Transcriptional Repressor E4-binding Protein 4 (E4BP4) Regulates Metabolic Hormone Fibroblast Growth Factor 21 (FGF21) during Circadian Cycles and Feeding

Abstract

Fibroblast growth factor 21 (FGF21) is a potent antidiabetic and triglyceride-lowering hormone whose hepatic expression is highly responsive to food intake. FGF21 induction in the adaptive response to fasting has been well studied, but the molecular mechanism responsible for feeding-induced repression remains unknown. In this study, we demonstrate a novel link between FGF21 and a key circadian output protein, E4BP4. Expression of Fgf21 displays a circadian rhythm, which peaks during the fasting phase and is anti-phase to E4bp4, which is elevated during feeding periods. E4BP4 strongly suppresses Fgf21 transcription by binding to a D-box element in the distal promoter region. Depletion of E4BP4 in synchronized Hepa1c1c-7 liver cells augments the amplitude of Fgf21 expression, and overexpression of E4BP4 represses FGF21 secretion from primary mouse hepatocytes. Mimicking feeding effects, insulin significantly increases E4BP4 expression and binding to the Fgf21 promoter through AKT activation. Thus, E4BP4 is a novel insulin-responsive repressor of FGF21 expression during circadian cycles and feeding.The mammalian circadian rhythm system plays a fundamental role in coordinating various physiological processes, which are manifested by a precise 24-h cycle and responsiveness to light or food cues (1-4). Recent genetic and biochemical studies of mammals, Drosophila, and bacteria have provided a general model of the circadian clock that is based on a transcriptional-translational feedback loop consisting of both positive and negative circadian clock proteins (1, 4). Besides controlling the core circadian oscillation loop, the clock proteins also actively participate in rhythmic expression of various output genes, which may account for the rhythmic activities in peripheral tissues (1, 3). As demonstrated in various microarray studies, genes important for gluconeogenesis, lipogenesis, and cholesterol synthesis are potential targets of clock proteins (5-8). Therefore, for drug administration and drug design, it becomes critical to understand how the cycling of individual metabolic genes is regulated in a 24-h rhythm (9, 10). E4BP4 (E4-binding protein 4), also called NFIL3, is a b-ZIP (basic leucine zipper) transcription factor initially identified as an IL-3-inducible factor in pro-B lymphocytes (11-13). The biological function of E4BP4 has been largely explored in the immune system, in which E4BP4 knock-out mice are defective in natural killer cell development and IgE class switch (14 -16). E4BP4 was first identified as a clock-controlled gene in mouse liver (17,18). Its mRNA and protein levels oscillate in a circadian fashion, which is anti-phase to DBP (D-site of albumin promoter-binding protein), another clock-controlled output gene (19). The mRNA of E4BP4 peaks at circadian time (CT) 2 0 and troughs at CT 12 (17,20). Although the role of E4BP4 in the mammalian circadian system is unclear, its homologue in Drosophila, vrille, serves as a key component of the core circadian network via a negative feedback loop (21-23). E...

Recruitment of Histone Methyltransferase G9a Mediates Transcriptional Repression of Fgf21 Gene by E4BP4 Protein*

“…Previously, we identified Fgf21 as a direct circadian target of E4BP4 in hepatocytes (9). In the present study, we demonstrate that E4BP4 is an in vivo repressor of Fgf21 transcription during fasting-refeeding cycles.…”

“…Plasmids and Adenovirus Generation-Both E4BP4 overexpression and shRNA vectors were reported before (9). The adenovirus for Ad-E4bp4 (overexpression), Ad-shE4bp4 (knockdown), and Ad-shG9a (knockdown) were generated using the pAdEasy system (Agilent) and pAd-Block-it system (Invitrogen).…”

“…4D). We previously showed that depletion of E4bp4 expression leads to dysregulated circadian oscillations of Fgf21 mRNA in synchronized Hepa1 cells (9). To examine the role of G9a in Fgf21 oscillations, we used Ad-shG9a to deplete G9a and synchronized Hepa1 cells for mRNA measurement.…”

“…So far, few repressors of Fgf21 gene expression are known. Our laboratory previously reported that the basic leucine zipper transcription factor E4BP4 represses Fgf21 upon insulin stimulation and is critical for circadian oscillations of Fgf21 (9). However, the epigenetic program underlying the E4BP4-dependent repression of Fgf21 remains unknown.…”

The Nuclear Receptor Rev-erbα Regulates Adipose Tissue-specific FGF21 Signaling

“…Previous studies have identified a clear link between the molecular clock and hepatic FGF21 signaling (60,61), including suppression of Fgf21 transcription by E4BP4, which is a direct Rev-erb␣ target in liver (60). In addition, Rev-erb␣ has been shown to modulate FGF21 expression in liver (62).…”