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. 2019 Oct 28;116(46):23232–23242. doi: 10.1073/pnas.1913199116

Fig. 4.

Fig. 4.

The upstream duplicated region mediates PPARγ activation of PM20D1. (A) The consensus motif logo for a PPRE is shown, along with the sequences of the second PPRE in the intronic and upstream putative PPARγ binding regions near PM20D1. There is 1 A:G difference (arrow), which predicts better PPARγ binding to the upstream G. (B) PPARγ ChIP was performed in SGBS cells, followed by qPCR to amplify a negative site, versus selective primers that distinguish the intronic or upstream sites in PM20D1. (C) Pyrosequencing analysis of PPARγ ChIP DNA, with the PCR not distinguishing the intronic and upstream paralogues, yet the ChIP but not input DNA showing enrichment of the upstream G nucleotide. (D) Luciferase reporter assay showing that mouse Pm20d1 intronic sequence is not activated by PPARγ, while the human upstream sequence is much more strongly activated than the intronic paralog. (E) Mutagenesis of luciferase reporters shows that the A:G difference in PPRE#2 is responsible for this difference. Mean and SEM, #P < 0.001; *P < 0.01; ^P < 0.05; NS, not significant by 2-tailed type 2 Student’s t-test.