Elizabeth E. Dudenhausen

Amino Acid Deprivation Induces the Transcription Rate of the Human Asparagine Synthetase Gene through a Timed Program of Expression and Promoter Binding of Nutrient-responsive Basic Region/Leucine Zipper Transcription Factors as Well as Localized Histone Acetylation

Expression of human asparagine synthetase (ASNS), which catalyzes asparagine and glutamate biosynthesis, is transcriptionally induced following amino acid deprivation. Previous overexpression and electrophoresis mobility shift analysis showed the involvement of the transcription factors ATF4, C/EBPβ, and ATF3-FL through the nutrient-sensing response element-1 (NSRE-1) within the ASNS promoter. Amino acid deprivation caused an elevated mRNA level for ATF4, C/EBPβ, and ATF3-FL, and the present study established that the nuclear protein content for ATF4 and ATF3-FL were increased during amino acid limitation, whereas C/EBPβ-LIP declined slightly. The total amount of C/EBPβ-LAP protein was unchanged, but changes in the distribution among multiple C/EBPβ-LAP forms were observed. Overexpression studies established that ATF4, ATF3-FL, and C/EBPβ-LAP could coordinately modulate the transcription from the human ASNS promoter. Chromatin immunoprecipitation demonstrated that amino acid deprivation increased ATF3-FL, ATF4, and C/EBPβ binding to the ASNS promoter and enhanced promoter association of RNA polymerase II, TATA-binding protein, and TFIIB of the general transcription machinery. A time course revealed a markedly different temporal order of interaction between these transcription factors and the ASNS promoter. During the initial 2 h, there was a 20-fold increase in ATF4 binding and a rapid increase in histone H3 and H4 acetylation, which closely paralleled the increased transcription rate of the ASNS gene, whereas the increase in ATF3-FL and C/EBPβ binding was considerably slower and more closely correlated with the decline in transcription rate between 2 and 6 h. The data suggest that ATF3-FL and C/EBPβ act as transcriptional suppressors for the ASNS gene to counterbalance the transcription rate activated by ATF4 following amino acid deprivation.

Human CCAAT/Enhancer-binding Protein β Gene Expression Is Activated by Endoplasmic Reticulum Stress through an Unfolded Protein Response Element Downstream of the Protein Coding Sequence

CCAAT/enhancer-binding protein β (C/EBPβ) is a member of the bZIP family of transcription factors that contribute to the regulation of a wide range of important cellular processes. The data in the present study document that transcription from the human C/EBPβ gene is induced in response to endoplasmic reticulum stress, such as glucose deprivation, or treatment of cells with tunicamycin or thapsigargin. Transient transfection of C/EBPβ genomic fragments linked to a luciferase reporter gene demonstrated that the C/EBPβ promoter plays no major regulatory role. Instead, by deletion analysis it was discovered that a 46-bp region, located at a genomic site that corresponds to the 3′-untranslated region of the C/EBPβ mRNA, harbored an element that was required for the stress response. Mutagenesis demonstrated that a cis-regulatory element located at nt +1614–1621 (5′-TGACGCAA-3′) is responsible for activation of the C/EBPβ gene. Electrophoresis mobility shift analysis revealed that proteins are bound to this element and that the amount of binding is increased following glucose deprivation. This element is homologous to a previously reported mammalian unfolded protein response element that binds XBP-1. Consistent with those data, overexpression of XBP-1 caused an increase in transcription that was mediated by the C/EBPβ mammalian unfolded protein response element.

Deprivation of protein or amino acid induces C/EBPβ synthesis and binding to amino acid response elements, but its action is not an absolute requirement for enhanced transcription

A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPbeta (CCAAT/enhancer-binding protein beta) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPbeta association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPbeta protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPbeta was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPbeta DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBPbeta binding to the AARE region in vivo, but at a time when transcription from the target gene was declining. A constant fraction of the basal and increased C/EBPbeta protein was phosphorylated on Thr(235) and the phospho-C/EBPbeta did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBPbeta-deficient MEFs (mouse embryonic fibroblasts) or C/EBPbeta siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBPbeta-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBPbeta is not required for transcriptional activation by the AAR pathway but, when present, acts in concert with ATF3 (activating transcription factor 3) to suppress transcription during the latter stages of the response.

Auto-activation of c-JUN Gene by Amino Acid Deprivation of Hepatocellular Carcinoma Cells Reveals a Novel c-JUN-mediated Signaling Pathway

Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.

