DAX-1 Acts as a Novel Corepressor of Orphan Nuclear Receptor HNF4α and Negatively Regulates Gluconeogenic Enzyme Gene Expression

Balachandar Nedumaran,Sungpyo Hong,Yuan-Bin Xie,Yong-Hoon Kim,Woo-Young Seo,Min-Woo Lee,Chul Ho Lee,Seung-Hoi Koo,Hueng-Sik Choi

doi:10.1074/jbc.m109.034660

Abstract

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is an atypical member of the nuclear receptor family and acts as a corepressor of a number of nuclear receptors. HNF4alpha (hepatocyte nuclear factor 4alpha) is a liver-enriched transcription factor that controls the expression of a variety of genes involved in cholesterol, fatty acid, and glucose metabolism. Here we show that DAX-1 inhibits transcriptional activity of HNF4alpha and modulates hepatic gluconeogenic gene expression. Hepatic DAX-1 expression is increased by insulin and SIK1 (salt-inducible kinase 1), whereas it is decreased in high fat diet-fed and diabetic mice. Coimmunoprecipitation assay from mouse liver samples depicts that endogenous DAX-1 interacts with HNF4alpha in vivo. In vivo chromatin immunoprecipitation assay affirms that the recruitment of DAX-1 on the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is inversely correlated with the recruitment of PGC-1alpha and HNF4alpha under fasting and refeeding, showing that DAX-1 could compete with the coactivator PGC-1alpha for binding to HNF4alpha. Adenovirus-mediated expression of DAX-1 decreased both HNF4alpha- and forskolin-mediated gluconeogenic gene expressions. In addition, knockdown of DAX-1 partially reverses the insulin-mediated inhibition of gluconeogenic gene expression in primary hepatocytes. Finally, DAX-1 inhibits PEPCK and glucose-6-phosphatase gene expression and significantly lowers fasting blood glucose level in high fat diet-fed mice, suggesting that DAX-1 can modulate hepatic gluconeogenesis in vivo. Overall, this study demonstrates that DAX-1 acts as a corepressor of HNF4alpha to negatively regulate hepatic gluconeogenic gene expression in liver.

Highlights

The nuclear receptor superfamily comprises a diverse group of transcription factors including conventional receptors with known ligands and orphan nuclear receptors without ligands [1, 2]
It is well established that DAX-1 functions as a coregulatory protein rather than a transcriptional factor, since it inhibits the transcriptional activity of other nuclear receptors, such as steroidogenic factor 1 [10], estrogen receptors (ERs) [9], androgen receptor [11], progesterone receptor [12], liver receptor homolog-1 [13], peroxisome proliferator-activated receptor ␥ (PPAR␥) [14], estrogen receptor-related receptor ␥ (ERR␥) [15], and Nur77 [16]
Our Western blot analysis in HepG2 cells showed that siRNA 2 significantly reduced the endogenous expression of DAX-1, whereas siRNA 1 did not alter the expression of endogenous DAX-1 (Fig. 1E)

Summary

Introduction

The nuclear receptor superfamily comprises a diverse group of transcription factors including conventional receptors with known ligands and orphan nuclear receptors without ligands [1, 2]. We have observed that DAX-1 inhibits the transcriptional activity of HNF4␣ to control hepatic gluconeogenic gene expression. To determine whether DAX-1 affects the transcriptional activity of HNF4␣, we performed transient transfection assay using a reporter gene containing multiple HNF4␣ binding site (8ϫ(HNF4␣)RE-Luc) and expression vectors for HA-hnf4␣ and FLAG-DAX-1 in HepG2 and 293T cells.

Results

Conclusion