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1
Fu, Lingchen, and Michael S. Kilberg. "Elevated cJUN expression and an ATF/CRE site within the ATF3 promoter contribute to activation of ATF3 transcription by the amino acid response." Physiological Genomics 45, no. 4 (2013): 127–37. http://dx.doi.org/10.1152/physiolgenomics.00160.2012.
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Mammalian cells respond to amino acid deprivation through multiple signaling pathways referred to as the amino acid response (AAR). Transcription factors mediate the AAR after their activation by several mechanisms; examples include translational control (activating transcription factor 4, ATF4), phosphorylation (p-cJUN), and transcriptional control (ATF3). ATF4 induces ATF3 transcription through a promoter-localized C/EBP-ATF response element (CARE). The present report characterizes an ATF/CRE site upstream of the CARE that also contributes to AAR-induced ATF3 transcription. ATF4 binds to the ATF/CRE and CARE sequences and both are required for a maximal response to ATF4 induction. ATF3, which antagonizes ATF4 and represses its own gene, also exhibited binding activity to the ATF/CRE and CARE sequences. The AAR resulted in elevated total cJUN and p-cJUN protein levels and both forms exhibited binding activity to the ATF/CRE and CARE ATF3 sequences. Knockdown of AAR-enhanced cJUN expression blocked induction of the ATF3 gene and mutation of either the ATF/CRE or the CARE site prevented the cJUN-dependent increase in ATF3-driven luciferase activity. The results indicate that both increased cJUN and the cis-acting ATF/CRE sequence within the ATF3 promoter contribute to the transcriptional activation of the gene during the AAR.
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Schindler, Maria, Sünje Fischer, René Thieme, Bernd Fischer, and Anne Navarrete Santos. "cAMP-Responsive Element Binding Protein: A Vital Link in Embryonic Hormonal Adaptation." Endocrinology 154, no. 6 (2013): 2208–21. http://dx.doi.org/10.1210/en.2012-2096.
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Abstract The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin, which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type 1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB, adiponectin expression was increased (diabetic 427.8 ± 59.3 pg/mL vs normoinsulinaemic 143.9 ± 26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may maintain glucose uptake in blastocysts grown in diabetic mothers.
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Fusakio, Michael E., Jeffrey A. Willy, Yongping Wang, et al. "Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver." Molecular Biology of the Cell 27, no. 9 (2016): 1536–51. http://dx.doi.org/10.1091/mbc.e16-01-0039.
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Disturbances in protein folding and membrane compositions in the endoplasmic reticulum (ER) elicit the unfolded protein response (UPR). Each of three UPR sensory proteins—PERK (PEK/EIF2AK3), IRE1, and ATF6—is activated by ER stress. PERK phosphorylation of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with preferential translation of ATF4 (CREB2). In cultured cells, ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterize whole-body and tissue-specific ATF4-knockout mice and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but instead ATF6 is a primary inducer. RNA-Seq analysis indicates that ATF4 is responsible for a small portion of the PERK-dependent UPR genes and reveals a requirement for expression of ATF4 for expression of genes involved in oxidative stress response basally and cholesterol metabolism both basally and under stress. Consistent with this pattern of gene expression, loss of ATF4 resulted in enhanced oxidative damage, and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera.
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Juliana, Christine A., Juxiang Yang, Andrea V. Rozo та ін. "ATF5 regulates β-cell survival during stress". Proceedings of the National Academy of Sciences 114, № 6 (2017): 1341–46. http://dx.doi.org/10.1073/pnas.1620705114.
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The stress response and cell survival are necessary for normal pancreatic β-cell function, glucose homeostasis, and prevention of diabetes. The homeodomain transcription factor and human diabetes gene pancreas/duodenum homeobox protein 1 (Pdx1) regulates β-cell survival and endoplasmic reticulum stress susceptibility, in part through direct regulation of activating transcription factor 4 (Atf4). Here we show that Atf5, a close but less-studied relative of Atf4, is also a target of Pdx1 and is critical for β-cell survival under stress conditions. Pdx1 deficiency led to decreased Atf5 transcript, and primary islet ChIP-sequencing localized PDX1 to the Atf5 promoter, implicating Atf5 as a PDX1 target. Atf5 expression was stress inducible and enriched in β cells. Importantly, Atf5 deficiency decreased survival under stress conditions. Loss-of-function and chromatin occupancy experiments positioned Atf5 downstream of and parallel to Atf4 in the regulation of eIF4E-binding protein 1 (4ebp1), a mammalian target of rapamycin (mTOR) pathway component that inhibits protein translation. Accordingly, Atf5 deficiency attenuated stress suppression of global translation, likely enhancing the susceptibility of β cells to stress-induced apoptosis. Thus, we identify ATF5 as a member of the transcriptional network governing pancreatic β-cell survival during stress.
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Lopez, Alex B., Chuanping Wang, Charlie C. Huang, et al. "A feedback transcriptional mechanism controls the level of the arginine/lysine transporter cat-1 during amino acid starvation." Biochemical Journal 402, no. 1 (2007): 163–73. http://dx.doi.org/10.1042/bj20060941.
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The adaptive response to amino acid limitation in mammalian cells inhibits global protein synthesis and promotes the expression of proteins that protect cells from stress. The arginine/lysine transporter, cat-1, is induced during amino acid starvation by transcriptional and post-transcriptional mechanisms. It is shown in the present study that the transient induction of cat-1 transcription is regulated by the stress response pathway that involves phosphorylation of the translation initiation factor, eIF2 (eukaryotic initiation factor-2). This phosphorylation induces expression of the bZIP (basic leucine zipper protein) transcription factors C/EBP (CCAAT/enhancer-binding protein)-β and ATF (activating transcription factor) 4, which in turn induces ATF3. Transfection experiments in control and mutant cells, and chromatin immunoprecipitations showed that ATF4 activates, whereas ATF3 represses cat-1 transcription, via an AARE (amino acid response element), TGATGAAAC, in the first exon of the cat-1 gene, which functions both in the endogenous and in a heterologous promoter. ATF4 and C/EBPβ activated transcription when expressed in transfected cells and they bound as heterodimers to the AARE in vitro. The induction of transcription by ATF4 was inhibited by ATF3, which also bound to the AARE as a heterodimer with C/EBPβ. These results suggest that the transient increase in cat-1 transcription is due to transcriptional activation caused by ATF4 followed by transcriptional repression by ATF3 via a feedback mechanism.
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Xu, Linyan, Xiang Gao, Wei Sang, et al. "EHMT2 Inhibitor BIX-01294 Induces Endoplasmic Reticulum Stress Mediated Apoptosis and Autophagy in Diffuse Large B Cell Lymphoma Cells." Blood 134, Supplement_1 (2019): 2787. http://dx.doi.org/10.1182/blood-2019-127653.
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Diffuse large B cell lymphoma (DLBCL) is the most common type lymphoma and the standard therapy R-CHOP regimen has made most patients complete remission, however, 30-40% patients still remains be refractory or relapsed and have a dismal outcome, indicating standard cytotoxic therapy also has limits in these patients. Therefore, it is essential to identify novel therapeutic targets and agents to understand the depth molecular pathogenesis mechanism of DLBCL and overcome the relapsed/refractory. EHMT2 abnormally expression has been discovered in various kinds of malignant cells, and its higher expression may be concerned with poor prognostic of these cancers. BIX-01294 is a small molecule compound which specifically inhibits EHMT2 activity and induces demethylation of H3K9. However, the role of BIX-01294 and the involvement of EHMT2 in DLBCL is not well study now. In the present study, we first examined the expression of EHMT2 and found EHMT2 were downregulated both in protein and mRNA levels. To determine the effect of BIX-01294 on DLBCL cells growth status, CCK-8 assay was preformed to detect the viability. BIX-01294 exhibited notable proliferation suppression in a dose-dependent manner in DLBCL cells, no matter the ABC type or GCB type cells. Then flow cytometry analysis was carried out to investigate the cell cycle distribution when treated with BIX-01294. The cells in G1 phase were increased in a dose-dependent fashion both in U2932 and SUDHL2 cells, and accompanied by the population in S phase was decreased. Furthermore, we preformed flow cytometric assay to elucidate apoptotic effect and found that BIX-01294 treatment induced U2932 and SUDHL2 apoptosis. By western blot and RT-qPCR, we also showed that the expression of anti-apoptotic protein c-FLIP was decreased and the level of DR4 and DR5 was upregulated. Conformably, BIX-01294 down-regulated anti-apoptotic protein Mcl-1 expression and up-regulated pro-apoptotic protein Bax level, indicating BIX-01294 activates exogenous and endogenous apoptotic signaling pathway in human DLBCL cells. We also showed both protein and mRNA levels of LC3B increased when cells treated with BIX-01294 in DLBCL cells, proves BIX-01294 also triggers autophagy. To elucidate the mechanism of BIX-01294-induced apoptosis and autophagy in DLBCL cells, we studied the active stage of ER (endoplasmic reticulum) stress. We examined the expression of GRP78, CHOP, ATF3 and ATF4, which were regarded as important protein markers of ER stress and found that their expression were all enhanced in a dose-dependent fashion, indicate BIX-01294 activates ER stress. We then wondered whether ATF3 and ATF4 influenced apoptosis and autophagy induced by BIX-01294. We conducted shRNA to inhibit ATF3 or ATF4 expression and found inhibition of ATF3 or ATF4 upregulation decreased the expression of LC3B, indicating ATF3 and ATF4 were contributed to BIX-01294 induced autophagy. CCK8 assay showed that the viability was increased in ATF3 or ATF4 abrogated cells after exposure to BIX-01294. Consistently, the percentage of apoptosis was significantly decreased in ATF3 or ATF4 knockdown cells than control cells by Annexin-V staining flow cytometry. In our experiments, we also showed that suppressed ATF4 expression inhibited ATF3 and CHOP expression in DLBCL cells. In conclusion, we showed that with the increased concentration of BIX-01294, the cell survival rate of DLBCL was obviously inhibited and cell cycle was arrested in G1 phase. BIX-01294 induced DLBCL cells apoptosis and autophagy. Furtherly, we explored one of the underlying mechanisms is through activating the ER stress pathway. We speculated that BIX-01294 treatment induces ATF4 upregulation, and then promotes ATF3 and CHOP expression, subsequently contributes to BIX-01294-mediated autophagy and apoptosis. Disclosures No relevant conflicts of interest to declare.
