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. 2013 Dec 25;42(5):3059–3072. doi: 10.1093/nar/gkt1323

Figure 4.

Figure 4.

Functional analysis of human meAP-1 sites. (A) Oligonucleotides containing meAP-1 binding sites of the PTK6, P2RY5 and SUV39H1 promoters. The meAP-1 sites are indicated in bold capital letters and the CpG pairs within the meAP-1 sites are underlined. (B) EMSAs showing the preferential binding of c-Jun/c-Fos to the CpG-methylated meAP-1 sites of the PTK6, P2RY5 and SUV39H1 promoters. EMSAs were performed with affinity-purified GFP-tagged c-Jun/c-Fos fusion protein transiently expressed in HEK293 cells. EMSAs of typical experiments are shown. (C) Determination of the apparent KD of c-Jun/c-Fos bound to meAP-1. ELISAs were performed with affinity-purified Strep/FLAG-tagged c-Jun/c-Fos fusion protein transiently expressed in HEK293 cells. Serial dilutions of Cy5 oligonucleotides were used to determine the KD values. The data were fitted to the Hill equation with one site-specific binding to determine the dissociation constants, which are indicated. Each data point indicates the mean and standard deviation of three independent experiments. (D) The c-Jun/c-Fos activation of promoters with meAP-1 binding sites is increased on CpG methylation. Pentamers of short oligonucleotides shown in (A), which contain the meAP-1 sites present in the promoters of the human PTK6, P2RY5 and SUV39H1 genes were introduced into a basic luciferase reporter plasmid as described in Figure 2E. Unmethylated and fully CpG-methylated reporter constructs were analyzed in the presence or absence of a cotransfected VP16:c-Jun/c-Fos expression plasmid. Each experiment was performed three times and means and standard deviations are depicted.