Sumoylated PPARα mediates sex-specific gene repression and protects the liver from estrogen-induced toxicity in mice
Nicolas Leuenberger, Sylvain Pradervand, Walter Wahli
Nicolas Leuenberger, Sylvain Pradervand, Walter Wahli
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Research Article Genetics

Sumoylated PPARα mediates sex-specific gene repression and protects the liver from estrogen-induced toxicity in mice

Abstract

As most metabolic studies are conducted in male animals, understanding the sex specificity of the underlying molecular pathways has been broadly neglected; for example, whether PPARs elicit sex-dependent responses has not been determined. Here we show that in mice, PPARα has broad female-dependent repressive actions on hepatic genes involved in steroid metabolism and immunity. In male mice, this effect was reproduced by the administration of a synthetic PPARα ligand. Using the steroid oxysterol 7α-hydroxylase cytochrome P450 7b1 (Cyp7b1) gene as a model, we elucidated the molecular mechanism of this sex-specific PPARα-dependent repression. Initial sumoylation of the ligand-binding domain of PPARα triggered the interaction of PPARα with GA-binding protein α (GABPα) bound to the target Cyp7b1 promoter. Histone deacetylase and DNA and histone methylases were then recruited, and the adjacent Sp1-binding site and histones were methylated. These events resulted in loss of Sp1-stimulated expression and thus downregulation of Cyp7b1. Physiologically, this repression conferred on female mice protection against estrogen-induced intrahepatic cholestasis, the most common hepatic disease during pregnancy, suggesting a therapeutic target for prevention of this disease.

Authors

Nicolas Leuenberger, Sylvain Pradervand, Walter Wahli

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Figure 5

AF-2–dependent interaction between PPARα and GABP proteins on the CYP7B1 promoter.

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AF-2–dependent interaction between PPARα and GABP proteins on the CYP7B1...
(A) Cotransfection experiments in NIH 3T3 cells using CYP7B1 promoter deletion constructs and a mouse PPARα expression vector or PSG5 as a control. Cells were then treated with WY-14643. (B) Schematic representation of the proximal CYP7B1 promoter region, which confers PPARα-dependent repression, with the positions of the GABP sites and the Sp1 site indicated. Sites conserved between mouse and human and sites only found in human are indicated. (C) NIH 3T3 cells were cotransfected with pCDNA3-flag-GABP expression vectors and a reporter gene driven by the –144/+189 CYP7B1 promoter region containing the GABP-binding sites or the –18/+176 promoter region, which lacks these sites. (A and C) Values are presented as mean ± SEM (n = 3 per group). **P ≤ 0.01. (D) NIH 3T3 cells were cotransfected with WT or mutant (Mut) PPARα (ΔC13 and K358R) expression vectors and the flag-GABP expression vector. Right: Reverse coimmunoprecipitation experiment. NT, nontransfected cells. (E) NIH 3T3 cells were cotransfected with a PPARα expression vector and Flag-GABPα expression vectors for WT or mutated LXXLL motif as indicated. (F) Same as in E, but IP was performed against Flag-tag, followed by Western blotting against PPARα. (G) Requirement of GABPα for mouse PPARα binding on the transfected human CYP7B1 promoter was determined by a ChiP assay and GABPα siRNA. (H) In vivo binding of PPARα and GABPα on the Cyp7b1 promoter was detected by a ChiP assay of hepatic nuclear protein using PPARα and GABPα antibodies. (I) Interaction of PPARα and GABPα on the Cyp7b1 promoter was detected by a re-ChIP assay (see Methods). Treated, WY-14643; Control, untreated.