolase product, individually. Although, overexpression of MBP-1 by transfected cells resulted in down regulation of c-myc expression and induction of apoptosis, constitutive overexpression of α-enolase did not alter c-myc expression or the rate of cellular proliferation. Cells grown in low glucose medium demonstrate increased MBP-1 levels which results in decreased c-myc expression and inhibition of cellular proliferation. A similar response was seen in hypoxic cells, indicating that enolase/MBP-1 plays an important role in cellular responses to external stress. These results suggest that MBP-1 down regulates c-myc expression by simultaneous binding to the P1 and P2 promoter sequences, while α-enolase binds only the P2 sequence. This suggests a very important regulatory role for α-enolase/MBP-1 in the relationship between cellular metabolism and growth regulation. Enolase/MBP-1 quadruplex-binding likely plays an important role in regulation of c-myc expression.