Figure 5.

Evidence for the 'buffering' of variation at conserved CTCF binding sites. (a) Proportion of homozygous polymorphic CTCF binding sites with 'buffered' levels of ChIP signal depending on the sites' evolutionary conservation (less conserved, BLS <0.5; more conserved, BLS ≥0.5). Sites at which the minor variant retained at least two-thirds of the major variant's signal were considered as 'buffered'. The P-value is from the Fisher test. Major and minor variants were defined on the basis of the global allele frequency data from [75,76]. (b) Differences in the CTCF binding signal (Δ ChIP signal) at homozygous polymorphic sites that show either 'low' (left) or 'high' (right) disparity in absolute motif match scores (Δ motif score) between the variants (<1 or >1, respectively). The ChIP signals are sign-adjusted relative to the direction of PWM score change. Site-specific signals from multiple individuals with the same genotype, where available, were summarized by mean. The P-value is from the Wilcoxon test. (c) Genotype-specific differences in the CTCF ChIP signal across individuals between homozygous polymorphic sites with appreciable differences in absolute PWM match scores (Δ motif score >1) at less conserved (BLS <0.5, left) and more conserved (BLS >0.5, right) CTCF motifs. The ChIP signals are sign-adjusted relative to the direction of PWM score change. Site-specific signals from multiple individuals with the same variant, where available, were summarized by mean. The P-value is from the Wilcoxon test. (d) An interaction linear model showing that interspecies motif conservation (expressed by branch length scores) reduces the effect of motif mutations on CTCF binding. Shown are the effect plots predicting the relationship between the change of PWM score (at the minor versus the major variant) and the change of the associated ChIP signal at three hypothetical levels of evolutionary conservation: BLS = 0 (low; left); BLS = 0.5 (medium; middle); and BLS = 1 (high; right). Major and minor variants were defined on the basis of the global allele frequency data from [75,76]. (e) An interaction linear model showing that interspecies motif conservation (BLS) reduces the effect of motif stringency on the binding signal. Shown are the effect plots predicting the relationship between motif scores and ranked ChIP signal at three hypothetical conservation levels: BLS = 0 (low; left); BLS = 0.5 (medium; middle); and BLS = 1 (high; right). (f) A schematic illustrating the observed effect of binding site mutations on CTCF binding signal at two polymorphic CTCF sites - one poorly conserved (BLS = 0.03, left) and one highly conserved (BLS = 0.84, right) - that have similar motif match scores (14.9 and 14.2, respectively). Sequences of higher- (top) and lower-scoring alleles (bottom) are shown on the figure. Mutations resulting in a similar loss of score (down to 12.5 and 11.8, respectively) resulted in a 53% loss of CTCF binding signal at the non-conserved site (left, compare the amplitudes of top (blue) to bottom (red) curves), in contrast to a mere 6% at the conserved site (right).