Insulator assay of the clustered YY1 binding sites. For insulator assays, the testing DNA fragments were positioned between an erythroid-specific enhancer (Enh) and a promoter directing the transcription of a reporter, neoR, as shown in B. This test used two control vectors: pNI-CD(-AscI) without any insulator and pNI-CD with a chicken insulator. The genomic fragments containing clustered YY1 binding sites were subcloned into the AscI site of the pNI-CD vector with two different orientations, forward (-F) and reverse (-R). The DNA fragments for the clustered sites of Xist and Tsix were derived from mouse (m), human (h), cow (b), and rat (r). Each fragment was tested more than three times, and the averaged values with standard errors are shown in A. The overall patterns observed in this series of experiments are summarized schematically in B with averaged values shown below the name of each construct.
