DNA mobility shift assays of two divergent motifs of the Tsix cluster. (A) The two motifs found in the Tsix cluster, AGACATTTT (mYY1–1) and AGGCATTTT (mYY1–2), were labeled as probes for gel shift assays. Each of two commercially available YY1 oligonucleotides, yy1 and yy2, were used as a competitor at two different molar ratios relative to a given probe (10×, lanes 3 and 9; 100×, lane 4 and 10). (B) Another series of the reciprocal competition assays using yy1 as a labeled probe were also performed. In this assay, the molar ratios of mYY1 and mYY2 to the labeled yy1 probe are 10× (lanes 3 and 6), 25× (lanes 4 and 7), and 100× (lanes 5 and 8), respectively. (C) Supershift assays were performed using two polyclonal antibodies, anti-YY1 antibody, and anti-H3 antibody as an unrelated negative control. The sequences of the four duplex oligonucleotide probes are shown at the bottom. The two motifs of the Tsix locus are positioned in the middle of these probes and marked with underlines. The nucleotide positions differing from the consensus sequence of YY1 binding sites are also indicated with boldface types.