ASSOCIATION OF HEPATIC SYSTEM A AMINO ACID TRANSPORTER WITH THE MEMBRANE-CYTOSKELETAL PROTEINS ANKYRIN AND FODRIN

System A activity is a highly regulated mechanism for the active transport of zwitterionic amino acids into mammalian cells. Monoclonal antibodies generated against a previously unidentified rat liver plasma membrane-associated protein were shown to immunoprecipitate solubilized System A transport activity. The immunoreactive protein was later determined by immunoblotting and peptide microsequencing to be rat liver alpha-fodrin (non-erythroid spectrin). Antibody against ankyrin, a protein that often serves as a bridge between integral membrane proteins and fodrin, also immunoprecipitated System A transport activity. Fractionation of solubilized plasma membrane proteins on sucrose gradients revealed that the System A transporter co-migrated as a complex with fodrin and ankyrin, even in the presence of detergent and urea. In contrast, the System N amino acid transporter does not co-migrate with ankyrin and fodrin, nor does the anti-fodrin antibody immunoprecipitate System N activity. The present data are the first to demonstrate an association between an organic solute transporter and the membranocytoskeletal proteins ankyrin and fodrin.

MAPK signaling triggers transcriptional induction of cFOS during amino acid limitation of HepG2 cells.

Amino acid (AA) deprivation in mammalian cells activates a collection of signaling cascades known as the AA response (AAR), which is characterized by transcriptional induction of stress-related genes, including FBJ murine osteosarcoma viral oncogene homolog (cFOS). The present study established that the signaling mechanism underlying the AA-dependent transcriptional regulation of the cFOS gene in HepG2 human hepatocellular carcinoma cells is independent of the classic GCN2-eIF2-ATF4 pathway. Instead, a RAS-RAF-MEK-ERK cascade mediates AAR signaling to the cFOS gene. Increased cFOS transcription is observed from 4-24 h after AAR-activation, exhibiting little or no overlap with the rapid and transient increase triggered by the well-known serum response. Furthermore, serum is not required for the AA-responsiveness of the cFOS gene and no phosphorylation of promoter-bound serum response factor (SRF) is observed. The ERK-phosphorylated transcription factor E-twenty six-like (p-ELK1) is increased in its association with the cFOS promoter after activation of the AAR. This research identified cFOS as a target of the AAR and further highlights the importance of AA-responsive MAPK signaling in HepG2 cells.

Activated transcription via mammalian amino acid response elements does not require enhanced recruitment of the Mediator complex

It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.

Characterization of a novel variant in siblings with Asparagine Synthetase Deficiency

Asparagine Synthetase Deficiency (ASD) is a recently described inborn error of metabolism caused by bi-allelic pathogenic variants in the asparagine synthetase (ASNS) gene. ASD typically presents congenitally with microcephaly and severe, often medically refractory, epilepsy. Development is generally severely affected at birth. Tone is abnormal with axial hypotonia and progressive appendicular spasticity. Hyperekplexia has been reported. Neuroimaging typically demonstrates gyral simplification, abnormal myelination, and progressive cerebral atrophy. The present report describes two siblings from consanguineous parents with a homozygous Arg49Gln variant associated with a milder form of ASD that is characterized by later onset of symptoms. Both siblings had a period of normal development before onset of seizures, and development regression. Primary fibroblast studies of the siblings and their parents document that homozygosity for Arg49Gln blocks cell growth in the absence of extracellular asparagine. Functional studies with these cells suggest no impact of the Arg49Gln variant on basal ASNS mRNA or protein levels, nor on regulation of the gene itself. Molecular modelling of the ASNS protein structure indicates that the Arg49Gln variant lies near the substrate binding site for glutamine. Collectively, the results suggest that the Arg49Gln variant affects the enzymatic function of ASNS. The clinical, cellular, and molecular observations from these siblings expand the known phenotypic spectrum of ASD.

Induction of early growth response gene 1 (EGR1) by endoplasmic reticulum stress is mediated by the extracellular regulated kinase (ERK) arm of the MAPK pathways

Endoplasmic reticulum (ER) stress activates three principal signaling pathways, collectively known as the unfolded protein response, leading to translational and transcriptional control mechanisms that dictate the cells response as adaptive or apoptotic. The present study illustrates that for HepG2 human hepatocellular carcinoma cells the signaling pathways triggered by ER stress extend beyond the three principal pathways to include mitogen-activated protein kinase (MAPK) signaling, leading to activation of transcription from the early growth response 1 (EGR1) gene. Analysis provided evidence for a SRC-RAS-RAF-MEK-ERK cascade mechanism that leads to enhanced phosphorylation of the transcription factor ELK1. ELK1 and serum response factor (SRF) are constitutively bound to the EGR1 promoter and are phosphorylated by nuclear localized ERK. The promoter abundance of both phospho-SRF and phopsho-ELK1 was increased by ER stress, but the SRF phosphorylation was transient. Knockdown of ELK1 had little effect on the basal EGR1 mRNA content, but completely blocked the increase in response to ER stress. Conversely, knockdown of SRF suppressed basal EGR1 mRNA content, but had only a small effect on the induction by ER stress. This research highlights the importance of MAPK signaling in response to ER stress and identifies ELK1 as a transcriptional mediator and the EGR1 gene as a target.