Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor that activates genes involved in de novo lipogenesis in mammals. The current model for glucose activation of ChREBP proposes that increased glucose metabolism triggers a cytoplasmic to nuclear translocation of ChREBP that is critical for activation. However, we find that ChREBP actively shuttles between the cytoplasm and nucleus in both low and high glucose in the glucose-sensitive β cell-derived line, 832/13. Glucose stimulates a three-fold increase in the rate of ChREBP nuclear entry, but trapping ChREBP in the nucleus by mutagenesis or with a nuclear export inhibitor does not lead to constitutive activation. In fact, mutational studies targeting the nuclear export signal of ChREBP also identified a distinct function essential for glucose-dependent transcriptional activation. From this, we conclude that an additional event independent of nuclear translocation is required for activation.
Glucose regulation of ChREBP has been mapped to its conserved N-terminal region of 300 amino acids, designated the MondoA Conserved Region (MCR). Within the MCR, five domains (MCRs 1-5) have a particularly high level of conservation and are likely to be important for glucose regulation. We carried out a large-scale deletion and substitution mutational analysis of the MCR domain of ChREBP. This analysis revealed that MCRs 1-4 function in a concerted fashion to repress ChREBP activity in basal (non-stimulatory) conditions. Deletion of the entire MCR 1-4 segment or combining four specific point mutations (Quad mutant) throughout this region lead to a highly active, glucose-independent form of ChREBP. However, deletion of any individual MCR domain and the majority of point mutations throughout MCRs 1-4 rendered ChREBP inactive. These observations suggest that MCRs 1-4 interact with a factor required for activation and that this interaction occurs after repression is relieved. This possibility is supported by the observation that the MCR 1-4 region can compete for activity with wild type ChREBP and the derepressed Quad mutant in both basal and stimulatory conditions. Thus, the MCR domains act in a complex and coordinated manner to regulate ChREBP activity in response to glucose.