Jixiu Shan

ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death

Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER.

ATF4-dependent transcription mediates signaling of amino acid limitation.

Mammals respond to dietary nutrient fluctuations; for example, deficiency of dietary protein or an imbalance of essential amino acids activates an amino acid response (AAR) signal transduction pathway, consisting of detection of uncharged tRNA by the GCN2 kinase, eIF2alpha phosphorylation and ATF4 expression. In concert with heterodimerization partners, ATF4 activates specific genes via a CCAAT-enhancer binding protein-activating transcription factor response element (CARE). This review outlines the ATF4-dependent transcriptional mechanisms associated with the AAR, focusing on progress during the past 5 years. Recent evidence suggests that maternal nutrient deprivation not only has immediate metabolic effects on the fetus, but also triggers gene expression changes in adulthood, possibly through epigenetic mechanisms. Therefore, understanding the transcriptional programs initiated by amino acid limitation is crucial and timely.

Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress

Loss of parkin function is responsible for the majority of autosomal recessive parkinsonism. Here, we show that parkin is not only a stress-protective, but also a stress-inducible protein. Both mitochondrial and endoplasmic reticulum (ER) stress induce an increase in parkin-specific mRNA and protein levels. The stress-induced upregulation of parkin is mediated by ATF4, a transcription factor of the unfolded protein response (UPR) that binds to a specific CREB/ATF site within the parkin promoter. Interestingly, c-Jun can bind to the same site, but acts as a transcriptional repressor of parkin gene expression. We also present evidence that mitochondrial damage can induce ER stress, leading to the activation of the UPR, and thereby to an upregulation of parkin expression. Vice versa, ER stress results in mitochondrial damage, which can be prevented by parkin. Notably, the activity of parkin to protect cells from stress-induced cell death is independent of the proteasome, indicating that proteasomal degradation of parkin substrates cannot explain the cytoprotective activity of parkin. Our study supports the notion that parkin has a role in the interorganellar crosstalk between the ER and mitochondria to promote cell survival under stress, suggesting that both ER and mitochondrial stress can contribute to the pathogenesis of Parkinsons disease.

The Transcription Factor Network Associated With the Amino Acid Response in Mammalian Cells

Mammals exhibit multiple adaptive mechanisms that sense and respond to fluctuations in dietary nutrients. Consumption of reduced total dietary protein or a protein diet that is deficient in 1 or more of the essential amino acids triggers wide-ranging changes in feeding behavior and gene expression. At the level of individual cells, dietary protein deficiency is manifested as amino acid (AA) deprivation, which activates the AA response (AAR). The AAR is composed of a collection of signal transduction pathways that terminate in specific transcriptional programs designed to catalyze adaptation to the nutrient stress or, ultimately, undergo apoptosis. Independently of the AAR, endoplasmic reticulum stress activates 3 signaling pathways, collectively referred to as the unfolded protein response. The transcription factor activating transcription factor 4 is one of the terminal transcriptional mediators for both the AAR and the unfolded protein response, leading to a significant degree of overlap with regard to the target genes for these stress pathways. Over the past 5 y, research has revealed that the basic leucine zipper superfamily of transcription factors plays the central role in the AAR. Formation of both homo- and heterodimers among the activating transcription factor, CCAAT enhancer-binding protein, and FOS/JUN families of basic leucine zipper proteins forms the nucleus of a highly integrated transcription factor network that determines the initiation, magnitude, and duration of the cellular response to dietary protein or AA limitation.

CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis

This study addresses the mechanisms by which CHOP directs gene regulatory networks and determines cell fate. Transcriptional expression of ATF5 is activated by both CHOP and ATF4 in the integrated stress response. CHOP and ATF5 control a switch to activate apoptotic genes and decrease cell survival in response to loss of proteostatic control.

A Novel DNMT3B Splice Variant Expressed in Tumor and Pluripotent Cells Modulates Genomic DNA Methylation Patterns and Displays Altered DNA Binding

DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe here the identification and characterization of a novel alternatively spliced form of DNMT3B lacking exon 5 within the NH2-terminal regulatory domain. This variant, which we term DNMT3B3Δ5 because it is closely related in structure to the ubiquitously expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain tissue, is downregulated during differentiation, and is conserved in the mouse. Creation of pluripotent iPS cells from fibroblasts results in marked induction of DNMT3B3Δ5. DNMT3B3Δ5 expression is also altered in human disease, with tumor cell lines displaying elevated or reduced expression depending on their tissue of origin. We then compared the DNA binding and subcellular localization of DNMT3B3Δ5 versus DNMT3B3, revealing that DNMT3B3Δ5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Δ5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results show that DNMT3B3Δ5 may play an important role in stem cell maintenance or differentiation and suggest that sequences encoded by exon 5 influence the functional properties of DNMT3B. (Mol Cancer Res 2009;7(10):1622–34)

