Characterization of the human insulin-induced gene 2 (INSIG2) promoter: the role of Ets-binding motifs

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.

FIGURE 1.

Alternative sequences at the 5′-end of INSIG2 mRNA from mouse or human liver. A, the nucleotide sequences of the first exons of mouse Insig2a and -2b were aligned with human sequence on the basis of coding exon 2 to predict the putative exons in the human gene. B, total RNA from the indicated tissues or cell lines was analyzed by RT-PCR analysis as described under “Experimental Procedures.” Specific primers were used to determine the presence of each variant in different organisms and cell types. Variant Insig2a is only present in mouse and rat liver and is not detected in human liver or cell lines. MW, molecular weight.

FIGURE 2.

Characterization of the human INSIG2 gene proximal promoter region. The sequences in the INSIG2 promoter shown on the left were inserted into the multiple cloning regions of the pGL3-basic vector and transfected into HEK293T (A) or HepG2 (B) cells. The results represent relative firefly/Renilla luciferase activities, considering the −347/−11 construct as 100% activation. Values are the mean ± S.E. from four separate experiments.

FIGURE 3.

Identification of conserved putative binding sites found in the human INSIG2 promoter. The INSIG2 promoter sequences from humans, mice, and rats are aligned, including the exon 1 situation (shaded sequence). Several highly conserved transcription factor binding sites were identified. The putative binding sites are shown in the figure and underlined on the sequences.

FIGURE 4.

Ets binding sites are critical for INSIG2 promoter activity. Mutation of diverse predicted binding sites in the proximal promoter was analyzed by luciferase assays. Activities of the mutated constructs on −347/−11 luc (A) and −152/−11 luc (B) transfected in HEK293T cells are shown. The results represent relative firefly/Renilla luciferase activities considering the wild type −347/−11 luc construct as 100% activation. Values are the mean ± S.E. from four separate experiments.

FIGURE 5.

ELK1 and SAP1a proteins bind to the INSIG promoter. A, DNA sequence for human INSIG2 proximal promoter. The arrows indicate the probes used in the experiment. B, representative autoradiogram from a typical electrophoretic mobility shift assay realized with recombinant human proteins incubated with ³²P-probes. Anti-ELK1, anti-SAP1a, anti-NRF2a, or anti-E2F antibodies (Ab) were used for supershift assays, and competitions were carried out with a 25-fold molar excess of non-radiolabeled wild type (WT) or mutant (M) probes. C, ChIP assay was performed with HEK293T and HepG2 cells. Immunoprecipitation of samples was performed with anti-ELK1, anti-SAP1a, or anti-SRF antibodies. A positive control of transcriptionally active genes was performed using anti-RNA-polymerase II antibody, and a negative reaction was included using a nonspecific IgG antibody or in the absence of antibody. The relative -fold enrichment of ELK1, SAP1a, or SRF binding sites is compared with the value of no antibody, which is set to 0. Representative quantitative PCRs of three independent experiments are shown.

FIGURE 6.

Knockdown experiments confirm that SAP1a regulates INSIG2 promoter activity. HepG2 cells were transfected with siRNA targeting ELK1 and SAP1a, and total mRNA was extracted. The level of knockdown was analyzed at the mRNA level using quantitative PCR for ELK1 or SAP1a (A). The effect of RNA interference was evaluated by measuring endogenous INSIG2 and SREBP1 gene expression (B). All data were normalized to GAPDH expression. The asterisks indicate a significant difference (*, p < 0.05) compared with cells with the negative siRNA control used as calibrator. The knockdown effect on ELK1 and SAP1a protein levels was analyzed by Western blot (C). Nuclear extracts from HepG2 cells transfected with siRNA targeting ELK1 or SAP1a were fractionated by SDS-PAGE, and immunoblots were developed using anti-ELK1, anti-SAP1a, and anti-actin antibodies. A representative blot of three experiments is shown. a.u., arbitrary units.

FIGURE 7.

The phosphorylated form of SAP1a regulates the human promoter. A construct containing the minimal human INSIG2 promoter region linked to a luciferase reporter plasmid was analyzed in HepG2 cells cotransfected with pcDNA-ELK1 or pcDNA-SAP1a expression vectors and increasing amounts of expression vector for a permanent active Ras protein (A) or pcDNA-SAP1a or SAP1a DBD (dominant negative form) in the presence or absence of 50 ng/well K-Ras expression vector (B). After cell lysis, firefly and Renillla luciferase activities were measured. Results represent relative firefly/Renilla luciferase activities. Values are the mean ± S.E. from four separate experiments.

FIGURE 8.

Insulin regulates human INSIG2 promoter activity. Wild type −347/−11 INSIG2 promoter construct (INSIG2–300) or a mutated construct where the SAP1a binding site is mutated by in vitro mutagenesis (mSAP1a) was transfected into rat hepatocytes for 24 h. Cells were incubated in Dulbecco's modified Eagle's medium containing low (1 g/liter) or high (4.5 g/liter) glucose in the absence (basal) or presence of 100 nm insulin (Ins). After 24 h, the cells were lysed for luciferase assays. The relative firefly/Renilla luciferase activities of the constructs as well as their response to glucose or insulin were compared with the activity of the wild-type construct with low glucose, which was set to 1 ± S.E. Values are the mean ± S.E. from four separate experiments. The asterisks indicate significant difference (*, p < 0.05), as determined using Student's t test.

FIGURE 9.

Palmitic acid prevents insulin stimulation of INSIG2 promoter activity. Human hepatocytes were treated with 100 nm insulin and/or 400 μm palmitic acid to induce insulin resistance. Total RNA was extracted, and endogenous INSIG2b and SREBP1c gene expression was analyzed by RT-quantitative PCR. Glucokinase (GK) expression was used as a control of insulin action. All data were normalized to GAPDH expression.