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Green, T. A., I. N. Alibhai, S. Unterberg, et al. "Induction of Activating Transcription Factors (ATFs) ATF2, ATF3, and ATF4 in the Nucleus Accumbens and Their Regulation of Emotional Behavior." Journal of Neuroscience 28, no. 9 (2008): 2025–32. http://dx.doi.org/10.1523/jneurosci.5273-07.2008.
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Teske, Brian F., Michael E. Fusakio, Donghui Zhou, et al. "CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis." Molecular Biology of the Cell 24, no. 15 (2013): 2477–90. http://dx.doi.org/10.1091/mbc.e13-01-0067.
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Environmental stresses that disrupt protein homeostasis induce phosphorylation of eIF2, triggering repression of global protein synthesis coincident with preferential translation of ATF4, a transcriptional activator of the integrated stress response (ISR). Depending on the extent of protein disruption, ATF4 may not be able to restore proteostatic control and instead switches to a terminal outcome that features elevated expression of the transcription factor CHOP (GADD153/DDIT3). The focus of this study is to define the mechanisms by which CHOP directs gene regulatory networks that determine cell fate. We find that in response to proteasome inhibition, CHOP enhances the expression of a collection of genes encoding transcription regulators, including ATF5, which is preferentially translated during eIF2 phosphorylation. Transcriptional expression of ATF5 is directly induced by both CHOP and ATF4. Knockdown of ATF5 increases cell survival in response to proteasome inhibition, supporting the idea that both ATF5 and CHOP have proapoptotic functions. Transcriptome analysis of ATF5-dependent genes reveals targets involved in apoptosis, including NOXA, which is important for inducing cell death during proteasome inhibition. This study suggests that the ISR features a feedforward loop of stress-induced transcriptional regulators, each subject to transcriptional and translational control, which can switch cell fate toward apoptosis.
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Gjymishka, Altin, Nan Su, and Michael S. Kilberg. "Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway." Biochemical Journal 417, no. 3 (2009): 695–703. http://dx.doi.org/10.1042/bj20081706.
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The UPR (unfolded protein response) pathway comprises three signalling cascades mediated by the ER (endoplasmic reticulum) stress-sensor proteins PERK [PKR (double-stranded RNA-activated protein kinase)-like ER kinase], IRE1 (inositol-requiring kinase 1) and ATF6 (activating transcription factor 6). The present study shows that ASNS (asparagine synthetase) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. ChIP (chromatin immunoprecipitation) analysis demonstrated that during ER stress, ATF4, ATF3 and C/EBPβ (CCAAT/enhancer-binding protein β) bind to the ASNS proximal promoter region that includes the genomic sequences NSRE (nutrient-sensing response element)-1 and NSRE-2, previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signalling resulted in enhanced recruitment of general transcription factors, including RNA Pol II (polymerase II), to the ASNS promoter. The ASNS gene is also activated by the AAR (amino acid response) pathway following amino acid deprivation of tissue or cells. Immunoblot analysis of HepG2 cells demonstrated that simultaneous activation of the AAR and UPR pathways did not further increase the ASNS or ATF4 protein abundance when compared with triggering either pathway alone. In addition, siRNA (small interfering RNA)-mediated knockdown of XBP1 (X-box-binding protein 1), ATF6α or ATF6β expression did not affect ASNS transcription, whereas siRNA against ATF4 suppressed ASNS transcription during UPR activation. Collectively, these results indicate that the PERK/p-eIF2α (phosphorylated eukaryotic initiation factor 2α)/ATF4 signalling cascade is the only arm of the UPR that is responsible for ASNS transcriptional induction during ER stress. Consequently, ASNS NSRE-1 and NSRE-2, in addition to ERSE (ER stress response element)-I, ERSE-II and the mUPRE (mammalian UPR element), function as mammalian ER-stress-responsive sequences.
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Kim, Wonil, Cary S. Koss, and Tanja A. Gruber. "KMT2A-Rearranged Infant Acute Lymphoblastic Leukemia Cells Undergo ER-Stress-Induced Apoptosis Following Exposure to Proteasome Inhibitors." Blood 134, Supplement_1 (2019): 1283. http://dx.doi.org/10.1182/blood-2019-127699.
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Infants diagnosed with KMT2A-rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) have a poor prognosis with an event free survival of 23-44%. To identify new treatment approaches we previously performed in vitro and in vivo assays to evaluate the activity of FDA approved compounds in 15 primary KMT2Ar infant leukemia samples. Three classes of agents were found to be active in these assays: proteasome inhibitors, anthracyclines, and histone deacetylase inhibitors (HDACi). KMT2Ar infant leukemia samples were exquisitely sensitive to the proteasome inhibitor bortezomib, requiring 10-100 fold less drug to achieve 50% toxicity when compared to non-KMT2Ar childhood ALL. Bortezomib is FDA approved for multiple myeloma and laboratory studies using this model system have previously demonstrated responses to be mediated through several mechanisms including NFKB inhibition, stabilization of cell cycle regulatory proteins, and perhaps most importantly the induction of an unfolded protein response (UPR) and endoplasmic reticulum (ER)-stress-induced apoptosis. To evaluate global protein dynamics in KMT2Ar ALL cells treated with bortezomib, we performed tandem mass tag (TMT) quantitative mass spectrometry on synchronized SEM cells exposed to either 50nM of bortezomib or DMSO at 0 hours (hr), 6hr, 12hr, 16hr, and 20hr. Applying pairwise comparison for 9232 unique proteins measured over the time course compared to untreated controls, we identified 1593 proteins with a log2 fold change >1.5 in bortezomib treated cells compared to 101 proteins in the DMSO control (FDR<0.01). Several proteins associated with ER-stress-induced apoptosis including ATF4, DDIT4, ATF3, TSC22D3 (GILZ), and PMAIP1 (NOXA) were upregulated more than 3-fold between 6 and 20hr, suggesting this pathway may play a role in bortezomib induced apoptosis of KMT2Ar cells (p<0.05 and log2 fold change of +/- 0.58). To validate this finding and further understand the role of the UPR and ER-stress-induced apoptosis, we evaluated seven key mediators of this pathway by western blot following bortezomib exposure on synchronized SEM cells over a 12 hour time course including ATF4, ATF6, CHOP, PERK, GADD34, CReP, and eIF2α as well as phosphorylated PERK (p-PERK) and eIF2α (p-eIF2α). This demonstrated a critical time point at 6hr where an increase in ATF4 (3.5 fold), CHOP (1.6 fold), and CReP (2.9 fold) protein levels was accompanied by a decrease in p-PERK (0.7 fold), and p-eIF2α (0.8 fold) whereas GADD34 levels remained constant. Although full-sized ATF6 (ATF6a) protein showed a considerable increase (1.9 fold), the levels of cleaved ATF6 (ATF6f) were only slightly increased (1.2 fold) consistent with ATF4-mediated upregulation of CHOP leading to increased protein synthesis along with ATP depletion, oxidative stress, and cell death. While GADD34 has been shown to be the main phosphatase that functions in a negative feedback loop to resolve cell stress, recent data suggests that stabilization of CReP mRNA by ER stress is able to reverse eIF2α phosphorylation at later stages of UPR leading to re-expression of key UPR proteins. Further, p-eIF2α-attenuated protein synthesis, and not ATF4 mRNA translation has been shown to promote cell survival. Our data support a model whereby the UPR and ER-stress in KMT2Ar ALL cells is induced upon exposure to bortezomib leading to increased levels of ATF4 and CHOP. Attenuation of p-eIF2α by CReP further contributes to cell death through the recovery of protein synthesis in a setting of limited protein folding capacity. These results support the use of proteasome inhibitors in KMT2Ar leukemia which is currently being formally evaluated in a Phase II clinical trial for newly diagnosed patients with infant ALL (NCT02553460). Disclosures Gruber: Bristol-Myers Squibb: Consultancy.
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Oskolkova, Olga V., Taras Afonyushkin, Alexander Leitner, et al. "ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response." Blood 112, no. 2 (2008): 330–39. http://dx.doi.org/10.1182/blood-2007-09-112870.
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Abstract We have shown previously that oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, stimulate angiogenesis via induction of autocrine mediators, such as vascular endothelial growth factor (VEGF). We now address the pathways mediating up-regulation of VEGF in human endothelial cells treated with OxPLs. Analysis of structure-function relationship using individual species of OxPLs demonstrated a close relation between induction of VEGF and activation of the unfolded protein response (UPR). Inducers of UPR up-regulated VEGF, whereas inhibition of UPR by chemical chaperones or knock-down of cochaperone HTJ-1 inhibited elevation of VEGF mRNA induced by OxPLs. OxPLs induced protein expression of activating transcription factor-4 (ATF4), an important effector of UPR. Expression levels of VEGF in OxPL-treated cells strongly correlated with induction of the ATF4 target genes ATF3 and TRB3. Knocking down ATF4 was paralleled by loss of VEGF induction by OxPLs. Chromatin immunoprecipitation demonstrated that OxPLs stimulated binding of ATF4 to a regulatory site in the VEGFA gene. Taken together, these data characterize UPR and more specifically its ATF4 branch as an important mechanism mediating up-regulation of VEGF by OxPLs, and allow hypothesizing that the UPR cascade might play a role in pathologic angiogenesis in atherosclerotic plaques.
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FAWCETT, Timothy W., Jennifer L. MARTINDALE, Kathryn Z. GUYTON, Tsonwin HAI, and Nikki J. HOLBROOK. "Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)–ATF composite site to regulate Gadd153 expression during the stress response." Biochemical Journal 339, no. 1 (1999): 135–41. http://dx.doi.org/10.1042/bj3390135.
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Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPβ and two uncharacterized protein complexes to the C/EBP–ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP–ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP–ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP–ATF composite site.
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Diaz-Bulnes, Paula, Maria Laura Saiz, Viviana Corte-Iglesias, et al. "Demethylation of H3K9 and H3K27 Contributes to the Tubular Renal Damage Triggered by Endoplasmic Reticulum Stress." Antioxidants 11, no. 7 (2022): 1355. http://dx.doi.org/10.3390/antiox11071355.
