Characterization of the Human Insulin-induced Gene 2 (INSIG2) Promoter: THE ROLE OF Ets-BINDING MOTIFS

Ana Fernández-Alvarez,María Soledad Alvarez,Carme Cucarella,Marta Casado

doi:10.1074/jbc.m109.067447

Abstract

Insulin-induced gene 2 (INSIG2) and its homolog INSIG1 encode closely related endoplasmic reticulum proteins that regulate the proteolytic activation of sterol regulatory element-binding proteins, transcription factors that activate the synthesis of cholesterol and fatty acids in animal cells. Several studies have been carried out to identify INSIG2 genetic variants associated with metabolic diseases. However, few data have been published regarding the regulation of INSIG2 gene expression. Two Insig2 transcripts have been described in rodents through the use of different promoters that produce different noncoding first exons that splice into a common second exon. Herein we report the cloning and characterization of the human INSIG2 promoter and the detection of an INSIG2-specific transcript homologous to the Insig2b mouse variant in human liver. Deletion analyses on 3 kb of 5'-flanking DNA of the human INSIG2 gene revealed the functional importance of a 350-bp region upstream of the transcription start site. Mutated analyses, chromatin immunoprecipitation assays, and RNA interference analyses unveiled the significance of an Ets-consensus motif in the proximal region and the interaction of the Ets family member SAP1a (serum response factor (SRF) accessory protein-1a) with this region of the human INSIG2 promoter. Moreover, our findings suggest that insulin activated the human INSIG2 promoter in a process mediated by phosphorylated SAP1a. Overall, these results map the functional elements in the human INSIG2 promoter sequence and suggest an unexpected regulation of INSIG2 gene expression in human liver.

Highlights

Metabolic syndrome, which is characterized by visceral obesity, dyslipidemia, hyperglycemia, and hypertension, has become one of the most significant public health challenges worldwide
Our results demonstrate for the first time that members of the Ets family of transcription factors are involved in the regulation of human Insulin-induced gene 2 (INSIG2) expression and of SREBP processing
INSIG2b was the only form of INSIG2 detected was added to the reaction mixture

Summary

Introduction

Metabolic syndrome, which is characterized by visceral obesity, dyslipidemia, hyperglycemia, and hypertension, has become one of the most significant public health challenges worldwide. SREBP1c (sterol regulatory element-binding protein 1c) is an essential transcription factor for the genomic actions of insulin on both carbohydrate and lipid metabolism (3). Changes in this transcription factor activity might be the key to linking insulin resistance with the metabolic disorders proposed by Lemieux et al (2) at the molecular level. An elevated expression of SREBP1c has been found in the liver of obese (ob/ob) mice, in insulin receptor substrate-2-deficient mice and in a transgenic mouse model of lipodystrophy (5, 6) After their synthesis on membranes of the endoplasmic reticulum (ER) in an inactive form, SREBPs move in vesicles to the Golgi complex, where they are processed sequentially by two proteases (7). INSIG1 is itself an obligatory SREBP target gene, whereas INSIG2 is expressed at a low but constitutive level, at least in cultured cells, and is not regulated by SREBPs (10)

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