Regulation of Rev-erbalpha's repressive activity and its physiological significance.

摘要

Rev-erbalpha is a constitutive transcriptional repressor and highly expressed in liver, skeletal muscle and fat. Rev-erbalpha has been found as a key negative regulator in circadian clock by directly repressing Bmal1 gene. Besides its function in regulating core clock genes, Rev-erbalpha also regulates time-specific expression of circadian output genes important for function and physiology of peripheral clocks. However, the role of Rev-erbalpha in peripheral clocks has not been fully characterized. Here we studied Rev-erbalpha's physiological function in liver via the gene expression profile in Rev-erbalpha-deficient HepG2 liver hepatoma cells. Our study shows that Rev-erbalpha influences the regulation of several metabolic pathways, such as bile acid and lipid metabolism. The importance of Rev-erbalpha in the control of metabolic homeostasis inspires us to study the regulation of Rev-erbalpha transcriptional activity. Firstly, we identified the endogenous ligand of Rev-erbalpha, heme, and found it can enhance Rev-erbalpha repressive effects on its target, Bmal1 and G6Pase. Rev-erbalpha 602HF mutant (His602 (H602) was mutated to phenylalanine) losing the ability to bind heme has less recruitment of NCoR/HDAC3 complex and impaired repression effects; indicating 602H is critical for the interaction between Rev-erbalpha and Heme. Secondly, we explore whether Rev-erbalpha is involved in a negative feedback loop for its own ligand's synthesis. We found heme binding to Rev-erbalpha recruits the NCoR/HDAC3 corepressor complex to repress the transcription of the coactivator PGC-1alpha, a potent inducer of heme synthesis. This is particularly important for maintaining intracellular heme level in a narrow physiological range and regulating cellular energy metabolism. Finally, to determine the role of post-translational modification of Rev-erbalpha in vivo, we generated C57BL/6 mice homozygous for two point mutations that mimic phosphorylation, form a constitutively phosphorylated mutant and increase Rev-erbalpha protein stability. Rev-erbalpha SD55/59 mice will allow us to probe, for the first time, the specific functions of post-translational modification of Rev-erbalpha in vivo. We expect theses modifications of Rev-erbalpha will contribute to circadian biology, as well as metabolism. These findings collectively indicate that Rev-erbalpha plays an important role in metabolism and its transcriptional activity can be regulated by the ligand availability (Chapter Two and Three) and post-translational modification (Chapter Four).