Figure 2

The cluster of YY1 binding sites as a transcriptional activator. The schematic diagram shows the promoter region of the zebrafish YY1 containing the 1^st exon and 4 perfect-matched YY1 binding sites (arrows). This promoter region was subcloned into the IRES-β-Geo promoterless vector, and subsequently modified to change the binding affinity and orientation of individual YY1 binding sites (A). Open arrows indicate the intact YY1 binding sites, while the gray arrows indicate the YY1 binding sites with lower affinity to the YY1 protein. All the YY1 binding sites in the Low affinity construct were modified from (A/C)G CCATnTT to AA CCATnTT. The orientation of the YY1 binding sites marked by closed arrows is reversed with respect to the endogenous binding sites. The Forward construct has all binding sites in the forward direction; the Reverse construct has all in a reverse direction; and the Both construct contains both pairs in a reverse direction. The binding potential of these sites was also completely abolished through changing from GCC ATnTT to ATT ATnTT (B). The numbers in the name of each construct indicate the position of mutated YY1 binding site. The promoter activity of each construct was analyzed more than three times, and the averaged value is shown along with S.D. (Standard Deviation). The averaged value for each construct was further compared with that of the Control construct. These promoter assays were performed using two different cell lines, Neuro2A and NIH3T3. Only the result set from the Neuro2A cell line is shown in graphs since the result set derived from NIH3T3 showed almost identical patterns.