Activation of the ATF3 gene through a co-ordinated amino acid-sensing response programme that controls transcriptional regulation of responsive genes following amino acid limitation

Abstract

Expression of ATF3 (activating transcription factor 3) is induced by a variety of environmental stress conditions, including nutrient limitation. In the present study, we demonstrate that the increase in ATF3 mRNA content following amino acid limitation of human HepG2 hepatoma cells is dependent on transcriptional activation of the ATF3 gene, through a highly co-ordinated amino acid-responsive programme of transcription factor synthesis and action. Studies using transient over-expression and knockout fibroblasts showed that several ATF and C/EBP (CCAAT/enhancer-binding protein) family members contribute to ATF3 regulation. Promoter analysis showed that a C/EBP-ATF composite site at -23 to -15 bp relative to the transcription start site of the ATF3 gene functions as an AARE (amino acid response element). Chromatin immunoprecipitation demonstrated that amino acid limitation increased ATF4, ATF3, and C/EBPbeta binding to the ATF3 promoter, but the kinetics of each was markedly different. Immediately following histidine removal, there was a rapid increase in histone H3 acetylation prior to an enhancement in ATF4 binding and in histone H4 acetylation. These latter changes closely paralleled the initial increase in RNA pol II (RNA polymerase II) binding to the promoter and in the transcription rate from the ATF3 gene. The increase in ATF3 and C/EBPbeta binding was considerably slower and more closely correlated with a decline in transcription rate. A comparison of the recruitment patterns between ATF and C/EBP transcription factors and RNA polymerase II at the AARE of several amino acid-responsive genes revealed that a highly co-ordinated response programme controls the transcriptional activation of these genes following amino acid limitation.

A Bifunctional Intronic Element Regulates the Expression of the Arginine/Lysine Transporter Cat-1 via Mechanisms Involving the Purine-rich Element Binding Protein A (Purα)

“…The element is identical to the AARE in the ATF3 promoter (30) and has only one mismatch with the Cat-1 AARE. The Cat-1 AARE and INE bind ATF4, but only the INE binds Pur␣ (data not shown), suggesting that Pur␣ binds to regions in the INE adjacent to the AARElike sequence.…”

“…The first intron also contains a TG repeat near the INE ((TG) 30 in the rat and (TG) 12 in the mouse) 59 and 57 bp downstream of the 5Ј-end of the first intron, respectively. Dinucleotide repeats of length Ն12 are found mostly in the introns of inefficiently transcribed genes.…”

Elevated ATF4 Expression, in the Absence of Other Signals, Is Sufficient for Transcriptional Induction via CCAAT Enhancer-binding Protein-activating Transcription Factor Response Elements

“…During the second stage of the ATF4 response, increased expression of ATF3, C/EBP␤, and CHOP leads to their binding at the CARE site, which causes a subsequent suppression of transcription back toward the basal level. This self-limiting model for ATF4 action, originally reported for asparagine synthetase (ASNS), has been confirmed for several other CARE-containing genes (14,18).…”

“…To determine whether the difference in ASNS control by ectopically expressed versus endogenous ATF4 was also true for other AAR element-driven genes, the mRNA content was analyzed for C/EBP␤ (15), sodium-dependent neutral amino acid transporter 2 (SNAT2) (26), ATF3 (14), and CHOP (20), all known to be induced by amino acid deprivation via ATF4 (Fig. 5).…”

“…This binding is associated with localized histone acetylation and subsequent recruitment of the general transcriptional machinery. Among the ATF4 target genes are the transcription factors ATF3 (13,14), CCAAT-enhancer binding protein ␤ (C/EBP␤) (15,16), and C/EBP homology protein (CHOP) (17). During the second stage of the ATF4 response, increased expression of ATF3, C/EBP␤, and CHOP leads to their binding at the CARE site, which causes a subsequent suppression of transcription back toward the basal level.…”