Figure 4.

A nuclear factor(s) binds to the CCTTG repeat. (A) Presence of CCTTG-specific factor in MEL cell nuclear extract. EMSA was performed with the MEL cell nuclear extract by using a probe containing four CCTTG repeats (WTX4). The shifted band is indicated with an arrowhead. Increasing amounts of unlabeled competitor probe WTX4 and a probe containing four mutated (CCGAG) repeats (MUTX4) at 10-, 50-, and 60-fold excess over the radioactively labeled probe were added to the binding mixture. The sequences of WTX4 and MUTX4 are shown below. (B) The repeat-binding sequence is conserved in mammalian fetal globin promoters. EMSA was performed with CCTTG containing probe JC454/5, which was derived from the γ-globin promoter, and JC493/4, which was derived from the sequence between the CACCC box and the CAAT box in the mouse bh1 promoter (shown below). Where indicated, 100-fold excess of the competitor was used. In the first lane, the MEL cell nuclear extract was left out of the binding reaction (−extr). AP2 binding sequence was used as negative control for competition. The TCTTG repeat in the bh1 promoter is marked with dashed arrows. (C) COUP TF II does not bind to the CCTTG repeat. EMSA was performed by using nuclear extract from COS-7 cells mock-transfected (−) or transfected with an expression vector for COUP TF II (+). WTX4 and RARE2 (sequence shown below), which contains binding sites for COUP TF II and related nuclear receptors, were used as probes. The COUP TF II complex is indicated with an arrowhead.