CCAAT/Enhancer-binding Protein-β Is a Mediator of the Nutrient-sensing Response Pathway That Activates the Human Asparagine Synthetase Gene

Fai Siu,Chin Chen,Can Zhong,Michael S Kilberg

doi:10.1074/jbc.m109533200

Abstract

Transcription from the human asparagine synthetase (AS) gene is increased in response to either amino acid (amino acid response) or glucose (unfolded protein response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the AS promoter, which are referred to as nutrient-sensing response element (NSRE)-1 and -2, both of which are absolutely necessary for gene activation. The NSRE-1 sequence was used to identify the corresponding transcription factor by yeast one-hybrid screening. Based on those results, electrophoretic mobility shift assays for individual CCAAT/enhancer-binding protein-beta (C/EBP) family members were performed to test for supershifting of complexes by specific antibodies. The results indicated that of all the family members, C/EBPbeta bound to the NSRE-1 sequence to the greatest extent and that the absolute amount of this complex was increased when extracts from amino acid- or glucose-deprived cells were tested. Using electrophoretic mobility shift assays, mutation of the NSRE-1 sequence completely prevented formation of the C/EBPbeta-containing complexes. In contrast, mutation of the NSRE-2 sequence did not block C/EBPbeta binding. Overexpression in HepG2 hepatoma cells of the activating isoform of C/EBPbeta increased AS promoter-driven transcription, whereas the inhibitory dominant-negative isoform of C/EBPbeta blocked enhanced transcription following amino acid or glucose deprivation. Collectively, the results provide both in vitro and in vivo evidence for a role of C/EBPbeta in the transcriptional activation of the AS gene in response to nutrient deprivation.

Highlights

Amino acid availability is clearly an important factor in general nutrition, during development [1], as well as in the progression of a wide range of diseases, including diabetes [2], kwashiorkor [3], and hepatic encephalopathy [4], and in cancer chemotherapy
Yeast One-hybrid Screening—To identify the transcription proteins that bound the nutrient-sensing response element (NSRE)-1 sequence, yeast one-hybrid screening was performed using three copies of the asparagine synthetase (AS) promoter NSRE-1 sequence placed in front of two independent reporter systems (HIS3 and LacZ) and a human pancreatic cDNA library fused to the Gal4 activation domain
The results presented in this report document that CCAAT/enhancer-binding protein-␤ (C/EBP)␤ binds to the NSRE-1 cis-element within the proximal promoter of the human AS gene and is involved in the activation of the gene in response to nutrient deprivation

Summary

Introduction

Amino acid availability is clearly an important factor in general nutrition, during development [1], as well as in the progression of a wide range of diseases, including diabetes [2], kwashiorkor [3], and hepatic encephalopathy [4], and in cancer chemotherapy. Induction of the AS activity occurs in temperature-sensitive Chinese hamster ovary cell mutants for asparaginyl-, leucyl-, methionyl-, and lysyl-tRNA synthetases [10] Consistent with these data, starvation of HeLa or HepG2 hepatoma cells for any one of a number of individual amino acids causes the accumulation of AS mRNA, documenting that this nutrient-sensing mechanism broadly detects amino acid availability [11,12,13]. Through the use of in vivo footprinting and single-nucleotide mutagenesis, it was demonstrated that the promoter sequence 5Ј-TGATGAAAC-3Ј (nt Ϫ68 to Ϫ60), the region first identified by Guerrini et al [14], is responsible for induction of the AS gene following activation of the UPR pathway [17]. Consistent with this proposal, Jousse et al [13] have documented that transcriptional control of a reporter gene driven by the AS and CHOP promoters responds differently when cells are deprived of selected individual amino acids

Methods

Results

Conclusion