| Mammals convert excess carbohydrates into the preferred energy storage form of triglycerides through the process of de novo lipogenesis for times of fasting. A number of genes encoding enzymes involved in this process are stimulated transcriptionally by increased glucose metabolism. The carbohydrate response element (ChoRE), which is responsible for the transcriptional response to glucose, has been mapped within the promoter regions of several lipogenic enzyme genes. The ChoRE contains two E box-like sequences that are separated by five base pairs. A basic helix-loop-helix/leucine zipper (bHLH/LZ) protein, Carbohydrate Response Element Binding Protein (ChREBP), was purified by DNA affinity chromatography using a ChoRE sequence. In my thesis, I demonstrated that ChREBP requires a binding partner Mlx (M&barbelow;ax l&barbelow;ike protein X&barbelow;) to function as the transcription factor in glucose signaling. ChREBP only bound to the ChoRE sequence in the presence of Mlx and binding of ChREBP/Mlx could discriminate between functional ChoREs and non-functional mutants. In addition, overexpressing dominant negative Mlx in hepatocytes inhibited the glucose response from a ChoRE-containing promoter as well as endogenous glucose-responsive genes. Using microarray analysis, I identified glucose-responsive genes and genes repressed by dominant negative Mlx in liver. Eighty percent of glucose-responsive genes overlapped with the list of genes repressed by dominant negative Mlx. These genes include not only all aspects of de novo lipogenesis, but also are involved in several other metabolic pathways. I conclude that ChREBP/Mlx acts as the major mediator that transcriptionally regulates glucose-responsive genes in hepatocytes. Moreover, I demonstrated that ChREBP/Mlx bound to the ChoRE as a tetramer with one heterodimer occupying each of the E boxes in the ChoRE. Molecular modeling and mutation studies indicated the residues in the loop region of Mlx, but not ChREBP, mediated the interaction between the two heterodimers. This tetramer formation was essential for supporting the glucose-response. Therefore, ChREBP/Mlx is the major transcription factor in glucose-signaling pathway and functions as a tetramer to mediate glucose-responsive transcriptional activation in liver. |
