FIG. 1.

The Rasgrf1 DMD has enhancer-blocking activity that depends upon CTCF binding sites. The enhancer-blocking test constructs shown at the left were prepared and transfected into K562 cells as described previously (3, 6). Each contained the chicken β-globin enhancer (Enh), the human ^Aγ-globin promoter directing transcription of a neo reporter (Neo), the chicken β-globin enhancer blocker (BE), and various test fragments. Each test was done 2 to 23 times. The colony number for each test is expressed relative to the number observed with the negative control plasmid (NI) (3), which lacked test sequences and produced an average of 226 colonies. JC5-4 containing the chick β-globin enhancer blocker served as a positive control (7). Rasgrf1 sequences tested included the DMD and repeats (two black triangles) in both orientations or downstream of the neo reporter, one copy of the DMD alone (1 × DMD), and a mutated DMD carrying mutations in the three known CTCF sites (designated 160, 230, and 320 in Fig. 2). Colony counts from 1 × DMD and the DMD with repeats are not significantly different. The difference between results for 1 × DMD and the mutant DMD 3 mut is significant (*, P = 0.008 by t test).