The classes of insulator protein binding sites. The composition of 16 co-binding groups detected by initial overlap comparison is indicated by the checkerboard pattern under the bar plot. The color code in log₂(IP/INPUT) units (indicated to the right) is used to show the number of sites of different binding strength within each class. For the multiprotein classes, the bars are divided from left to right corresponding to the top to bottom positions of the proteins in the chart below. The numbers of sites of each class that bind all corresponding proteins within 60% of their ChIP-chip signal dynamic range are indicated above the bars. Only those sites were used for further analysis. The dashed line on each bar indicates the position of the 60% cutoff. The classes representing robust co-binding combinations are numbered in red.

The sequence determinants and interdependence of the insulator protein binding to chromatin. (A) The logo representations of sequence motifs characteristic of SU(HW), CTCF, and CP190 binding sites defined by the MEME algorithm and used in the analysis in D. (B) The effects of the RNAi knock-down on the target protein and its co-binding partners. The sites at which ChIP-chip signal was consistently reduced judged from the comparison of two replicate mock RNAi experiments and two specific RNAi experiments (z-scores < −3, unpaired t-test) were counted and their fractions plotted. Here and in C and D, the error bars indicate the 95% confidence interval. The bar plots show that the binding of CP190 to some of the class 9 but not at gypsy-like sites depends on CTCF. However, the binding of CTCF to class 9 sites does not depend on CP190. In contrast, the binding of SU(HW) and CP190 to gypsy-like sites is interdependent. (C) As illustrated by this bar plot, BEAF-32 and CP190 bind to common sites independently. (D) The presence of SU(HW) and CTCF recognition sequences within indicated classes of sites demonstrates that the coincidence of the two motifs is responsible for the co-binding of SU(HW) and CTCF to class 12 sites.

The effects of RNAi knock-down on the binding of insulator proteins to chromatin. BG3 cells were subjected to RNAi against key insulator proteins followed by ChIP-chip. (A) Western blots of threefold serial dilutions of nuclear protein from cells treated with specific and mock dsRNA (indicated above the panels) show 10-fold or greater knock-down of the corresponding proteins. The antibodies used for detection are indicated to the right, and the loading controls are shown in Supplemental Figure S4. The comparison of average binding for (B) SU(HW), (C) CTCF, (D) CP190, and (E) BEAF-32 after mock and specific RNAi shows that the binding is reduced at the majority of sites (data points below red dashed line). (Blue dots) The sites with consistent reduction in both replicate experiments (estimated conservatively with unpaired t-test; z-scores < −3); (green dots) others. (F) scs′ is one of the BEAF-32 high-affinity binding sites resistant to RNAi. The BEAF-32 ChIP-chip signals after BEAF-32 and mock RNAi are plotted along the segment of chromosome 3R. (White circles) Peaks affected by BEAF-32 knock-down; (red circles) peaks that remain unchanged. The genes shown above the coordinate scale are transcribed from left to right, those below the scale from right to left.

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.

Functional effects of insulator protein withdrawal. (A) Affymetrix GeneChip expression analysis of cells from the RNAi experiments described in Figure 3. The average fold change between the two specific and two mock RNAi experiments (y-axes) was plotted against the highest average expression value detected in the mock or specific RNAi samples (x-axes). Each graph point represents one transcript interrogated by the microarray. Transcripts robustly expressed before or after specific RNAi treatment are to the right of the vertical dashed lines. Of these, those showing consistent twofold or greater change after specific RNAi treatment in both replicate experiments are circled. (B) As evident from ChIP-chip of H3K27me3 from mock RNAi-treated BG3 cells, the sens-2 gene is repressed by PcG. The right border of the corresponding H3K27me3 domain is sharp and coincides with a standalone CP190 site (marked by a vertical green dashed line) and with the Rca1 transcript. The ChIP-chip with H3K4me3 and H3K36me3 indicates that Rca1 is transcriptionally active. The left side of the H3K27me3 domain declines gradually with no obvious border. It harbors gypsy-like and CTCF+CP190 binding sites marked by orange and purple dashed lines, respectively. The knock-downs of insulator proteins have no effect on the position of the right border of the H3K27me3 domain but change the shape of its left tail. The changes in histone methylation profile are best seen on the relative difference browser tracks. (C) twi is also repressed by PcG mechanisms in BG3 cells. The right border of the corresponding H3K27me3 domain is set by the presence of an active transcript. The left border is maintained by a gypsy-like (class 3) insulator (vertical orange dashed line), as evident from the extension of K27 trimethylation after SU(HW) or CP190 knock-down. (D) The pie chart shows the frequencies of various genomic features associated with definable H3K27me3 domain borders.