Title

In vivo mechanism of retinoid regulation of phosphoenolpyruvate carboxykinase gene expression in liver

Date of Completion

January 2004

Keywords

Biology, Molecular|Health Sciences, Nutrition

Degree

Ph.D.

Abstract

Vitamin A and its metabolites regulate the expression of many genes involved in their own metabolism and transport and thus have a global impact on growth, development and cellular differentiation. Phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme whose gene is responsive to numerous hormonal signals, including retinoids. Retinoid regulation of a metabolic gene is a relatively new finding, suggesting a novel role for this nutrient's involvement in intermediary metabolism. ^ Within the complex promoter/regulatory domain of the PEPCK gene, three retinoic acid response elements (RAREs) mediate PEPCK's response to retinoids. However, the specific protein/DNA and protein/protein binding events that occur at the RAREs in vivo remain undefined. ^ In these studies, the mechanism of nuclear protein assembly at the PEPCK promoter was examined in vitro by electrophoretic mobility shift assay (EMSA) and in vivo by chromatin immunoprecipitation (ChIP) in mouse liver. Differences in nuclear receptor and coregulator binding patterns, histone acetylation and subsequent linkage to the transcription start site were observed in vitamin A-sufficient (VAS) and vitamin A-deficient (VAD) mice. Specifically, steroid receptor coactivator-1 (SRC-1), CREB binding protein (CBP) and peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), were associated with PEPCK RARE1/RARE2, likely as part of a larger multiprotein complex. SRC-1 and PGC-1α association was decreased with vitamin A-deficiency. This was correlated with a decrease in acetylation of specific lysines on histone H3. Additionally, PPARα binding to upstream and downstream RAREs was decreased with vitamin A-deficiency, indicative of the potential loss of a coactivator docking site. RNA polymerase II association with the PEPCK promoter was significantly decreased in VAD mice, and was restored, as was PEPCK mRNA expression, with physiological doses of all-trans and 9-cis retinoic acid. ^ This is the first report of an in vivo mechanism of retinoid regulation of a gene involved in carbohydrate metabolism, from nuclear receptors binding specific RAREs to the localized histone code of the PEPCK gene. These data suggest that disruption of the multiprotein coactivator complex that bridges the PEPCK RAREs and the preinitiation complex is the cause of decreased histone acetylation, the interruption in RNA polymerise II association and decreased PEPCK gene expression in vitamin A-deficiency. ^