Widespread plasticity in CTCF occupancy linked to DNA methylation

Abstract

CTCF is a ubiquitously expressed regulator of fundamental genomic processes including transcription, intra- and interchromosomal interactions, and chromatin structure. Because of its critical role in genome function, CTCF binding patterns have long been assumed to be largely invariant across different cellular environments. Here we analyze genome-wide occupancy patterns of CTCF by ChIP-seq in 19 diverse human cell types, including normal primary cells and immortal lines. We observed highly reproducible yet surprisingly plastic genomic binding landscapes, indicative of strong cell-selective regulation of CTCF occupancy. Comparison with massively parallel bisulfite sequencing data indicates that 41% of variable CTCF binding is linked to differential DNA methylation, concentrated at two critical positions within the CTCF recognition sequence. Unexpectedly, CTCF binding patterns were markedly different in normal versus immortal cells, with the latter showing widespread disruption of CTCF binding associated with increased methylation. Strikingly, this disruption is accompanied by up-regulation of CTCF expression, with the result that both normal and immortal cells maintain the same average number of CTCF occupancy sites genome-wide. These results reveal a tight linkage between DNA methylation and the global occupancy patterns of a major sequence-specific regulatory factor.

Figure 1.

CTCF in vivo binding exhibits widespread plasticity. (A–C) Constitutive and variable CTCF sites. (A) The H19/IGF2 imprinted locus in multiple human cell types. Note the total silencing in two cell lines of the seven CTCF sites in the differentially methylated region (DMR; yellow box at left), and the complex pattern of cell-selective CTCF binding flanked by constitutive sites. Location (hg19), chr11:2,015,000–2,184,000. (B,C) Additional examples of variable sites. (D) Genome-wide analysis of CTCF binding in 19 cell types reveals 77,811 distinct binding sites; 27,662 sites are constitutively present in all cell types; 50,149 variable sites exhibiting a wide range of selectivity are present in a subset of one to 18 cell types (below). (E) Genomic distribution of variable sites is similar to constitutive sites (Supplemental Fig. S2A).

Figure 2.

CTCF occupancy distinguishes similar cell types. (A) Unsupervised hierarchical clustering of binding at all CTCF sites. (B) CTCF occupancy at 4146 variable binding sites that distinguish immortal cell lines, epithelia, fibroblasts and endothelia (Methods). x-axis, CTCF binding sites in chromosomal order, separated into sites that are up-regulated and down-regulated (arrows) in each of the three groups (immortal, epithelial, fibroblast, and endothelial). Color corresponds to Z-score of normalized ChIP-seq density.

Figure 3.

Impact of DNA methylation on cell-selective CTCF binding. (A) Example CTCF binding sites, where occupancy (above) quantitatively increases as local CpG methylation decreases (below). Green indicates CpG is 0% methylated; yellow, 50%; and red, 100%. (B) Quantitative analysis of methylation at the boxed CTCF binding site in A. (C) Global impact of methylation at variable CTCF sites monitored by RRBS. Sixty-five percent of sites with cell-type selective patterns of methylation also exhibited differences in occupancy. (D) At methylated binding sites, occupancy was reduced on average by 87% compared with cell lines without methylation at the same site. Shown are sites where increased methylation was associated with decreased occupancy (98% of all significant sites).

Figure 4.

Sites significantly affected by methylation are enriched for CpGs at two positions. Frequency of a CpG (y-axis) at positions relative to the CTCF motif (x-axis) is shown for sites with variable methylation that is associated (red) and is not associated (gray) with occupancy changes. Note that at positions 1 and 11, there is a 2.2- and 1.8-fold enrichment, respectively, for the presence of a CpG at sites where the variable methylation was not associated with occupancy. Twenty-nine percent of CTCF motifs genome-wide contain a CpG at one or both of these positions.

Figure 5.

Cell-selective patterns of methylation associated with occupancy differences. (A) Methylation status at 1969 CTCF sites where differential methylation is significantly associated with occupancy differences. Color corresponds to the percentage of bisulfite sequencing tags at each site overlapping methylated CpG positions. Dendrogram (left) highlights pattern of hypermethylation in immortal cell lines. (Right) Smoothed plot of number of immortal lines exhibiting hypermethylation at each site. (B) Immortal lines show no significant difference in number of occupied CTCF sites (y-axis, mean). Error bars, SD. (C) immortal lines demonstrate increased CTCF transcript levels (y-axis, mean). Error bars, SD. (D) Immortal lines exhibit increased methylation relative to the other cell types, though significant promoter methylation is rarely observed in normal lines. y-axis, genome-wide median of per-site methylation. P-values, Wilcoxon. Promoter, ±2.5 kb of RefSeq transcription start site.