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Loss of protein homeostasis (proteostasis) in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR), restoring correct protein folding. Sustained ER stress exacerbates activation of the major UPR branches (IRE1α/XBP1, PERK/ATF4, ATF6), inducing expression of numerous genes involved in inflammation, cell death, autophagy, and oxidative stress. We investigated whether epigenetic dynamics mediated by histone H3K9 and H3K27 methylation might help to reduce or inhibit the exacerbated and maladaptive UPR triggered in tubular epithelial cells. Epigenetic treatments, specific silencing, and chromatin immunoprecipitation assays were performed in human proximal tubular cells subjected to ER stress. Pharmacological blockage of KDM4C and JMJD3 histone demethylases with SD-70 and GSKJ4, respectively, enhanced trimethylation of H3K9 and H3K27 in the ATF4 and XBP1 genes, inhibiting their expression and that of downstream genes. Conversely, specific G9a and EZH2 knockdown revealed increases in ATF4 and XBP1 expression. This is a consequence of the reduced recruitment of G9a and EZH2 histone methylases, diminished H3K9me3 and H3K27me3 levels, and enhanced histone acetylation at the ATF4 and XBP1 promoter region. G9a and EZH2 cooperate to maintain the repressive chromatin structure in both UPR-induced genes, ATF4 and XBP1. Therefore, preserving histone H3K9 and H3K27 methylation could ameliorate the ER stress, and consequently the oxidative stress and the triggered pathological processes that aggravate renal damage.
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Quirós, Pedro M., Miguel A. Prado, Nicola Zamboni, et al. "Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals." Journal of Cell Biology 216, no. 7 (2017): 2027–45. http://dx.doi.org/10.1083/jcb.201702058.
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Mitochondrial stress activates a mitonuclear response to safeguard and repair mitochondrial function and to adapt cellular metabolism to stress. Using a multiomics approach in mammalian cells treated with four types of mitochondrial stressors, we identify activating transcription factor 4 (ATF4) as the main regulator of the stress response. Surprisingly, canonical mitochondrial unfolded protein response genes mediated by ATF5 are not activated. Instead, ATF4 activates the expression of cytoprotective genes, which reprogram cellular metabolism through activation of the integrated stress response (ISR). Mitochondrial stress promotes a local proteostatic response by reducing mitochondrial ribosomal proteins, inhibiting mitochondrial translation, and coupling the activation of the ISR with the attenuation of mitochondrial function. Through a trans–expression quantitative trait locus analysis, we provide genetic evidence supporting a role for Fh1 in the control of Atf4 expression in mammals. Using gene expression data from mice and humans with mitochondrial diseases, we show that the ATF4 pathway is activated in vivo upon mitochondrial stress. Our data illustrate the value of a multiomics approach to characterize complex cellular networks and provide a versatile resource to identify new regulators of mitochondrial-related diseases.
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Bastola, Prabhakar, Gary S. Leiserowitz, and Jeremy Chien. "Multiple Components of Protein Homeostasis Pathway Can Be Targeted to Produce Drug Synergies with VCP Inhibitors in Ovarian Cancer." Cancers 14, no. 12 (2022): 2949. http://dx.doi.org/10.3390/cancers14122949.
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Protein quality control mechanisms play an important role in cancer progression by providing adaptive responses and morphologic stability against genome-wide copy number alterations, aneuploidy, and conformation-altering somatic mutations. This dependency on protein quality control mechanisms creates a vulnerability that may be exploited for therapeutic benefits by targeting components of the protein quality control mechanism. Recently, valosin-containing protein (VCP), also known at p97 AAA-ATPase, has emerged as a druggable target in cancer cells to affect their dependency on protein quality control. Here, we show that VCP inhibitors induce cytotoxicity in several ovarian cancer cell lines and these compounds act synergistically with mifepristone, a drug previously shown to induce an atypical unfolded protein response. Although mifepristone at a clinically achievable dose induces a weak unfolded protein response, it enhances the cytotoxic effects of VCP inhibitor CB-5083. Mechanistically, mifepristone blocks the cytoprotective effect of ATF6 in response to endoplasmic reticulum (ER) stress while activating the cytotoxic effects of ATF4 and CHOP through the HRI (EIF2AK1)-mediated signal transduction pathway. In contrast, CB-5083 activates ATF4 and CHOP through the PERK (EIF2AK3)-mediated signaling pathway. This combination activates ATF4 and CHOP while blocking the adaptive response provided by ATF6, resulting in increased cytotoxic effects and synergistic drug interaction.
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Yuniati, Laurensia, Laurens T. van der Meer, Geert JV Poelmans, et al. "The Leukemia-Associated Protein BTG1 Is Required for ATF4-Mediated Cellular Stress Responses." Blood 124, no. 21 (2014): 3587. http://dx.doi.org/10.1182/blood.v124.21.3587.3587.
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Abstract During the course of tumorigenesis and subsequent chemotherapeutic intervention, cancer cells experience various kinds of physiological stress, including hypoxia and nutrient limitation. Escaping cell death is one of the routes utilized by these malignant cells to allow continued growth and to acquire therapy resistance. B-cell Translocation Gene 1 (BTG1) is recurrently affected by genomic deletion in pediatric acute lymphoblastic leukemia (ALL) patients. Here, we define BTG1 as a mediator of the cellular stress response. When challenged with cellular stressors, such as amino acid or glucose deprivation as well as drug induced Endoplasmic Reticulum (ER) stress, mouse embryonic fibroblasts (MEFs) lacking Btg1 expression show a 20-30% increased survival rate relative to wildtype cells (Figure 1). Similarly, bone marrow B-cell progenitors isolated from Btg1 knockout mice are more resistant to Asparaginase (ASNase), a drug widely used in the treatment of ALL. Activating Transcription Factor 4 (ATF4) is the master regulator of the stress response pathway that is activated upon nutrient limitation and ER stress. Importantly, loss of ATF4 function results in an enhanced survival almost identical to the effects we measured in Btg1 knockout cells. While ATF4 protein expression itself is not different between the genotypes, gene expression analysis revealed that the induction of a subset of ATF4 target genes (Ddit3, Atf3, Trib3, Gadd34, and Ndrg1) is significantly reduced in Btg1 knockout cells. As these genes are effectors of the apoptosis machinery, increased survival in the Btg1 knockout cells may reflect an attenuation of ATF4 function. We hypothesized that BTG1 complexes with ATF4 to modify its function by recruiting Protein Arginine Methyl Transferase 1 (PRMT1). This enzyme, known to cooperate with BTG1, marks its substrate proteins with a post translational modification but has not been previously implicated in the regulation of ATF4 activity. Co-immunoprecipitation experiments indeed revealed a direct interaction between BTG1 and ATF4. We used purified proteins in an in vitro methylation assay to show that ATF4 is directly methylated by PRMT1 on arginine residue 239. Expression of the mutant ATF4 R239K, which cannot be methylated, in an ATF4 knockout background resulted in reduced transcriptional activity in response to stress relative to wildtype ATF4. In addition, we aimed to mimic the effect of BTG1 loss on the regulation ATF4 function by the addition of PRMT1 inhibitor AMI-1. Treatment of cells with this selective inhibitor faithfully recapitulates BTG1 loss by attenuating the induction of ATF4 target genes upon stress. Our findings establish the interplay of BTG1-ATF4-PRMT1 as a part of the cellular stress response. Taken together, our data indicate that BTG1 is necessary to maintain normal ATF4 function under cellular stress conditions. Loss of BTG1 expression, as it occurs during lymphoid leukemia development, may therefore provide a selective advantage for leukemic cells to survive and to resist treatment at a later stage of disease. Figure 1 Btg1 is required for survival under cellular stress. Wildtype (WT) and Btg1-/- MEFs were challenged with different treatments that cause nutrient limitation and ER stress. A MTT based assay was used to study the metabolic activity of the cells as a measure of viability. The relative cell survival as compared to untreated cells (set as 100%) is shown. Bars represent average data from four independent experiments ± SEM. 2-tailed t-test was used to test for significance: * p<0.05, ** p<0.01. Figure 1. Btg1 is required for survival under cellular stress. Wildtype (WT) and Btg1-/- MEFs were challenged with different treatments that cause nutrient limitation and ER stress. A MTT based assay was used to study the metabolic activity of the cells as a measure of viability. The relative cell survival as compared to untreated cells (set as 100%) is shown. Bars represent average data from four independent experiments ± SEM. 2-tailed t-test was used to test for significance: * p<0.05, ** p<0.01. Disclosures No relevant conflicts of interest to declare.
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Teske, Brian F., Sheree A. Wek, Piyawan Bunpo, et al. "The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress." Molecular Biology of the Cell 22, no. 22 (2011): 4390–405. http://dx.doi.org/10.1091/mbc.e11-06-0510.
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Disruptions of the endoplasmic reticulum (ER) that perturb protein folding cause ER stress and elicit an unfolded protein response (UPR) that involves translational and transcriptional changes in gene expression aimed at expanding the ER processing capacity and alleviating cellular injury. Three ER stress sensors (PERK, ATF6, and IRE1) implement the UPR. PERK phosphorylation of the α subunit of eIF2 during ER stress represses protein synthesis, which prevents further influx of ER client proteins. Phosphorylation of eIF2α (eIF2α∼P) also induces preferential translation of ATF4, a transcription activator of the integrated stress response. In this study we show that the PERK/eIF2α∼P/ATF4 pathway is required not only for translational control, but also for activation of ATF6 and its target genes. The PERK pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the ER to the Golgi for intramembrane proteolysis and activation of ATF6. As a consequence, liver-specific depletion of PERK significantly reduces both the translational and transcriptional phases of the UPR, leading to reduced protein chaperone expression, disruptions of lipid metabolism, and enhanced apoptosis. These findings show that the regulatory networks of the UPR are fully integrated and help explain the diverse biological defects associated with loss of PERK.
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Fox, Daniel K., Scott M. Ebert, Kale S. Bongers, et al. "p53 and ATF4 mediate distinct and additive pathways to skeletal muscle atrophy during limb immobilization." American Journal of Physiology-Endocrinology and Metabolism 307, no. 3 (2014): E245—E261. http://dx.doi.org/10.1152/ajpendo.00010.2014.
