Figure 4.

Functional evaluation of classes of insulator protein binding sites. (A,B) Sites bound by different combinations of insulator proteins show distinct biases in their distribution relative to genes and gene activity. Class 2–4 sites are rarely close to TSSs, while class 5–7 sites are primarily TSS-proximal. In rare cases when class 2–4 sites are TSS proximal, these promoters tend to be inactive. In contrast, BEAF-32 (classes 5 and 7) binds predominantly next to active TSSs. While many standalone CP190 sites are next to active TSSs, some are not. The proximity to TSSs and genes in A is defined based on a 2-kb margin, and the binding to TSSs in B is defined on a 1-kb margin. The background distribution expected by chance is shown as the rightmost bar in A and is derived from 10 times the number of positions sampled randomly but with the same chromosome representation. (C) The schematic of the transgenic enhancer blocking assay. A DNA fragment of interest (black rectangle) is cloned in the FRT cassette positioned between the upstream wing and body enhancers (green ovals) and the promoter of the reporter yellow gene (yellow rectangle). The resulting construct is injected into yellow minus flies. DNA fragments capable of enhancer blocking (red rectangle) prevent the activation of the reporter yellow gene by upstream enhancers but allow the activation of the gene by the downstream bristle enhancer (“br” green oval). This yields transgenic flies with pigmented bristles but a yellow body and wings. Ineffectual DNA fragments (green rectangle) allow activation of the reporter gene in all tissues and yield wild-type transgenic flies. The fragments harboring repressive activity (blue rectangle) block the expression of transgenic yellow in all tissues, which results in flies devoid of any pigmentation. The results of transgenic tests are summarized in D.