TG-interacting factor 1 acts as a transcriptional repressor of sterol O-acyltransferase 2

Abstract

Acat2 [gene name: sterol O-acyltransferase 2 (SOAT2)] esterifies cholesterol in enterocytes and hepatocytes. This study aims to identify repressor elements in the human SOAT2 promoter and evaluate their in vivo relevance. We identified TG-interacting factor 1 (Tgif1) to function as an important repressor of SOAT2. Tgif1 could also block the induction of the SOAT2 promoter activity by hepatocyte nuclear factor 1α and 4α. Women have ∼ 30% higher hepatic TGIF1 mRNA compared with men. Depletion of Tgif1 in mice increased the hepatic Soat2 expression and resulted in higher hepatic lipid accumulation and plasma cholesterol levels. Tgif1 is a new player in human cholesterol metabolism.

Keywords: hepatocyte nuclear factor 1α and 4α; human; liver; triglyceride.

Fig. 1.

Tgif1 is a repressor of SOAT2. (A) Putative repressor elements in the human SOAT2 promoter (p-1305) were mutated, and these mutated constructs [i.e., T-cell factor 4E (Tcf4e), Gata, and Tgif] were used for transient transfections of HuH7 cells. (B) The human SOAT2 promoter and an expression vector for Tgif1 were used to transiently cotransfect HuH7 cells. (C) Transient cotransfections with the SOAT2 promoter construct in which the identified Tgif site was mutated and the expression vector for Tgif1. (D) Transient cotransfections with deletion constructs of the SOAT2 promoter and the Tgif1 vector. (E) Three putative binding sites for Tgif, located at −1,270/−1,264 bp (Mut-1270), −484/−478 bp (Mut-484), and −15/−10 bp (Mut-15) upstream of the transcription start site in the SOAT2 promoter, were mutated individually or in combination with the −1,270/−1,264 bp site and used along with the Tgif1 vector. (F) Soluble chromatin, prepared from human liver, was immunoprecipitated with antibodies against either Tgif1 or IgG and amplified using primers designed to target the three putative Tgif binding sites in the human SOAT2 promoter. Data are expressed as mean ± SEM (n = 4). Differences were tested using the Dunnett test. *** P < 0.001.

Fig. 2.

Tgif1 blocks the induction by Hnf1α and Hnf4α of the SOAT2 promoter activity. HuH7 cells were transiently cotransfected with the SOAT2 promoter (p-1305) or with constructs of the promoter in which binding sites for Tgif were mutated along with vectors for Hnf1α (A) or Hnf4α (B), with and without Tgif1. Caco2 cells were transiently cotransfected with the SOAT2 promoter and vectors for Tgif1 (C) or Hnf4α (D). Caco2 cells were transiently cotransfected with the human SOAT2 promoter along with vectors for Hnf1α (E) or Hnf4α (F), with and without Tgif1. Data are expressed as mean ± SEM (n = 4).

Fig. 3.

Effects of Meis2d on the SOAT2 promoter. Transient cotransfections of HuH7 cells with the human SOAT2 promoter (p-1305) (A) or with a construct of the promoter in which a binding site for Tgif was mutated (Mut-15) (B) along with a vector for Meis2d. (C) Transient cotransfections of HuH7 cells with the SOAT2 promoter and vectors for Meis2d and Tgif1. (D, E) Transient cotransfections of Caco2 cells with the SOAT2 promoter and vectors for Meis2d and Tgif1. Data are expressed as mean ± SEM (n = 4).

Fig. 4.

Gene expression in humans. Hepatic TGIF1 mRNA levels were measured in 33 fertile and 24 postmenopausal female and 41 male normolipidemic Chinese subjects using real-time RT PCR. Gene expression was normalized to GAPDH mRNA, and data are expressed as mean ± SEM. Differences among patients were tested using multiway ANOVA, followed by post hoc comparisons according to the least significant difference test. *** P < 0.001.