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Immobilization causes skeletal muscle atrophy via complex signaling pathways that are not well understood. To better understand these pathways, we investigated the roles of p53 and ATF4, two transcription factors that mediate adaptations to a variety of cellular stresses. Using mouse models, we demonstrate that 3 days of muscle immobilization induces muscle atrophy and increases expression of p53 and ATF4. Furthermore, muscle fibers lacking p53 or ATF4 are partially resistant to immobilization-induced muscle atrophy, and forced expression of p53 or ATF4 induces muscle fiber atrophy in the absence of immobilization. Importantly, however, p53 and ATF4 do not require each other to promote atrophy, and coexpression of p53 and ATF4 induces more atrophy than either transcription factor alone. Moreover, muscle fibers lacking both p53 and ATF4 are more resistant to immobilization-induced atrophy than fibers lacking only p53 or ATF4. Interestingly, the independent and additive nature of the p53 and ATF4 pathways allows for combinatorial control of at least one downstream effector, p21. Using genome-wide mRNA expression arrays, we identified p21 mRNA as a skeletal muscle transcript that is highly induced in immobilized muscle via the combined actions of p53 and ATF4. Additionally, in mouse muscle, p21 induces atrophy in a manner that does not require immobilization, p53 or ATF4, and p21 is required for atrophy induced by immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as essential and complementary mediators of immobilization-induced muscle atrophy and discover p21 as a critical downstream effector of the p53 and ATF4 pathways.
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Straub, Isabella R., Woranontee Weraarpachai, and Eric A. Shoubridge. "Multi-OMICS study of a CHCHD10 variant causing ALS demonstrates metabolic rewiring and activation of endoplasmic reticulum and mitochondrial unfolded protein responses." Human Molecular Genetics 30, no. 8 (2021): 687–705. http://dx.doi.org/10.1093/hmg/ddab078.
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Abstract Mutations in CHCHD10, coding for a mitochondrial intermembrane space protein, are a rare cause of autosomal dominant amyotrophic lateral sclerosis. Mutation-specific toxic gain of function or haploinsufficiency models have been proposed to explain pathogenicity. To decipher the metabolic dysfunction associated with the haploinsufficient p.R15L variant, we integrated transcriptomic, metabolomic and proteomic data sets in patient cells subjected to an energetic stress that forces the cells to rely on oxidative phosphorylation for ATP production. Patient cells had a complex I deficiency that resulted in an increased NADH/NAD+ ratio, diminished TCA cycle activity, a reorganization of one carbon metabolism and an increased AMP/ATP ratio leading to phosphorylation of AMPK and inhibition of mTORC1. These metabolic changes activated the unfolded protein response (UPR) in the ER through the IRE1/XBP1 pathway, upregulating downstream targets including ATF3, ATF4, CHOP and EGLN3, and two cytokine markers of mitochondrial disease, GDF15 and FGF21. Activation of the mitochondrial UPR was mediated through an upregulation of the transcription factors ATF4 and ATF5, leading to increased expression of mitochondrial proteases and heat shock proteins. There was a striking transcriptional up regulation of at least seven dual specific phosphatases, associated with an almost complete dephosphorylation of JNK isoforms, suggesting a concerted deactivation of MAP kinase pathways. This study demonstrates that loss of CHCHD10 function elicits an energy deficit that activates unique responses to nutrient stress in both the mitochondria and ER, which may contribute to the selective vulnerability of motor neurons.
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Freundt, Johanna K., Gerrit Frommeyer, Fabian Wötzel, et al. "The Transcription Factor ATF4 Promotes Expression of Cell Stress Genes and Cardiomyocyte Death in a Cellular Model of Atrial Fibrillation." BioMed Research International 2018 (May 29, 2018): 1–15. http://dx.doi.org/10.1155/2018/3694362.
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Introduction. Cardiomyocyte remodelling in atrial fibrillation (AF) has been associated with both oxidative stress and endoplasmic reticulum (ER) stress and is accompanied by a complex transcriptional regulation. Here, we investigated the role the oxidative stress and ER stress responsive bZIP transcription factor ATF4 plays in atrial cardiomyocyte viability and AF induced gene expression. Methods. HL-1 cardiomyocytes were subjected to rapid field stimulation. Forced expression of ATF4 was achieved by adenoviral gene transfer. Using global gene expression analysis and chromatin immunoprecipitation, ATF4 dependent transcriptional regulation was studied, and tissue specimen of AF patients was analysed by immunohistochemistry. Results. Oxidative stress and ER stress caused a significant reduction in cardiomyocyte viability and were associated with an induction of ATF4. Accordingly, ATF4 was also induced by rapid field stimulation mimicking AF. Forced expression of wild type ATF4 promoted cardiomyocyte death. ATF4 was demonstrated to bind to the promoters of several cell stress genes and to induce the expression of a number of ATF4 dependent stress responsive genes. Moreover, immunohistochemical analyses showed that ATF4 is expressed in the nuclei of cardiomyocytes of tissue specimen obtained from AF patients. Conclusion. ATF4 is expressed in human atrial cardiomyocytes and is induced in response to different types of cell stress. High rate electrical field stimulation seems to result in ATF4 induction, and forced expression of ATF4 reduces cardiomyocyte viability.
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Ebert, Scott M., Blake B. Rasmussen, Andrew R. Judge, et al. "Biology of Activating Transcription Factor 4 (ATF4) and Its Role in Skeletal Muscle Atrophy." Journal of Nutrition 152, no. 4 (2021): 926–38. http://dx.doi.org/10.1093/jn/nxab440.
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ABSTRACT Activating transcription factor 4 (ATF4) is a multifunctional transcription regulatory protein in the basic leucine zipper superfamily. ATF4 can be expressed in most if not all mammalian cell types, and it can participate in a variety of cellular responses to specific environmental stresses, intracellular derangements, or growth factors. Because ATF4 is involved in a wide range of biological processes, its roles in human health and disease are not yet fully understood. Much of our current knowledge about ATF4 comes from investigations in cultured cell models, where ATF4 was originally characterized and where further investigations continue to provide new insights. ATF4 is also an increasingly prominent topic of in vivo investigations in fully differentiated mammalian cell types, where our current understanding of ATF4 is less complete. Here, we review some important high-level concepts and questions concerning the basic biology of ATF4. We then discuss current knowledge and emerging questions about the in vivo role of ATF4 in one fully differentiated cell type, mammalian skeletal muscle fibers.
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Wang, Yang, Muhammad Ali, Qi Zhang, et al. "ATF4 Transcriptionally Activates SHH to Promote Proliferation, Invasion, and Migration of Gastric Cancer Cells." Cancers 15, no. 5 (2023): 1429. http://dx.doi.org/10.3390/cancers15051429.
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Activating transcription factor 4 (ATF4) is a DNA-binding protein widely generated in mammals, which has two biological characteristics that bind the cAMP response element (CRE). The mechanism of ATF4 as a transcription factor in gastric cancer affecting the Hedgehog pathway remains unclear. Here, we observed that ATF4 was markedly upregulated in gastric cancer (GC) using immunohistochemistry and Western blotting assays in 80 paraffin-embedded GC samples and 4 fresh samples and para-cancerous tissues. ATF4 knockdown using lentiviral vectors strongly inhibited the proliferation and invasion of GC cells. ATF4 upregulation using lentiviral vectors promoted the proliferation and invasion of GC cells. We predicted that the transcription factor ATF4 is bound to the SHH promoter via the JASPA database. Transcription factor ATF4 is bound to the promoter region of SHH to activate the Sonic Hedgehog pathway. Mechanistically, rescue assays showed that ATF4 regulated gastric cancer cells’ proliferation and invasive ability through SHH. Similarly, ATF4 enhanced the tumor formation of GC cells in a xenograft model.
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Zong, Ying, Shijie Feng, Jinwei Cheng, Chenlin Yu, and Guocai Lu. "Up-Regulated ATF4 Expression Increases Cell Sensitivity to Apoptosis in Response to Radiation." Cellular Physiology and Biochemistry 41, no. 2 (2017): 784–94. http://dx.doi.org/10.1159/000458742.
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Background/Aims: Activating transcription factor 4 (ATF4) is a member of the activating transcription factor family which regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses. ATF4 is also over-expressed in human solid tumors, although its effect on responsiveness to radiation is largely unexplored. Methods: Real-time PCR was used to detect ATF4 mRNA levels in cells treated with different doses of 60Coγ radiation. Cell viability was assayed using a cell counting kit. The cell cycle was analyzed using flow cytometry, and cell apoptosis was assayed using Annexin V-PI double labeling. Small interfering RNA (siRNA) against ATF4 was transfected into ECV304 cells using Lipofectamine 2000. An ATF4 over-expression plasmid (p-ATF4-CGN) was transfected into HEK293 cells that endogenously expressed low levels of ATF4. The levels of intracellular reactive oxygen species (ROS) were measured using CM-H2DCFDA as a probe. Results: ATF4 mRNA and protein expression levels were higher after radiation and increased in a dose- and time-dependent manner in AHH1 lymphoblast cells (P < 0.05). An increase in ATF4 levels was also observed after radiation in primary murine spleen cells, human endothelial ECV304 cells, human liver LO2 cells, breast cancer MCF7 cells, and human hepatocellular carcinoma HEPG2 cells. No change was observed in human embryonic kidney 293 (HEK293) cells. Over-expressing ATF4 in HEK293 cells inhibited cell proliferation, increased cell apoptosis and significantly increased the proportion of cells in G1 phase. Conversely, when ATF4 expression was knocked down using siRNA in ECV304 cells, it protected the cells from radiation-induced apoptosis. These findings suggest that ATF4 may play a role in radiation-induced cell killing by inhibiting cell proliferation and promoting cell apoptosis. Conclusions: In this study, we found that radiation up-regulated the expression of ATF4. We used ATF4 knockdown and over-expression systems to show that ATF4 may play a role in radiation-induced cellular apoptosis.
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STEINMÜLLER, Lars, Giuseppe CIBELLI, Jonathan R. MOLL, Charles VINSON, and Gerald THIEL. "Regulation and composition of activator protein 1 (AP-1) transcription factors controlling collagenase and c-Jun promoter activities." Biochemical Journal 360, no. 3 (2001): 599–607. http://dx.doi.org/10.1042/bj3600599.
