Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals demarcation of active and repressive domains

Abstract

Insulators are DNA elements that prevent inappropriate interactions between the neighboring regions of the genome. They can be functionally classified as either enhancer blockers or domain barriers. CTCF (CCCTC-binding factor) is the only known major insulator-binding protein in the vertebrates and has been shown to bind many enhancer-blocking elements. However, it is not clear whether it plays a role in chromatin domain barriers between active and repressive domains. Here, we used ChIP-seq to map the genome-wide binding sites of CTCF in three cell types and identified significant binding of CTCF to the boundaries of repressive chromatin domains marked by H3K27me3. Although we find an extensive overlapping of CTCF-binding sites across the three cell types, its association with the domain boundaries is cell-type-specific. We further show that the nucleosomes flanking CTCF-binding sites are well positioned. Interestingly, we found a complementary pattern between the repressive H3K27me3 and the active H2AK5ac regions, which are separated by CTCF. Our data indicate that CTCF may play important roles in the barrier activity of insulators, and this study provides a resource for further investigation of the CTCF function in organizing chromatin in the human genome.

Figure 1.

CTCF-binding sites overlap among different cell types. (A) Genome-wide distribution of CTCF-binding sites in CD4⁺ T cells, Jurkat cells, and HeLa cells. (B) CTCF-binding sites in CD4⁺ T cells, HeLa cells, and Jurkat cells overlap extensively. (C) Overlap of CTCF-binding sites in CD4⁺ T cells, HeLa cells, and Jurkat cells in an 850-kb region in chromosome 1, shown as custom tracks on the UCSC genome browser (Karolchik et al. 2008). (D) The densities of genes and CTCF-binding sites per 2 Mbp in chromosome 1. X-axis, number of genes/2 Mbp; y-axis, number of CTCF-binding sites/2 Mbp. (E) CTCF-binding motif in CD4⁺ T cells, HeLa cells, and Jurkat cells.

Figure 2.

CTCF is enriched at the H3K27me3 domain boundaries. (A) Histone H3K27me3 pattern at the HOXD locus in CD4⁺ T cells; (B) beta-globin locus in HeLa cells shown as custom tracks on the UCSC genome browser (Karolchik et al. 2008). (Green) Expressed genes; (pink) silent genes. (C) A 400-kb region in chromosome 1 in CD4⁺ T cells. The barrier CTCF sites, shown as red bars, could be involved in maintaining the FOXJ3 gene free of H3K27me3, thus keeping it active, while the GUCA2A and GUCA2B genes that are within the H3K27me3 domain are silent. (Green) Expressed genes; (pink) silent genes. (D) In HeLa and CD4⁺ T cells, colocalization of the barrier CTCF sites with H3K27me3 domain boundaries (green lines) is higher compared with the colocalization between the randomly distributed sites and H3K27me3 domain boundaries (blue curve). The colocalization of the STAT1 and E2F4-binding sites (red line) with the H3K27me3 domain boundaries is lower than the colocalization between the randomly distributed sites and H3K27me3 domain boundaries (blue curve) in HeLa and CD4⁺ T cells. (E) Distribution of the barrier CTCF sites in HeLa and CD4⁺ T cells.

Figure 3.

Barrier CTCF sites are cell-type-specific. (A) While over 50% and 74% of the CTCF-binding sites in CD4⁺ T cells and HeLa cells, respectively, overlap (open circles), only 5% and 11% of the barrier CTCF sites in the respective cell types overlap. The barrier CTCF sites are shaded. (B) A 2.5-Mbp region in chromosome 2 in CD4⁺ T cells (top) and HeLa cells (bottom). (Green) Expressed genes; (pink) silent genes. (HC1, HC2) Barrier CTCF sites in HeLa cells. A locus, which is occupied by CTCF in both CD4⁺ T cells and HeLa cells, could be functioning as a barrier in HeLa cells (HC1), but not in CD4⁺ T cells (TC1). (TC2, TC3) CTCF-binding sites, which could potentially be playing the role of domain barriers in CD4⁺ T cells. (Inset) An enlargement of the 30-kb region (TC2) in CD4⁺ T cells. The H3K27me3 domain is shaded gray.

Figure 4.

CTCF-binding sites separate active and repressive domains. (A) CTCF-binding sites separate the chromatin domains marked by H3K27me3 and H2AK5ac in a 900-kb region in chromosome 1. The H3K27me3 and H2AK5ac loci are complementary to each other and appear to be separated by CTCF-binding sites (green bars). The active genes are shaded green and the silent genes are shaded pink. (Inset) The transition between the H3K27me3 and H2AK5ac domains separated by CTCF sites. (B) The H3K27me3 profiles were plotted surrounding the nonpromoter (at least 5 kb away from the promoter) CTCF-binding sites in CD4⁺ T cells. (BS) CTCF-binding site. Red line indicates the reads from the sense strand and green line indicates reads from the antisense strand of DNA. (C) The H2AK5ac profiles were plotted surrounding the nonpromoter CTCF-binding sites in CD4⁺ T cells. (BS) CTCF-binding site.

Figure 5.

Nucleosomes are strongly positioned at the CTCF-binding sites and the positioning is cell-type-specific. (A) Nucleosomes at the CTCF-binding sites in CD4⁺ T cells are phased. CTCF binds to the linker region surrounded by well-positioned nucleosomes. The deduced nucleosomes are indicated as ovals. (BS) CTCF-binding site. (B) Nucleosome positions in CD4⁺ T cells at the HeLa cell-specific CTCF-binding sites. A nucleosome occludes the CTCF-binding site where no binding is detected in CD4⁺ T cells. The possible nucleosome positions on the CTCF-binding site are indicated as ovals.