Regulation of the human GLUT4 gene promoter: interaction between a transcriptional activator and myocyte enhancer factor 2A

Abstract

The GLUT4 gene is subject to complex tissue-specific and metabolic regulation, with a profound impact on insulin-mediated glucose disposal. We have shown, by using transgenic mice, that the human GLUT4 promoter is regulated through the cooperative function of two distinct regulatory elements, domain 1 and the myocyte enhancer factor 2 (MEF2) domain. The MEF2 domain binds transcription factors MEF2A and MEF2D in vivo. Domain I binds a transcription factor, GLUT4 enhancer factor (GEF). In this report, we show a restricted pattern of GEF expression in human tissues, which overlaps with MEF2A only in tissues expressing high levels of GLUT4, suggesting the hypothesis that GEF and MEF2A function together to activate GLUT4 transcription. Data obtained from transiently transfected cells support this hypothesis. Neither GEF nor MEF2A alone significantly activated GLUT4 promoter activity, but increased promoter activity 4- to 5-fold when expressed together. Deletion of the GEF-binding domain (domain I) and the MEF2-binding domain prevented activation, strengthening the conclusion that promoter regulation occurs through these elements. GEF and MEF2A, isolated from nuclei of transfected cells, bound domain I and the MEF2 domain, respectively, which is consistent with activation through these regulatory elements. Finally, GEF and MEF2A coimmunoprecipitated in vivo, strongly supporting a mechanism of GLUT4 transcription activation that depends on this protein-protein interaction.

Fig. 3.

Transcription from the human GLUT4 promoter in a cell-culture system. (A) COS 7 cells were transfected according to Materials and Methods. Each transfection included 500 ng of the human GLUT4 promoter fused to firefly luciferase (hG4-luc), 100 ng of a plasmid used to control transfection efficiency (pRLTk-luc), and 500 ng of various combinations of plasmids encoding transcription factors GEF and/or members of the MEF2 family. Light units expressed from the firefly luciferase gene (GLUT 4 promoter) were corrected for light units expressed from the sea pansy luciferase gene (transfection efficiency reporter, pRLTk-luc). Data were analyzed by using two-way ANOVA. *, statistically significant (P < 0.05) interaction between factors (GEF and MEF2A). (B) Immunoblots of lysates obtained from COS 7 cells transfected with plasmids encoding transcription factors GEF (G), MEF2A (2A), MEF2D (2D), or empty vector (V). Transfected cell lysates were probed by using corresponding antibodies specific for MEF2A, MEF2D, and GEF, respectively.

Fig. 4.

Transcription of human GLUT4 reporter (hG4-luc) or mutant GLUT4 promoter (ΔΔ-hG4-luc) containing deletions of the GEF- and MEF2-binding sites. Transfections were carried out as described in Materials and Methods and Fig. 3. Differences between the reporter constructs were determined with a Student's t test. *, significant difference (P < 0.01).

Fig. 6.

Coprecipitation of GEF and MEF2A in vitro and in vivo. (A) Purified GST-GEF or GST alone were incubated with nuclear extracts from Cos cells expressing either MEF2A or MEF2D. GST fusion proteins were captured by using glutathione agarose beads and washed, and the associated proteins were fractionated by SDS/10% PAGE and Western blot analysis by using anti-MEF2A polyclonal or anti-MEF2D antibody. Lane 7 is 25% of input of the MEF2A-containing lysate and lane 8 is 10% input of the MEF2D-containing lysate. (B) Coimmunoprecipitation of MEF2A and MEF2D using anti-GEF IgG in nuclear extracts expressing exogenous GEF and MEF2A or GEF and MEF2D. As a control for specificity, lysates were precipitated with nonimmune IgG (lanes 2 and 5). Antigen–antibody complexes were captured by using protein A/G plus agarose, washed, and fractionated on SDS/10% PAGE. A sample of each lysate representing 10% of the sample was loaded in lanes 1 and 4 of each Western blot. Blots were labeled with mouse monoclonal antibodies specific for MEF2A or MEF2D. (C) COS 7 cells were transfected as described in Fig. 3. Plasmids encoding GEF, MEF2A, or MEF2D were transfected at a combined total of 500 ng. The data were analyzed by two-way ANOVA. **, the contribution of MEF2A (P < 0.0001); *, significance of the interaction term (MEF2A and MEF2D) (P = 0.0186).

Fig. 1.

Distribution of GEF mRNA using a human multitissue Northern blot probed with a radiolabeled GEFdb cDNA. The blot was stripped and reprobed with an actin probe to control for loading. The commercially obtained Northern blot (Clontech) was loaded with mRNA from the following tissues: lane 1, pancreas; lane 2, kidney; lane 3, skeletal muscle; lane 4, liver; lane 5, lung; lane 6, placenta; lane 7, brain; and lane 8, heart.

Fig. 2.

GEF protein distribution in mouse. Fifty micrograms of mouse white adipose tissue (W), brown adipose tissue (B), heart (H), and skeletal muscle (S) lysates were immunoblotted by using antiserum raised against the N-terminal 161 amino acids of GEF. Control lanes 1 and 2 were obtained from COS 7 cells transfected with vector plasmid (V) or full length GEF cDNA (G).

Fig. 5.

Nuclear localization of transfected GEF and MEF2. A plasmid encoding GFP-GEF or GFP alone was transfected into COS 7 cells. Fixed cells expressing the protein were scanned by using confocal microscopy, and Z sections were compiled to obtain the images shown in A. To-Pro-3 (Molecular Probes) was included during staining to demarcate the nucleus. EMSA of lysates obtained from cells transfected with plasmids expressing vector only (V), GEF (G), MEF2A (2A), and MEF2D (2D) were performed with radiolabeled oligonucleotides corresponding to the domain I-binding site (B) or GLUT4 MEF2-binding site (C). Lysates in B were incubated with anti-GEF IgG (lanes 3 and 5), or with a nonspecific IgG (lanes 2 and 4).