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The activator protein 1 (AP-1) transcription factor is composed of heterodimers of the Fos/activating transcription factor (ATF) and Jun subfamilies of basic-region leucine-zipper (B-ZIP) proteins. In order to determine the identities of individual B-ZIP proteins in various AP-1 complexes we tested the effect of dominant-negative mutants to the B-ZIP proteins c-Fos, ATF2, ATF4 and CCAAT-enhancer-binding protein (C/EBP) on the activities of the collagenase and c-Jun promoters. These dominant-negative mutants inhibit DNA binding of wild-type B-ZIP proteins in a leucine-zipper-dependent fashion. Transcription of a collagenase promoter/reporter gene was induced in HepG2 hepatoma cells by expression of c-Fos and c-Jun, administration of PMA (‘TPA’) or by expression of a truncated form of MEK (mitogen-activated/extracellular-signal-regulated kinase kinase) kinase-1, MEKK1Δ. In all cases, the dominant-negative mutants A-Fos and A-ATF2 decreased collagenase promoter activity. However, A-ATF4 and A-C/EBP had no effect. A-Fos and A-ATF2 also reduced MEKK1Δ-induced stimulation of the c-Jun promoter. In contrast, constitutive c-Jun promoter activity was blocked solely by A-ATF2, strongly suggesting that ATF2 and/or an ATF2-dimerizing protein are of major importance for c-Jun transcription in unstimulated cells. These results demonstrate that AP-1 transcription factors of different compositions control c-jun gene transcription in resting or stimulated cells.
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Li, Ruohan, Huaixiang Zhou, Mingzhe Li, et al. "Gremlin-1 Promotes Colorectal Cancer Cell Metastasis by Activating ATF6 and Inhibiting ATF4 Pathways." Cells 11, no. 14 (2022): 2136. http://dx.doi.org/10.3390/cells11142136.
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Cancer cell survival, function and fate strongly depend on endoplasmic reticulum (ER) proteostasis. Although previous studies have implicated the ER stress signaling network in all stages of cancer development, its role in cancer metastasis remains to be elucidated. In this study, we investigated the role of Gremlin-1 (GREM1), a secreted protein, in the invasion and metastasis of colorectal cancer (CRC) cells in vitro and in vivo. Firstly, public datasets showed a positive correlation between high expression of GREM1 and a poor prognosis for CRC. Secondly, GREM1 enhanced motility and invasion of CRC cells by epithelial–mesenchymal transition (EMT). Thirdly, GREM1 upregulated expression of activating transcription factor 6 (ATF6) and downregulated that of ATF4, and modulation of the two key players of the unfolded protein response (UPR) was possibly through activation of PI3K/AKT/mTOR and antagonization of BMP2 signaling pathways, respectively. Taken together, our results demonstrate that GREM1 is an invasion-promoting factor via regulation of ATF6 and ATF4 expression in CRC cells, suggesting GREM1 may be a potential pharmacological target for colorectal cancer treatment.
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Ziolkowski, Leah M., and Kay F. Macleod. "Abstract C100: Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis." Cancer Research 84, no. 2_Supplement (2024): C100. http://dx.doi.org/10.1158/1538-7445.panca2023-c100.
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Abstract Biological stresses that are cell-intrinsic (e.g., oncogene-induced imbalances in protein translation, lipid synthesis, mitochondrial dysfunction) or cell-extrinsic (e.g., nutrient deprivation, hypoxia, drug exposure) disrupt cellular function and viability if not mitigated. The Integrated Stress Response (ISR) consists of upstream regulatory kinases (PERK, GCN2, HRI, PKR) that respond to such stresses in part by activating the ATF4 transcription factor. ATF4 then rewires nutrient uptake, cellular metabolism, mitochondrial function, and protein chaperone networks to restore cellular homeostasis. We have shown that ATF4 is upregulated in mouse and human pancreatic ductal adenocarcinoma (PDAC), even compared to normal acinar cells where ATF4 is expressed at relatively high levels. To investigate the role of ATF4 in PDAC, we generated mice with inducible acinar-specific deletion of Atf4 using Ptf1a-CreERT to delete Atf4 following treatment of adult Atf4-floxed mice with tamoxifen. In contrast to previous studies showing germline deletion of Atf4 in mice caused premature mortality at approximately 3 weeks of age due to pancreatic insufficiency, inducible deletion of Atf4 in adult acinar cells does not cause pancreatic deficiency or animal demise. Rather, adult Ptf1a-CreERT;Atf4fl/fl (CA) mice are viable although more sensitive to cerulein-induced pancreatitis. Specifically, compared to control (WT) mice, CA mice exhibit more extensive acinar-ductal metaplasia (ADM), including more extensive expression of cytokeratin-19 (CK19), increased cell death and take longer to recover normal pancreatic architecture following in vivo cerulein challenge. Using ex vivo acinar explant assays derived from WT and CA mice, we further show that induction of ADM in WT acinar cells with TGF-a is associated with down-regulation of Atf4 and Atf4 target genes in WT acinar explants while CA explants undergo accelerated ADM. Furthermore, expression of exogenous Atf4 in acinar cells inhibited ADM. These in vivo and ex vivo findings are consistent with down-regulation of Atf4 being required for ADM. Given the tumor-promoting role of ADM in the early stages of PDAC, we next crossed the CA mice to LSL-KRasG12D mice to generate LSL-KRasG12D;Ptf1a-CreERT;Atf4fl/fl (KCA) mice to examine how loss of Atf4 affected pancreatic intraepithelial neoplasia (PanIN) and PDAC formation. Surprisingly, given the critical need to down-regulate Atf4 in ADM, we show for the first time that genetic deletion of Atf4 in pancreatic acinar cells blocks both PanIN and PDAC formation in KCA mice compared to age-matched KC mice. Rather, KCA mice developed a fatty pancreas devoid of tumor with very few CK19-positive cells forming. These results suggest that Atf4 is critical for PanIN formation and prevents PDAC. This also suggests that Atf4 plays a different role in PanIN formation than in ADM where its down-regulation was critical and indicates a dynamic role for Atf4 depending on oncogenic versus proteotoxic stress, as will be discussed. Citation Format: Leah M. Ziolkowski, Kay F. Macleod. Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C100.
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Ahmad, Hafiz Ishfaq, Asia Iqbal, Nabeel Ijaz, et al. "Molecular Evolution of the Activating Transcription Factors Shapes the Adaptive Cellular Responses to Oxidative Stress." Oxidative Medicine and Cellular Longevity 2022 (July 13, 2022): 1–13. http://dx.doi.org/10.1155/2022/2153996.
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Reactive oxygen species (ROS) play an essential part in physiology of individual cell. ROS can cause damage to various biomolecules, including DNA. The systems that have developed to harness the impacts of ROS are antique evolutionary adaptations that are intricately linked to almost every aspect of cellular function. This research reveals the idea that during evolution, rather than being largely conserved, the molecular pathways reacting to oxidative stress have intrinsic flexibility. The coding sequences of the ATF2, ATF3, ATF4, and ATF6 genes were aligned to examine selection pressure on the genes, which were shown to be very highly conserved among vertebrate species. A total of 33 branches were explicitly evaluated for their capacity to diversify selection. After accounting for multiple testing, significance was determined using the likelihood ratio test with a threshold of p ≤ 0.05 . Positive selection signs in these genes were detected across vertebrate lineages. In the selected test branches of our phylogeny, the synonymous rate variation revealed evidence (LRT, p value = 0.011 ≤ 0.05) of gene-wide episodic diversifying selection. As a result, there is evidence that diversifying selection occurred at least once on at least one test branch. These findings indicate that the activities of ROS-responsive systems are also theoretically flexible and may be altered by environmental selection pressure. By determining where the genes encoding these processes are “targeted” during evolution, we may better understand the mechanism of adaptation to oxidative stress during evolution.
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Zheng, Zhaofeng, Shangda Yang, Fanglin Gou, et al. "The ATF4-RPS19BP1 Axis Modulates Ribosome Biogenesis to Promote Erythropoiesis." Blood 142, Supplement 1 (2023): 142. http://dx.doi.org/10.1182/blood-2023-184592.
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The function of hematopoietic stem cells (HSCs) and the process of hematopoiesis are intricately governed by a complex interplay of intrinsic programs and extrinsic signals from the microenvironment (Crane et al., Nat Rev Immunol, 2017). Previous work reported by our group and others has demonstrated that ATF4, a basic region-leucine zipper transcription factor, plays pivotal roles in fetal liver hematopoiesis, HSC maintenance, bone formation and tumorigenesis (Zhao et al., Blood, 2015; Sun et al., Sci Adv, 2021; Yang et al., Cell, 2004). However, the precise function of ATF4 in the bone marrow niche and how ATF4 regulates adult hematopoiesis remain largely unknown. Here, we employ four cell-type-specific mouse Cre lines to conditionally knock out Atf4 in Cdh5 + endothelial cells (ECs), Prx1 + bone marrow stromal cells (BMSCs), Osx + osteo-progenitor cells, and Mx1 + hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. We found that Atf4 deletion in BMSCs reduced BMSC numbers and impaired their CFU-F activity as well as differentiation toward osteoblasts, but spares hematopoiesis and HSC function in adult mice. Furthermore, Atf4 depletion in the ECs or osteo-progenitors of adult mice has a very mild effect on the BM hematopoiesis and HSC maintenance. Intriguingly, Atf4 depletion in the hematopoietic system resulted in severe anemia. More than 70% of the Mx1cre; Atf4 fl/fl (Δ/Δ) mice gradually died from anemia. Atf4 deficiency markedly reduced the frequency of erythroid progenitor cells (EPCs) (including MEP, PreCFU-E, and CFU-E cells) and impaired their CFU-E and BFU-E colony-forming ability. Atf4 depletion severely impaired the repopulation ability of BM cells and the self-renewal capacity of HSCs. Single cell RNA-seq (scRNA-seq) analysis of BM Lin -cKit + cells confirmed the defects of erythropoiesis after Atf4 depletion. The absence of ATF4 compelled early erythroid progenitors to enter the S phase, which led to replication stress and in turn activated both the DNA damage response and apoptosis. Subsequently, we conducted an integrative analysis of the genes that were identified as downregulated in MEPs by RNA-seq, ATAC-seq, and H3K4me3 Cut&Tag and genes that were identified as downregulated in erythroid progenitors (i.e., PreMegE, Ery1, and Ery2) by scRNA-seq, which predicted Rps19bp1 as the top target gene of Atf4. Luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that ATF4 was a transcriptional activator of Rps19bp1. Downregulation of Rps19bp1 caused by Atf4 deletion led to decreased assembles of 40S proteins, such as RPS3, RPS6 and RPS19. O-propargyl-puromycin (OP-Puro) incorporation and SUnSET assay further confirmed the global protein synthesis defect in Atf4-deficient MEP cells. The protein synthesis defects and impaired erythropoiesis were rescued upon RPS19BP1 overexpression in Atf4 knockdown MEL cell line or BM cKit + cells from Atf4-deficient mice. Ribosome profiling of CMP cells from Δ/Δ and fl/fl mice further demonstrated that the expression of proteins involved in ribosome biogenesis was significantly lower in Atf4-depleted than in wildtype CMP cells. The expression of gene sets linked to PreCFU-E signature was also lower in Atf4-depleted than in wildtype CMPs, suggesting that the reduction in ribosome biogenesis significantly affected the translation efficiency of erythroid-related pathway components, thereby impeding erythroid lineage commitment. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, we demonstrated that, unlike in the fetal liver, ATF4 governs adult HSC function and erythropoiesis in a cell-intrinsic manner. We revealed a novel role for ATF4 in erythropoiesis, which links it to RPS19BP1, ribosome biogenesis and protein translation. Our findings have highlighted the crucial importance of protein synthesis regulation during erythroid lineage commitment. This discovery will likely have extended implications for understanding and treating ribosomopathy-associated erythroid failure.
