G-quadruplex-forming GGA repeat region functions as a negative regulator of the Ccnb1ip1 enhancer

An enhancer located upstream of the transcriptional start site of Ccnb1ip1 containing two GGA-rich regions and a 14-GGA repeat (GGA)₁₄ region has been previously identified. Three copies of four GGA repeats in the c-myb promoter that form a tetrad:heptad:heptad:tetrad (T:H:H:T) dimerized G-quadruplex (G4) structure reportedly functions as both a transcriptional repressor and activator. Here, the secondary structures of the two GGA-rich and (GGA)₁₄ regions were analyzed using circular dichroism spectral analysis, which indicated that the two GGA-rich DNAs formed parallel-type G4 structures, whereas (GGA)₁₄ DNA formed the T:H:H:T dimerized G4 structure. Reporter assays demonstrated that individual regions did not show enhancer activity; however, the deletion of the (GGA)₁₄ region resulted in 1.5-fold higher enhancer activity than that of the whole enhancer. These results indicate that the (GGA)₁₄ region that forms the T:H:H:T dimerized G4 structure functions as a negative regulator of the Ccnb1ip1 enhancer.