Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions

Abstract

Insulators are protein-bound DNA elements that are thought to play a role in chromatin organization and the regulation of gene expression by mediating intra- and interchromosomal interactions. Suppressor of Hair-wing [Su(Hw)] and Drosophila CTCF (dCTCF) insulators are found at distinct loci throughout the Drosophila melanogaster genome and function by recruiting an additional protein, Centrosomal Protein 190 (CP190). We performed chromatin immunoprecipitation (ChIP) and microarray analysis (ChIP-chip) experiments with whole-genome tiling arrays to compare Su(Hw), dCTCF, boundary element-associated factor (BEAF), and CP190 localization on DNA in two different cell lines and found evidence that BEAF is a third subclass of CP190-containing insulators. The DNA-binding proteins Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization. Notably, cell line-specific localization sites for all three DNA-binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression. These findings suggest a model in which insulator subclasses may have distinct functions that together organize the genome in a cell type-specific manner, resulting in differential regulation of gene expression.

Figure 1.

Genome-wide localization of insulator proteins. (A) Representative ChIP–chip data for Su(Hw), dCTCF, CP190, and BEAF over a 250-kb region of Chromosome 2L. The Y-axis contains the log₂ value of the ratio of ChIP/input signal. For each antibody, the top row represents the normalized data from one biological replicate and the bottom row represents the analyzed peak data that summarizes three biological replicates. Gene data that depict mRNA, 5′UTR, and exon information are shown in black, such that boxes above the line represent genes on the plus strand and genes below the line represent genes on the minus strand. (B) A summary of the whole-genome peak analysis for Su(Hw), dCTCF, CP190, and BEAF using the more stringent cutoff of <1% FDR. (C) A Venn diagram indicating the number of binding sites that overlap between Su(Hw), dCTCF, BEAF, and CP190.

Figure 2.

Genomic distribution of insulator protein-binding sites. (A) Pie charts representing the distribution of Su(Hw), dCTCF, BEAF, and CP190 in relation to intergenic regions, introns, and exons. Exonic distribution is then further broken down into protein-coding exons, 5′UTRs, and 3′UTRs. (B) The distribution of the insulator proteins in relation to the 5′ and 3′ ends of annotated genes. The graph depicts 200-bp fragments encompassing1 kb upstream of and downstream from the start and end of genes as well as 600 bp into the start and end of genes. (C) Percent of insulator protein-binding sites within 200 bp of the 5′ end of genes that localize to genes with high, medium, and low/no expression. Line colors correspond to the same proteins as denoted in B.

Figure 3.

Insulator protein DNA binding is a cell type-specific process. (A) Summary of the whole-genome comparison between Su(Hw), dCTCF, BEAF, and CP190 in Kc and Mbn2 cells. Bars represent the percentage of binding sites that are cell type-unique using the data sets defined with the more stringent cutoff of a 1% FDR. (B) Validation of four Su(Hw) (left), four dCTCF (middle), and four BEAF (right) cell type-specific binding sites by real-time PCR. For each protein, two Kc cell type-specific binding sites (columns a,b) and two Mbn2 cell type-specific binding sites (columns c,d) were tested. Each binding site was analyzed by conventional ChIP for the DNA-binding protein as well as CP190 in each cell type. Percent input values were normalized to a well-known binding site for each insulator protein.

Figure 4.

The role of CP190 insulators in chromatin organization. (A) Venn diagram depicting the overlap of CP190-binding sites between Kc cells and Mbn2 cells. Roughly 17% of CP190 sites in Kc cells and 14% of sites in Mbn2 cells were found to be cell type-specific. Of these cell type-specific binding sites, a subset was found to be dependent on differential DNA-binding protein localization (top boxes) and a subset was found to be independent of DNA-binding protein localization (bottom boxes). Cartoons indicate the state of DNA-binding protein and CP190 localization in Kc cells (orange) and Mbn2 cells (blue) at sites represented by each region. (B) Model depicting the functional specialization of CP190 insulator subclasses. Su(Hw) and dCTCF may orchestrate the primary level of chromatin organization. BEAF and dCTCF could then fine-tune this organization around highly expressed genes (yellow arrows). The interactions between insulator protein-binding sites may be facilitated by CP190.