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Pan, Yuan-Xiang, Hong Chen, Michelle M. Thiaville, and Michael S. Kilberg. "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." Biochemical Journal 401, no. 1 (2006): 299–307. http://dx.doi.org/10.1042/bj20061261.
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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/EBPβ 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/EBPβ 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.
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Ebert, Scott M., Steven A. Bullard, Nathan Basisty та ін. "Activating transcription factor 4 (ATF4) promotes skeletal muscle atrophy by forming a heterodimer with the transcriptional regulator C/EBPβ". Journal of Biological Chemistry 295, № 9 (2020): 2787–803. http://dx.doi.org/10.1074/jbc.ra119.012095.
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Skeletal muscle atrophy is a highly-prevalent and debilitating condition that remains poorly understood at the molecular level. Previous work found that aging, fasting, and immobilization promote skeletal muscle atrophy via expression of activating transcription factor 4 (ATF4) in skeletal muscle fibers. However, the direct biochemical mechanism by which ATF4 promotes muscle atrophy is unknown. ATF4 is a member of the basic leucine zipper transcription factor (bZIP) superfamily. Because bZIP transcription factors are obligate dimers, and because ATF4 is unable to form highly-stable homodimers, we hypothesized that ATF4 may promote muscle atrophy by forming a heterodimer with another bZIP family member. To test this hypothesis, we biochemically isolated skeletal muscle proteins that associate with the dimerization- and DNA-binding domain of ATF4 (the bZIP domain) in mouse skeletal muscle fibers in vivo. Interestingly, we found that ATF4 forms at least five distinct heterodimeric bZIP transcription factors in skeletal muscle fibers. Furthermore, one of these heterodimers, composed of ATF4 and CCAAT enhancer-binding protein β (C/EBPβ), mediates muscle atrophy. Within skeletal muscle fibers, the ATF4–C/EBPβ heterodimer interacts with a previously unrecognized and evolutionarily conserved ATF–C/EBP composite site in exon 4 of the Gadd45a gene. This three-way interaction between ATF4, C/EBPβ, and the ATF–C/EBP composite site activates the Gadd45a gene, which encodes a critical mediator of muscle atrophy. Together, these results identify a biochemical mechanism by which ATF4 induces skeletal muscle atrophy, providing molecular-level insights into the etiology of skeletal muscle atrophy.
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Smith, Spencer G., Kathryn A. Haynes, and Ashok N. Hegde. "Degradation of Transcriptional Repressor ATF4 during Long-Term Synaptic Plasticity." International Journal of Molecular Sciences 21, no. 22 (2020): 8543. http://dx.doi.org/10.3390/ijms21228543.
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Maintenance of long-term synaptic plasticity requires gene expression mediated by cAMP-responsive element binding protein (CREB). Gene expression driven by CREB can commence only if the inhibition by a transcriptional repressor activating transcription factor 4 (ATF4; also known as CREB2) is relieved. Previous research showed that the removal of ATF4 occurs through ubiquitin-proteasome-mediated proteolysis. Using chemically induced hippocampal long-term potentiation (cLTP) as a model system, we investigate the mechanisms that control ATF4 degradation. We observed that ATF4 phosphorylated at serine-219 increases upon induction of cLTP and decreases about 30 min thereafter. Proteasome inhibitor β-lactone prevents the decrease in ATF4. We found that the phosphorylation of ATF4 is mediated by cAMP-dependent protein kinase. Our initial experiments towards the identification of the ligase that mediates ubiquitination of ATF4 revealed a possible role for β-transducin repeat containing protein (β-TrCP). Regulation of ATF4 degradation is likely to be a mechanism for determining the threshold for gene expression underlying maintenance of long-term synaptic plasticity and by extension, long-term memory.
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Li, Houkai, Qingshu Meng, Fei Xiao, et al. "ATF4 deficiency protects mice from high-carbohydrate-diet-induced liver steatosis." Biochemical Journal 438, no. 2 (2011): 283–89. http://dx.doi.org/10.1042/bj20110263.
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Chronic feeding of HCD (high-carbohydrate diet) is one of the major contributors to the prevailing of metabolic diseases. ATF4 (activating transcription factor 4) has been shown to play an important role in the regulation of glucose metabolism and obesity development; however, it is unclear how ATF4−/− mice respond to HCD. In the present study, we show that 8 weeks of HCD results in significant higher accumulation of TAGs (triacylglycerols) in livers and impairment in glucose tolerance in ATF4+/+ mice, but not in ATF4−/− mice, compared with those on a normal diet. Meanwhile, energy expenditure is further enhanced by HCD in ATF4−/− mice. Moreover, we show that ATF4 deficiency suppresses HCD-induced SCD1 (stearoyl-CoA desaturase 1) expression, furthermore, oral supplementation of the main product of SCD1 oleate (18:1) increases TAG accumulation in livers of ATF4−/− mice. Taken together, these results suggest that ATF4 deficiency is protective for HCD-induced hepatic steatosis and impairment of glucose tolerance and insulin sensitivity. Furthermore, the resistance to hepatic steatosis is at least in part due to suppression of SCD1 expression under HCD.
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Lange, Philipp S., Juan C. Chavez, John T. Pinto, et al. "ATF4 is an oxidative stress–inducible, prodeath transcription factor in neurons in vitro and in vivo." Journal of Experimental Medicine 205, no. 5 (2008): 1227–42. http://dx.doi.org/10.1084/jem.20071460.
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Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of oxidative stress–inducible transcription factors and their role in propagating the death cascade are not well known. In an in vitro model of oxidative stress, the expression of the bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathione depletion and localized to the promoter of a putative death gene in neurons. Germline deletion of ATF4 resulted in a profound reduction in oxidative stress–induced gene expression and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative death by ATF4 deletion was associated with decreased consumption of the antioxidant glutathione. Forced expression of ATF4 was sufficient to promote cell death and loss of glutathione. In ATF4−/− neurons, restoration of ATF4 protein expression reinstated sensitivity to oxidative death. In addition, ATF4−/− mice experienced significantly smaller infarcts and improved behavioral recovery as compared with wild-type mice subjected to the same reductions in blood flow in a rodent model of ischemic stroke. Collectively, these findings establish ATF4 as a redox-regulated, prodeath transcriptional activator in the nervous system that propagates death responses to oxidative stress in vitro and to stroke in vivo.
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Yu, Shibing, Renny T. Franceschi, Min Luo, et al. "Parathyroid Hormone Increases Activating Transcription Factor 4 Expression and Activity in Osteoblasts: Requirement for Osteocalcin Gene Expression." Endocrinology 149, no. 4 (2008): 1960–68. http://dx.doi.org/10.1210/en.2007-1573.
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PTH is an important peptide hormone regulator of calcium homeostasis and osteoblast function. However, its mechanism of action in osteoblasts is poorly understood. Our previous study demonstrated that PTH activates mouse osteocalcin (Ocn) gene 2 promoter through the osteoblast-specific element 1 site, a recently identified activating transcription factor-4 (ATF4) -binding element. In the present study, we examined effects of PTH on ATF4 expression and activity as well as the requirement for ATF4 in the regulation of Ocn by PTH. Results show that PTH elevated levels of ATF4 mRNA and protein in a dose- and time-dependent manner. This PTH regulation requires transcriptional activity but not de novo protein synthesis. PTH also increased binding of nuclear extracts to osteoblast-specific element 1 DNA. PTH stimulated ATF4-dependent transcriptional activity mainly through protein kinase A with a lesser requirement for protein kinase C and MAPK/ERK pathways. Lastly, PTH stimulation of Ocn expression was lost by small interfering RNA down-regulation of ATF4 in MC-4 cells and Atf4−/− bone marrow stromal cells. Collectively, these studies for the first time demonstrate that PTH increases ATF4 expression and activity and that ATF4 is required for PTH induction of Ocn expression in osteoblasts.
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Yang, Xi, Xin Gao, Yibei Zhu, et al. "ATF4 is induced by ROS and regulates Th1 and Th17 cells (P6219)." Journal of Immunology 190, no. 1_Supplement (2013): 115.5. http://dx.doi.org/10.4049/jimmunol.190.supp.115.5.
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Abstract Reactive oxygen species (ROS) play a major role in the pathogenesis of chronic inflammatory and autoimmune diseases. It is well established that ROS modulate T cell-mediated immune responses. However, little is known about underlying molecular mechanisms of how ROS regulate T cell immune responses and how T cells adapt to high levels of ROS. We demonstrated that ROS induced ATF4 in activated T cells. ATF4 is a basic leucine-zipper (bZip) transcription factor, which regulates cellular redox state and amino acid metabolism. We found that, in the high ROS condition, ATF4-/- Th1 cells failed to produce IFN-γ whereas WT Th1 cells still made large amounts of IFN-γ. Inhibition of ROS by addition of antioxidants recovered the IFN-γ production by ATF4-/- Th1 cells, suggesting ATF4 is critically required for the function of Th1 cells in the presence of high levels of ROS. ATF4-/- antigen presenting cells (APC) promoted IL-17 production, suggesting ATF4 plays an important role in APCs in suppressing Th17-driving cytokines. In addition, we demonstrated that experimental allergic encephalomyelitis (EAE) was exacerbated in ATF4 deficient mice compared with wild type (WT) mice. The number of autoreactive Th1 cells was decreased whereas that of autoreactive Th17 cells was elevated in ATF4-/- mice compared with WT mice. Our study establishes that ATF4 promotes Th1 and suppresses Th17 immune responses and likely plays an important role in autoimmunity and cancer immunology.