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

Protein limitation in vivo or amino acid deprivation of cells in culture causes a signal transduction cascade consisting of activation of the kinase GCN2 (general control nonderepressible 2), phosphorylation of eukaryotic initiation factor 2, and increased synthesis of activating transcription factor (ATF) 4 by a translational control mechanism. In a self-limiting transcriptional program, ATF4 transiently activates a wide range of downstream target genes involved in transport, cellular metabolism, and other cell functions. Simultaneous activation of other signal transduction pathways by amino acid deprivation led to the question of whether or not the increased abundance of ATF4 alone was sufficient to trigger the transcriptional control mechanisms. Using 293 cells that ectopically express ATF4 in a tetracycline-inducible manner showed that ATF4 target genes were activated in the absence of amino acid deprivation. Ectopic expression of ATF4 alone resulted in effective recruitment of the general transcription machinery, but some reduction in histone modification was observed. These data document that ATF4 alone is sufficient to trigger the amino acid-responsive transcriptional control program. However, the absolute amount of ectopic ATF4 required to achieve the same degree of transcriptional activation observed after amino acid limitation was greater, suggesting that other factors may serve to enhance ATF4 function.

Expression profiling after activation of amino acid deprivation response in HepG2 human hepatoma cells

Dietary protein malnutrition is manifested as amino acid deprivation of individual cells, which activates an amino acid response (AAR) that alters cellular functions, in part, by regulating transcriptional and posttranscriptional mechanisms. The AAR was activated in HepG2 human hepatoma cells, and the changes in mRNA content were analyzed by microarray expression profiling. The results documented that 1,507 genes were differentially regulated by P < 0.001 and by more than twofold in response to the AAR, 250 downregulated and 1,257 upregulated. The spectrum of altered genes reveals that amino acid deprivation has far-reaching implications for gene expression and cellular function. Among those cellular functions with the largest numbers of altered genes were cell growth and proliferation, cell cycle, gene expression, cell death, and development. Potential biological relationships between the differentially expressed genes were analyzed by computer software that generates gene networks. Proteins that were central to the most significant of these networks included c-myc, polycomb group proteins, transforming growth factor β1, nuclear factor (erythroid-derived 2)-like 2-related factor 2, FOS/JUN family members, and many members of the basic leucine zipper superfamily of transcription factors. Although most of these networks contained some genes that were known to be amino acid responsive, many new relationships were identified that underscored the broad impact that amino acid stress has on cellular function.

Despite increased ATF4 binding at the C/EBP-ATF composite site following activation of the unfolded protein response, system A transporter 2 (SNAT2) transcription activity is repressed in HepG2 cells.

The activated amino acid response (AAR) and unfolded protein response (UPR) stress signaling pathways converge at the phosphorylation of translation initiation factor eIF2α. This eIF2α modification suppresses global protein synthesis but enhances translation of selected mRNAs such as that for activating transcription factor 4 (ATF4). An ATF4 target gene, SNAT2 (system A sodium-dependent neutral amino acid transporter 2), contains a C/EBP-ATF site that binds ATF4 and triggers increased transcription during the AAR. However, the present studies show that despite increased ATF4 binding to the SNAT2 gene during UPR activation in HepG2 human hepatoma cells, transcription activity was not enhanced. Hyperacetylation of histone H3 and recruitment of the general transcription factors at the HepG2 SNAT2 promoter occurred in response to the AAR but not the UPR. In contrast, the UPR did enhance transcription from a plasmid-based reporter gene driven by a SNAT2 genomic fragment containing the C/EBP-ATF site. Simultaneous activation of the AAR and the UPR pathways revealed that the UPR actually suppressed the increased SNAT2 transcription by the AAR pathway, demonstrating that the UPR pathway generates a repressive signal that acts downstream of ATF4 binding.

Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element

Background: CHAC1 is associated with the stress response in atherosclerosis. Results: ATF4, ATF3, and CEBPβ regulate CHAC1 transcription. Human CHAC1 protein overexpression depletes glutathione. Conclusion: CHAC1 is induced following multiple cell stress signals and leads to depletion of glutathione. Significance: CHAC1 may be an essential link between stress signaling and the oxidative status of the cell, contributing to multiple diseases. Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the −267 ATF/cAMP response element (CRE) site and a novel −248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein β binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease.