CTCF physically links cohesin to chromatin

Abstract

Cohesin is required to prevent premature dissociation of sister chromatids after DNA replication. Although its role in chromatid cohesion is well established, the functional significance of cohesin's association with interphase chromatin is not clear. Using a quantitative proteomics approach, we show that the STAG1 (Scc3/SA1) subunit of cohesin interacts with the CCTC-binding factor CTCF bound to the c-myc insulator element. Both allele-specific binding of CTCF and Scc3/SA1 at the imprinted IGF2/H19 gene locus and our analyses of human DM1 alleles containing base substitutions at CTCF-binding motifs indicate that cohesin recruitment to chromosomal sites depends on the presence of CTCF. A large-scale genomic survey using ChIP-Chip demonstrates that Scc3/SA1 binding strongly correlates with the CTCF-binding site distribution in chromosomal arms. However, some chromosomal sites interact exclusively with CTCF, whereas others interact with Scc3/SA1 only. Furthermore, immunofluorescence microscopy and ChIP-Chip experiments demonstrate that CTCF associates with both centromeres and chromosomal arms during metaphase. These results link cohesin to gene regulatory functions and suggest an essential role for CTCF during sister chromatid cohesion. These results have implications for the functional role of cohesin subunits in the pathogenesis of Cornelia de Lange syndrome and Roberts syndromes.

Fig. 1.

Detection of CTCF-interacting factors by ICAT quantitative mass spectrometry. Protein samples recovered from wild-type and mutant CTCF-binding sites (Fig. S1) were labeled with the heavy and normal ICAT reagents, respectively, and prepared for μLC-MS/MS (14). SEQUEST database searching matched an MS/MS spectrum to the indicated ICAT-labeled peptide sequence derived from Scc3/SA1 as the best match. The relative abundance of isotopically heavy (Lower) and normal (Upper) ICAT-labeled peptides corresponding to Scc3/SA1 was calculated by reconstructing single-ion chromatograms for each peptide using XPRESS (14). A peptide derived from the Scc3/SA1 was enriched to a similar level (1:5.0) as peptides corresponding to CTCF (1:7.7).

Fig. 2.

Detection and colocalization of CTCF and Scc3/SA1 in ChIP-Chip experiments. (A) Plot of intensity ratios of individual probes within a region of chromosome 6 and detection of a CTCF-binding site by MeDiCHi (see Methods). (B) Plot of intensity ratios of the Scc3/SA1 ChIP-Chip experiment within the same chromosomal region as in A. (C) Frequency histogram of the distances of Scc3/SA1 peaks (P ≤ 0.01) to all CTCF peaks (P ≤ 0.01) within 2,000 bp.

Fig. 3.

Allele-specific association of cohesin and CTCF with the Igf2/H19 ICR is detected by methylation-sensitive PCR. (A) Position of AciI restriction sites within the amplified region of the ICR. (B) The CpG methylation sensitive restriction enzyme AciI, but not EcoRI, reduces the number of genomic templates available for PCR amplification of the ICR recovered after chromatin immunoprecipitation with either anti-CTCF or -Scc3/SA1 but not after immunoprecipitation with anti-3mK9 H3 (histone H3 trimethylated at lysine 9). As an internal control, a genomic region of the beta-globin gene promoter that lacks both EcoRI and AciI sites was coamplified. (C) Quantitation of the methylation sensitive PCR. Ratios of ICR/beta-globin signals were determined for each immunoprecipitation (dark bars) and compared with the signal ratio after digestion with EcoRI, which was set to 100. Although the paternal allele is CpG methylated and associated with histone H3 trimethylated at lysine 9, the maternal allele is unmethylated and associated with CTCF and Scc3/SA1.

Fig. 4.

Association of cohesin with the myotonic dystrophy gene DM1 requires CTCF. (A) Scheme of the human DM1 sequences integrated via RMCE into murine 3T3 cells. Sequences of the wild-type and mutant CTCF-binding sites 1 and 2 are shown in Fig. S5. (B) ChIP experiments at the wild-type and mutant DM1 loci reveal that mutations in CTCF motifs 1 and 2 [mut (site1,2)] abrogate binding of both CTCF and Scc3/SA1, whereas binding of CTCF and Scc3/SA1 to myc-N and myc-A, both binding sites for CTCF at the endogenous murine c-myc gene (8, 13), remains unaffected. Bars represent the average and standard deviation of three independent experiments.

Fig. 5.

Selective association of Scc3/SA1 with genomic regions. (A) Schematic description of the human HoxA locus. CTCF-binding sites are indicated by vertical bars hx1–hx5. (B) Expression pattern of Hox genes A2–A11 in HBL100 and PC3 cells. Expression of GAPDH is used as a reference. (C) Relative enrichment of genomic regions hx1–hx5 after ChIP with anti-CTCF or -Scc3/SA1. Site hx4 associates with CTCF but does not recruit Scc3/SA1. Relative enrichments are representative data from three independent experiments. (D) ChIP reveals selective binding of CTCF and Scc3/SA1 to genomic regions on chromosome 7 [position 89620788, hg17, University of California, Santa Cruz (UCSC)], chromosome 19 (position 59310952, hg17, UCSC), and chromosome 2 (position 220232167, hg17, UCSC). Genomic regions on chromosomes 7 and 19 associate with Scc3/SA1 but not with CTCF. In contrast, position 220232167 on chromosome 2 binds CTCF but not Scc3/SA1. Error bars indicate standard deviation of enrichment observed in three independent ChIP.

Fig. 6.

Association of CTCF with chromosomal sites and centromeres during mitosis. (A) Example of a CTCF site on chromosome 6 occupied in both asynchronously growing cells (Upper, CTCF Cyc) and cells arrested in mitosis (Lower, CTCF-Mit). The calculated distance between the peaks is indicated (distance = 0). A complete list of all sites detected in both asynchronously growing and mitotic cells is shown in Table S4. (B) Immunofluorescence detection of CTCF bound to centromeres of human mitotic chromosomes. A representative example of a mitotic chromosome preparation is shown. All cells examined from each preparation showed centromeric staining by CTCF.