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Huang, Peng, Scott A. Peslak, Xianjiang Lan, et al. "Heme-Regulated Inhibitor (HRI) Activates Transcription Factor ATF4 to Promote BCL11A Transcription and Fetal Hemoglobin Silencing." Blood 134, Supplement_1 (2019): 814. http://dx.doi.org/10.1182/blood-2019-123837.
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Reactivation of fetal hemoglobin in adult red blood cells benefits patients with sickle cell disease and β-thalassemia. BCL11A is one of the predominant repressors of fetal γ-globin transcription and stands as an appealing target for therapeutic genome manipulation. However, pharmacologic perturbation of BCL11A function or its co-regulators remains an unmet challenge. Previously, we reported the discovery of the erythroid-enriched protein kinase HRI as a novel regulator of γ-globin transcription and found that HRI functions in large part via controlling the levels of BCL11A transcription (Grevet et al., Science, 2018). However, the specific mechanisms underlying HRI-mediated modulation of BCL11A levels remain unknown. To identify potential HRI-controlled transcription factors that regulate BCL11A, we performed a domain-focused CRISPR screen that targeted the DNA binding domains of 1,447 genes in the human erythroid cell line HUDEP2. Activating transcription factor 4 (ATF4) emerged as a novel γ-globin repressor. Prior studies reported that ATF4 production is under positive influence of HRI. Specifically, HRI phosphorylates translation factor EIF2α which in turn augments translation of ATF4 mRNA. As expected, HRI deficiency reduced ATF4 protein amounts in HUDEP2 and primary erythroid cells. We further found that the degree of γ-globin reactivation was similar in ATF4 and HRI-depleted cells. ATF4 ChIP-seq in both HUDEP2 and primary erythroblast identified 4,547 and 3,614 high confidence binding sites, respectively. Notably, we did not observe significant enrichment of ATF4 binding or even the presence of an ATF4 consensus motif at the γ-globin promoters, suggesting that ATF4 regulates the γ-globin genes indirectly. However, ATF4 specifically bound to one of the three major BCL11A erythroid enhancers (+55) in both cell types. This was the sole binding site within the ~0.5Mb topologically associating domain that contains the BCL11A gene. Eliminating this ATF4 motif via CRISPR guided genome editing lowered BCL11A mRNA levels and increased γ-globin transcription. Capture-C showed that ATF4 knock-out or removal of the ATF4 site at the BCL11A (+55) enhancer decreased chromatin contacts with the BCL11A promoter. Forced expression of BCL11A largely restored γ-globin silencing in cells deficient for ATF4 or lacking the ATF4 motif in the BCL11A (+55) enhancer. An unexplained observation from our prior study was that HRI loss did not significantly lower Bcl11a levels in murine erythroid cells. Therefore, we mutated the analogous ATF4 motif in the Bcl11a enhancer in the murine erythroid cell line G1E. Unlike in human cells, Bcl11a mRNA synthesis was decreased only very modestly, and there was no effect on the murine embryonic globin genes whose silencing requires Bcl11a. This suggests that the species specific regulation of BCL11A by HRI results from divergent functional roles of ATF4 binding at the BCL11A (+55) enhancer. In sum, our studies uncover a major pathway that extends linearly from HRI to ATF4 to BCL11A to γ-globin. Moreover, these results further support HRI as a pharmacologic target for the selective regulation of BCL11A and γ-globin. Disclosures Blobel: Pfizer: Research Funding; Bioverativ: Research Funding.
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Zeng, Peng, Shengnan Sun, Rui Li, Zhi-Xiong Xiao, and Hu Chen. "HER2 Upregulates ATF4 to Promote Cell Migration via Activation of ZEB1 and Downregulation of E-Cadherin." International Journal of Molecular Sciences 20, no. 9 (2019): 2223. http://dx.doi.org/10.3390/ijms20092223.
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HER2 (human epidermal growth factor receptor 2) activation is critical in breast cancer development. HER2 promotes cell proliferation, angiogenesis, survival, and metastasis by activation of PI3K/Akt, Ras/MEK/ERK, and JAK/STAT pathways. However, beyond these signaling molecules, the key proteins underlining HER2-mediated metastasis remain elusive. ATF4 (Activating transcription factor 4), a critical regulator in unfolded protein response (UPR), is implicated in cell migration and tumor metastasis. In this study, we demonstrate that HER2 upregulated ATF4 expression at both mRNA and protein levels, resulting in cell migration increased. In addition, ATF4 upregulated ZEB1 (Zinc finger E-box-binding homeobox 1) and suppressed E-cadherin expression resulting in promoting cell migration. Restoration of E-cadherin expression effectively inhibited HER2- or ATF4-mediated cell migration. In addition, upregulated expression of ATF4 was found in HER2-positive breast cancer specimens. Together, this study demonstrates that ATF4-ZEB1 is important for HER2-mediated cell migration and suggests that ATF4-ZEB1 may be potential therapeutic targets for breast cancer metastasis.
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Barrera-Lopez, Juan F., Guadalupe Cumplido-Laso, Marcos Olivera-Gomez, et al. "Early Atf4 activity drives airway club and goblet cell differentiation." Life Science Alliance 7, no. 3 (2024): e202302284. http://dx.doi.org/10.26508/lsa.202302284.
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Activating transcription factor 4 (Atf4), which is modulated by the protein kinase RNA-like ER kinase (PERK), is a stress-induced transcription factor responsible for controlling the expression of a wide range of adaptive genes, enabling cells to withstand stressful conditions. However, the impact of the Atf4 signaling pathway on airway regeneration remains poorly understood. In this study, we used mouse airway epithelial cell culture models to investigate the role of PERK/Atf4 in respiratory tract differentiation. Through pharmacological inhibition and silencing of ATF4, we uncovered the crucial involvement of PERK/Atf4 in the differentiation of basal stem cells, leading to a reduction in the number of secretory cells. ChIP-seq analysis revealed direct binding of ATF4 to regulatory elements of genes associated with osteoblast differentiation and secretory cell function. Our findings provide valuable insights into the role of ATF4 in airway epithelial differentiation and its potential involvement in innate immune responses and cellular adaptation to stress.
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Yang, Hsiang-Yu, Jhao-Ying Chen, Yen-Nien Huo, et al. "The Role of Sirtuin 1 in Palmitic Acid-Induced Endoplasmic Reticulum Stress in Cardiac Myoblasts." Life 12, no. 2 (2022): 182. http://dx.doi.org/10.3390/life12020182.
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Background: Lipotoxicity causes endoplasmic reticulum (ER) stress, leading to cell apoptosis. Sirtuin 1 (Sirt1) regulates gene transcription and cellular metabolism. In this study, we investigated the role of Sirt1 in palmitate-induced ER stress. Methods: Both H9c2 myoblasts and heart-specific Sirt1 knockout mice fed a palmitate-enriched high-fat diet were used. Results: The high-fat diet induced C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) expression in both Sirt1 knockout mice and controls. The Sirt1 knockout mice showed higher CHOP and ATF4 expression compared to those in the control. Palmitic acid (PA) induced ATF4 and CHOP expression in H9c2 cells. PA-treated H9c2 cells showed decreased cytosolic NAD+/NADH alongside reduced Sirt1′s activity. The H9c2 cells showed increased ATF4 and CHOP expression when transfected with plasmid encoding dominant negative mutant Sirt1. Sirt1 activator SRT1720 did not affect CHOP and ATF4 expression. Although SRT1720 enhanced the nuclear translocation of ATF4, the extent of the binding of ATF4 to the CHOP promoter did not increase in PA treated-H9c2 cells. Conclusion: PA-induced ER stress is mediated through the upregulation of ATF4 and CHOP. Cytosolic NAD+ concentration is diminished by PA-induced ER stress, leading to decreased Sirt1 activity. The Sirt1 activator SRT1720 promotes the nuclear translocation of ATF4 in PA-treated H9c2 cells.
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van der Meer, Laurens T., Laurensia Yuniati, Esther J. H. Tijchon, et al. "Loss Of Tumor Suppressor BTG1 Enhances ATF4 Function and Promotes Cell Survival." Blood 122, no. 21 (2013): 3796. http://dx.doi.org/10.1182/blood.v122.21.3796.3796.
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Abstract We and others have shown that the B cell Translocation Gene 1 (BTG1) locus is affected by genomic deletions in 9% of pediatric acute lymphoblastic leukemia (ALL) patients. The fact that multiple subclones carrying distinct deletions can be present in individual patients suggests that interfering with normal BTG1 function provides a selective growth advantage to leukemic cells. However, it remains unclear how loss of BTG1 promotes clonal outgrowth. We detected an up to 15-fold increases of BTG1 expression when lymphoid cells were exposed to various challenge conditions, including nutrient limitation and ER stress induction. To test for a functional role for BTG1 in the cellular response to stress, we cultured BTG1 knockout cells in medium without glucose or amino acid (Figure 1) and found that BTG1 knockout cells show a 20-30% improved survival rate as compared to wildtype cells.Figure 1BTG1 knockout cells are resistant to Asparaginase treatment.Figure 1. BTG1 knockout cells are resistant to Asparaginase treatment. As Activating Transcription Factor 4 (ATF4) is a master regulator of cellular stress signaling, we hypothesized that the improved survival after BTG1 loss is regulated via ATF4. By immunoprecipitation experiments, we showed that BTG1 complexes with ATF4. In addition, co-expression of BTG1 attenuates ATF4 transcriptional activity on target gene promoters and suppresses both recombinant and endogenous ATF4 function in these promoter reporter assays (Figure 2).Figure 2BTG1 attenuates ATF4 transcriptional activity.Figure 2. BTG1 attenuates ATF4 transcriptional activity. Although BTG1 possesses no catalytic activity, it functions as a transcriptional co-regulator that acts by recruiting Protein Arginine Methyl Transferase 1 (PRMT1) to transcription factor complexes. By in vitro methylation assays with purified proteins we showed that ATF4 is directly methylated by PRMT1 on a single arginine residue. In addition we found that the PRMT1 interacting domain in BTG1, while dispensable for the BTG1-ATF4 interaction, is essential for the BTG1 mediated suppression of ATF4 function. In search for additional evidence for the functional interaction between BTG1 and ATF4 we performed global expression analysis on murine cells expressing the B cell marker B220. This revealed a significant deregulation of ATF4 target genes in BTG1 knockout cells when compared to wildtype cells. Together, our data indicate that BTG1 suppresses activation of ATF4 in response to cellular stress. Loss of BTG1 function, as it occurs during leukemia development, enhances ATF4 activity, thereby promoting cell survival under cellular stress conditions such as nutrient deprivation or ER stress. Leukemic cells carrying BTG1 deletions may thus benefit from this increased resistance to cellular stress, not only during leukemia development but also during treatment. Hence, targeting the ATF4 stress response pathway may prevent relapse of therapy-resistant leukemic clones. Cells were treated with 2 IU/L Asparaginase for 24 hours. After treatment, cell viability was measured using an MTT assay. The average of 4 independent experiments is plotted with error bars representing the standard error of the mean. A luciferase reporter gene controlled by the ATF4 responsive ASNS promoter region was transfected into HEK293 cells. Asparaginase treatment induces endogenous ATF4 expression, which results in an increase in luciferase signal (Mock transfected cells). Co-expression of BTG1 represses both endogenous ATF4 activity as well as ectopically expressed ATF4 activity as detected by a decrease in luciferase signal. The average of 2 independent experiments is plotted with error bars representing the standard deviation. Disclosures: No relevant conflicts of interest to declare.
