Differences in the Molecular Mechanisms Involved in the Transcriptional Activation of the CHOP and Asparagine Synthetase Genes in Response to Amino Acid Deprivation or Activation of the Unfolded Protein Response

Alain Bruhat,Julien Averous,Valérie Carraro,Can Zhong,Andreas M Reimold,Michael S Kilberg,Pierre Fafournoux

doi:10.1074/jbc.m206149200

Abstract

A promoter element called the amino acid response element (AARE), which is essential for the induction of CHOP (a CCAAT/enhancer-binding protein-related gene) transcription by amino acid depletion, has been previously characterized. Conversely, the human asparagine synthetase (AS) promoter contains two cis-acting elements termed nutrient-sensing response elements (NSRE-1 and NSRE-2) that are required to activate the gene by either amino acid deprivation or the endoplasmic reticulum stress response. The results reported here document the comparison between CHOP and AS transcriptional control elements used by the amino acid pathway. We first establish that the AS NSRE-1 sequence shares nucleotide sequence and functional similarities with the CHOP AARE. However, we demonstrate that the CHOP AARE can function independently, whereas AS NSRE-1 is functionally weak by itself and instead requires the presence of NSRE-2. Furthermore, AS NSRE-2 can confer endoplasmic reticulum stress responsiveness to the CHOP AARE. Using activating transcription factor-2-deficient mouse embryonic fibroblasts, we also show that lack of this transcription factor does not abolish the amino acid inducibility of AS transcription, but this transcription factor is necessary to obtain the full AS response to amino acid starvation. Collectively, these results document that there are significant differences in the molecular mechanisms involved in the transcriptional activation of CHOP and AS by amino acid limitation.

Highlights

The molecular mechanisms involved in the control of gene expression in response to amino acid deprivation have been extensively studied in yeast [1, 2]
The human CHOP gene 5Ј-flanking region contains an amino acid response element (AARE) that is required for increasing transcription of the gene following amino acid deprivation
Our single-nucleotide mutagenesis affecting the 9-bp core sequence demonstrated that the T residues at positions 2, 3, and 7; the C at position 5; and the A at position 9 are required to confer amino acid responsiveness and established that the minimum core consensus sequence is 5Ј-(R/C)TT(R/ T)CRTCA-3Ј

Summary

Introduction

The molecular mechanisms involved in the control of gene expression in response to amino acid deprivation have been extensively studied in yeast [1, 2]. Most of the results have been obtained studying the transcriptional regulation of asparagine synthetase (AS) and CHOP (C/EBP homologous protein) gene expression in response to amino acid deprivation. Gel shift assays, and in vivo footprinting experiments, Barbosa-Tessmann et al demonstrated that two ciselements termed nutrient-sensing response elements (NSRE-1, 5Ј-TGATGAAAC-3Ј, nt Ϫ68 to Ϫ60; and NSRE-2, 5Ј-GTTACA3Ј, nt Ϫ48 to Ϫ43) in the AS promoter sequence are essential for transcriptional activation by amino acid limitation or the ERSR. An amino acid response element (AARE) was localized between nucleotides Ϫ313 and Ϫ295 in the CHOP promoter This 19-bp DNA control element, which is essential for amino acid activation of the CHOP promoter, can regulate a basal promoter in response to starvation of several individual amino acids [25]. It has recently been hypothesized that the transcription factor ATF-4 could be involved in the amino acid regulation of CHOP expression [26]

Methods

Results

Conclusion