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. Author manuscript; available in PMC: 2021 Mar 30.
Published in final edited form as: Science. 2017 May 5;356(6337):eaaj2239. doi: 10.1126/science.aaj2239

Fig. 4. Examples of effects of mCpG on TF binding.

Fig. 4.

(A) Bisulfite-SELEX. Two models for POU5F1 (OCT4) are recovered from different stages of the bisulfite-SELEX process. OCT4 can bind to both unmethylated and methylated sequences corresponding to the indicated motifs, but it prefers to bind the sequences when the indicated CpG is methylated (remains CpG after bisulfite treatment, indicated in the box). Lightning bolts represent bisulfite treatment, and blue shading highlights dinucleotides affected by methylation. Numbers at the bottom show the increased percentage of mCpG from cycle 3 to cycle 4. (B) Example of a type A methyl-minus TF, MAX (Myc-associated factor X). The scatterplot (left) shows the counts of all 8-mer subsequences from methyl-SELEX (y axis) and HT-SELEX (x axis) at cycle 4. Filled circles indicate subsequences that are more enriched than any other subsequence within a Huddinge distance (25) of 1.The most enriched sequence (CCACGTGC) is also indicated. Because methylation of CpG inhibits MAX binding, the population of the red circles (sequences with CpG) forms an elongated pattern that is located below the population of the black circles (sequences without CpG); this is also shown by the simplified glyph (top). When binding to the optimal site is blocked, other sequences (CACATGGC) that bind more weakly enrich more strongly. The logo of the MAX motif is also shown (right), with the effect of methylation of the CpG in bisulfite-SELEX shown below it. MAX is classified as type A because the consensus of its motif contains a CpG (bracket). (C) A type B methyl-minus TF, DMRTC2, for which the primary motif (right, top) is not affected by methylation, but a CpG in the secondary motif (right, bottom) is. Sequences matching the consensus of its two motifs are indicated on the scatterplot. (D and E) As in (B) and (C), but for the type A methyl-plus TF HOXB13 (D) and the type B methyl-plus TF POU5F1 (OCT4) (E). The subsequence ATGCGCAT is much more enriched by POU5F1 (OCT4) in the presence of CpG methylation. OCT4 also enriches the subsequence ATGCTAAT, which does not contain a CpG and is not affected by methylation.