Glucose controls nuclear accumulation, promoter binding, and transcriptional activity of the MondoA-Mlx heterodimer

Abstract

Maintenance of energy homeostasis is a fundamental requirement for organismal fitness: defective glucose homeostasis underlies numerous metabolic diseases and cancer. At the cellular level, the ability to sense and adapt to changes in intracellular glucose levels is an essential component of this strategy. The basic helix-loop-helix-leucine zipper (bHLHZip) transcription factor complex MondoA-Mlx plays a central role in the transcriptional response to intracellular glucose concentration. MondoA-Mlx complexes accumulate in the nucleus in response to high intracellular glucose concentrations and are required for 75% of glucose-induced transcription. We show here that, rather than simply controlling nuclear accumulation, glucose is required at two additional steps to stimulate the transcription activation function of MondoA-Mlx complexes. Following nuclear accumulation, glucose is required for MondoA-Mlx occupancy at target promoters. Next, glucose stimulates the recruitment of a histone H3 acetyltransferase to promoter-bound MondoA-Mlx to trigger activation of gene expression. Our experiments establish the mechanistic circuitry by which cells sense and respond transcriptionally to various intracellular glucose levels.

FIG. 1.

MondoA is regulated by glucose in L6 myoblast cells. (A) TXNIP promoter occupancy by MondoA in L6 cells grown in complete media (Con) or glucose-starved overnight and treated for 3 h with glucose-free media (−), 20 mM 2-deoxyglucose (2DOG), or 20 mM glucose (Glu). (B) Activity of a TXNIP-luciferase reporter plasmid in L6 cells cotransfected with expression plasmids as indicated, starved overnight for glucose, and incubated for 7 h in the absence or presence of 25 mM glucose. (C) TXNIP protein expression in whole-cell lysates from L6 cells treated overnight in the absence or presence of 25 mM glucose. (D) Glucose uptake in L6 cells infected with retroviral vector alone or ΔN237NLS MondoA.

FIG. 2.

Nuclear accumulation of MondoA is regulated by Mlx binding and glucose. (A to C) Quantified subcellular localization of V5 tag in L6 cells transfected with the indicated MondoA-V5 constructs and treated as indicated. (A) wt MondoA or MondoA(I766P) treated for 4 h with leptomycin B (LMB). (B) wt MondoA or MondoA(M133A/I766P), starved overnight for glucose and treated for 3 h in the presence or absence of 2DOG. (C) wt MondoA, starved overnight for glucose and treated for 4 h in the presence or absence of 2DOG and LMB. (D) wt MondoA or MondoA(M133A) and Gal4 vector alone or Gal4-Crm1, starved overnight for glucose and treated for 3 h with 2DOG. Only cells with predominantly nuclear MondoA were scored.

FIG. 3.

Glucose-dependent activities of MondoA require the MCR and bHLHZip domains. (A) Asterisks denote point mutations in MondoA MCRI (H78A/H81A/H88A), MCRII (M133A), MCRIII (I166A/W167A/R168A), MCRIV (Y210D/W211D/K212D), or the bHLHZip domain (I766P or H724P). MCRV is a deletion of amino acids 282 to 324. DCD, dimerization and cytoplasmic localization domain. (B to D) Localization or activity of MondoA-V5 mutants in L6 cells, normalized to wt. (B) Nuclear localization of the indicated mutants, with 2DOG treatment. (C) TXNIP luciferase activity of the indicated mutants, with glucose treatment. (D) Nuclear localization of the indicated mutants, with LMB treatment.

FIG. 4.

MCRII controls localization and activity of MondoA-Mlx. MEFs from control mice (wt), MondoA KO, and KO cells rescued with retrovirally expressed wt MondoA (KO + wt) or MCRII mutants (KO + L129A mutant, KO + F130A mutant, KO + M133A mutant, and KO + ΔMCRII mutant) were measured for protein expression with the indicated antibodies (A), glucose uptake (B), and subcellular localization of MondoA in the presence or absence of 2DOG (C). Asterisk indicates nonspecific background band.

FIG. 5.

Nuclear accumulation of MondoA is not sufficient for promoter occupancy. (A) MondoA occupancy at the TXNIP promoter E box in wt MondoA, KO, and KO + rescue MEFs treated overnight in the presence or absence of glucose. (B) Experiments were performed as described for panel A except for the use of ARRDC4 promoter primers.

FIG. 6.

Glucose-dependent activation of TXNIP involves MondoA MCRII-dependent acetylation of histone H3. MondoA KO, KO + wt MondoA, and KO + MondoA(ΔMCRII) MEFs incubated overnight in the presence or absence of glucose and assayed for TXNIP luciferase activity (A) and H3K4me3 (B) or acetylated histone H3 (C) modifications at TXNIP exon 1.

FIG. 7.

Model of glucose-dependent regulation of MondoA. MondoA-Mlx heterodimers shuttle between cytoplasm and nucleus in the presence and absence of glucose (dashed lines). Intracellular glucose is metabolized to glucose-6-phosphate (G6P), which we propose as a MondoA signaling molecule that promotes nuclear accumulation (A), promoter occupancy (B), and recruitment of HAT coactivators (C) by MondoA-Mlx, in turn activating target genes, such as TXNIP.