Identification of factors that interact with the E1A-inducible adenovirus E3 promoter.

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.

Section: Discussionmentioning

confidence: 99%

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

Bruhat¹,

Avérous²,

Carraro³

et al. 2002

“…5B), which corresponds to Ϫ327/Ϫ322 (3Ј-AGTCAC-5Ј) of the mGnRHR gene promoter. These data therefore define a novel AP-1 binding element in the 5Ј-flanking sequence of the mGnRHR gene (Table I) (42)(43)(44)(45)(46)(47)(48)(49)(50).…”

Section: Discussionmentioning

confidence: 99%

Direct Binding of AP-1 (Fos/Jun) Proteins to a SMAD Binding Element Facilitates Both Gonadotropin-releasing Hormone (GnRH)- and Activin-mediated Transcriptional Activation of the Mouse GnRH Receptor Gene

Norwitz¹,

Xu²,

Xu³

et al. 2002

The response of pituitary gonadotropes to gonadotropin-releasing hormone (GnRH) correlates directly with the concentration of GnRH receptors (GnRHR) on the cell surface, which is mediated in part at the level of gene expression. Several factors are known to affect expression of the mouse GnRHR (mGnRHR) gene, including GnRH and activin. We have previously shown that activin augments GnRH-mediated transcriptional activation of mGnRHR gene, and that region ؊387/؊308 appears to be necessary to mediate this effect. This region contains two overlapping cis-regulatory elements of interest: GnRHR activating sequence (GRAS) and a putative SMAD-binding element (SBE). This study investigates the role of these elements and their cognate transcription factors in transactivation of the mGnRHR gene. Transfection studies confirm the presence of GnRH-and activin-response elements within ؊387/؊308 of mGnRHR gene promoter. Competition electrophoretic mobility shift assay experiments using ؊335/ ؊312 as probe and ␣T3-1 nuclear extract or SMAD, Jun, and Fos proteins demonstrate direct binding of AP-1 (Fos/Jun) protein complexes to ؊327/؊322 and SMAD proteins to ؊329/؊328. Further transfection studies using mutant constructs of these cis-regulatory elements confirm that both are functionally important. These data define a novel cis-regulatory element comprised of an overlapping SBE and newly characterized non-consensus AP-1 binding sequence that integrates the stimulatory transcriptional effects of both GnRH and activin on the mGnRHR gene.

“…Each mobility shift experiment was repeated three times to confirm the reproducibility of the results. The sequences of the probes were: CHOP-AARE (5Ј-aacattgcatcatccccgc-3Ј), ATF binding site (5Ј-atccggtgacgtcacggggg-3Ј) (23), and C/EBP binding site (5Ј-tgcagattgcgcaatctgca-3Ј) (24).…”

Section: Methodsmentioning

confidence: 99%

Induction of CHOP Expression by Amino Acid Limitation Requires Both ATF4 Expression and ATF2 Phosphorylation

Avérous

Bruhat

Jousse

et al. 2004

241

214

The CHOP gene is transcriptionally induced by amino acid starvation. We have previously identified a genomic cis-acting element (amino acid response element (AARE)) involved in the transcriptional activation of the human CHOP gene by leucine starvation and shown that it binds the activating transcription factor 2 (ATF2). The present study was designed to identify other transcription factors capable of binding to the CHOP AARE and to establish their role with regard to induction of the gene by amino acid deprivation. Electrophoretic mobility shift assay and transient transfection experiments show that several transcription factors that belong to the C/EBP or ATF families bind the AARE sequence and activate transcription. Among all these transcription factors, only ATF4 and ATF2 are involved in the amino acid control of CHOP expression. We show that inhibition of ATF2 or ATF4 expression impairs the transcriptional activation of CHOP by amino acid starvation. The transacting capacity of ATF4 depends on its expression level and that of ATF2 on its phosphorylation state. In response to leucine starvation, ATF4 expression and ATF2 phosphorylation are increased. However, induction of ATF4 expression by the endoplasmic reticulum stress pathway does not fully activate the AARE-dependent transcription. Taken together our results demonstrate that at least two pathways, one leading to ATF4 induction and one leading to ATF2 phosphorylation, are necessary to induce CHOP expression by amino acid starvation. This work was extended to the regulation of other amino acid regulated genes and suggests that ATF4 and ATF2 are key components of the amino acid control of gene expression.