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Zuo, Meng-yu, Tong-juan Tang, Xiang Wang, et al. "Atractylenolide III Attenuates Apoptosis in H9c2 Cells by Inhibiting Endoplasmic Reticulum Stress through the GRP78/PERK/CHOP Signaling Pathway." Evidence-Based Complementary and Alternative Medicine 2022 (September 14, 2022): 1–12. http://dx.doi.org/10.1155/2022/1149231.
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The objective of this study was to determine the effect of atractylenolide III (ATL-III) on endoplasmic reticulum stress (ERS) injury, H9c2 cardiomyocyte apoptosis induced by tunicamycin (TM), and the GRP78/PERK/CHOP signaling pathway. Molecular docking was applied to predict the binding affinity of ATL-III to the key proteins GRP78, PERK, IREα, and ATF6 in ERS. Then, in vitro experiments were used to verify the molecular docking results. ERS injury model of H9c2 cells was established by TM. Cell viability was detected by MTT assay, and apoptosis was detected by Hoechst/PI double staining and flow cytometry. Protein expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax, Bcl-2, and Caspase-3 were detected by Western blot. And mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4 were detected by RT-qPCR. Moreover, the mechanism was further studied by using GRP78 inhibitor (4-phenylbutyric acid, 4-PBA), and PERK inhibitor (GSK2656157). The results showed that ATL-III had a good binding affinity with GRP78, and the best binding affinity was with PERK. ATL-III increased the viability of H9c2 cells, decreased the apoptosis rate, downregulated Bax and Caspase-3, and increased Bcl-2 compared with the model group. Moreover, ATL-III downregulated the protein and mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4, consistent with the inhibition of 4-PBA. ATL-III also decreased the expression levels of PERK, eIF2α, ATF4, CHOP, Bax, and Caspase-3, while increasing the expression of Bcl-2, which is consistent with GSK2656157. Taken together, ATL-III could inhibit TM-induced ERS injury and H9c2 cardiomyocyte apoptosis by regulating the GRP78/PERK/CHOP signaling pathway and has myocardial protection.
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Nishikawa, Sakiko, Yuka Itoh, Muneshige Tokugawa, et al. "Kurarinone from Sophora Flavescens Roots Triggers ATF4 Activation and Cytostatic Effects Through PERK Phosphorylation." Molecules 24, no. 17 (2019): 3110. http://dx.doi.org/10.3390/molecules24173110.
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In response to cellular stresses, activating transcriptional factor 4 (ATF4) regulates the expression of both stress-relieving genes and apoptosis-inducing genes, eliciting cell fate determination. Since pharmacological activation of ATF4 exerts potent anti-tumor effects, modulators of ATF4 activation may have potential in cancer therapy. We herein attempted to identify small molecules that activate ATF4. A cell-based screening to monitor TRB3 promoter activation was performed using crude drugs used in traditional Japanese Kampo medicine. We found that an extract from Sophora flavescens roots exhibited potent TRB3 promoter activation. The activity-guided fractionation revealed that kurarinone was identified as the active ingredient. Intriguingly, ATF4 activation in response to kurarinone required PKR-like endoplasmic reticulum kinase (PERK). Moreover, kurarinone induced the cyclin-dependent kinase inhibitor p21 as well as cytostasis in cancer cells. Importantly, the cytostatic effect of kurarinone was reduced by pharmacological inhibition of PERK. These results indicate that kurarinone triggers ATF4 activation through PERK and exerts cytostatic effects on cancer cells. Taken together, our results suggest that modulation of the PERK-ATF4 pathway with kurarinone has potential as a cancer treatment.
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Wu, Shengyu, Xiaolu Zhu, Biechuan Guo, et al. "Unfolded Protein Response Pathways Correlatively Modulate Endoplasmic Reticulum Stress Responses in Rat Retinal Müller Cells." Journal of Ophthalmology 2019 (February 24, 2019): 1–12. http://dx.doi.org/10.1155/2019/9028483.
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Background. Endoplasmic reticulum stress (ERS) in the retinal Müller cells is a key factor contributing to the retinal inflammation and vascular leakage in diabetic retinopathy (DR). This study was to investigate the underlying mechanisms through which the 3 main unfolded protein response (UPR) pathways regulate ERS and to examine the expression levels of vascular endothelial growth factor (VEGF) in Müller cells in vitro. Methods. Rat Müller cell lines were stimulated with high glucose to mimic a diabetic environment in vitro. PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) were downregulated or upregulated with shRNA or overexpression plasmids. The transfected Müller cells were cultivated in high glucose medium for 48 hours. Expression of glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP1), ATF6, and VEGF was examined with immunofluorescence and western blot. Results. Our data indicated that ERS was found in both high glucose and osmotic control groups. Overexpression or downregulation of UPR pathways effectively increased or reduced the production of GRP78, ATF4, XBP1, ATF6, and VEGF, respectively. These 3 signaling pathways had similar regulatory effects on VEGF. Conclusion. The 3 UPR-mediated inflammatory pathways were dependent on each other. Inhibition any of these signaling pathways in UPR might be a potential therapeutic target for DR.
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He, Li, Meng, Wu, Zhao, and Zhao. "Parkin-Dependent Mitophagy is Required for the Inhibition of ATF4 on NLRP3 Inflammasome Activation in Cerebral Ischemia-Reperfusion Injury in Rats." Cells 8, no. 8 (2019): 897. http://dx.doi.org/10.3390/cells8080897.
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Background: Nod-like receptor protein 3 (NLRP3) inflammasome is a crucial contributor in the inflammatory process during cerebral ischemia/reperfusion (I/R) injury. ATF4 plays a pivotal role in the pathogenesis of cerebral I/R injury, however, its function and underlying mechanism are not fully characterized yet. In the current study, we examined whether ATF4 ameliorates cerebral I/R injury by inhibiting NLRP3 inflammasome activation and whether mitophagy is involved in this process. In addition, we explored the role of parkin in ATF4-mediated protective effects. Method: To address these issues, healthy male adult Sprague-Dawley rats were exposed to middle cerebral artery occlusion for 1 h followed by 24 h reperfusion. Adeno-associated virus (AAV) and siRNA were injected into rats to overexpress and knockdown ATF4 expression, respectively. After pretreatment with AAV, mdivi-1(mitochondrial division inhibitor-1) was injected into rats to block mitophagy activity. Parkin expression was knockdown using specific siRNA after AAV pretreatment. Result: Data showed that ATF4 overexpression induced by AAV was protective against cerebral I/R injury, as evidenced by reduced cerebral infraction volume, decreased neurological scores and improved outcomes of HE and Nissl staining. In addition, overexpression of ATF4 gene was able to up-regulate Parkin expression, enhance mitophagy activity and inhibit NLRP3 inflammasome-mediated inflammatory response. ATF4 knockdown induced by siRNA resulted in the opposite effects. Furthermore, ATF4-mediated inhibition of NLRP3 inflammasome activation was strongly affected by mitophagy blockage upon mdivi-1 injection. Besides, ATF4-mediated increase of mitophagy activity and inhibition of NLRP3 inflammasome activation were effectively reversed by Parkin knockdown using siRNA. Conclusion: Our study demonstrated that ATF4 is able to alleviate cerebral I/R injury by suppressing NLRP3 inflammasome activation through parkin-dependent mitophagy activity. These results may provide a new strategy to relieve cerebral I/R injury by modulating mitophagy-NLRP3 inflammasome axis.
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Ma, Shihong, Hui Wang, Wanling Li, Zhe Yan, Xuanming Luo, and Pinxiang Lu. "The correlation between the expression of ATF4 and procalcitonin combined with the detection of RET mutation and the pathological stage and clinical prognosis of medullary thyroid carcinoma." Canadian Journal of Physiology and Pharmacology 100, no. 1 (2022): 19–25. http://dx.doi.org/10.1139/cjpp-2021-0316.
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To explore the correlation between the activating transcription factor 4 (ATF4) and procalcitonin (PCT) expressions combined with RET mutation and the pathological staging and clinical prognosis of sporadic medullary thyroid carcinoma (SMTC). Fifty cases (tumor tissue) of SMTC diagnosed by clinicopathology were collected and the patients with nodular goiter were selected as normal control. The RET mutation site was analyzed by detection kit and expressions of PCT and ATF4 in SMTC were analyzed by Western blot and immunohistochemistry. Multiple linear regression was used to analyze the correlation of risk factors (PCT or ATF4 expression, RET mutation, tumor differentiation, SMTC stage, lymphatic metastasis) for 5-year recurrence and survival of SMTC. The ATF4 and PCT expressions were significantly decreased and increased, respectively, with the increase of the SMTC stage. The most frequent mutation of RET gene in cancer tissue was M 22458A in exon 16. The ATF4 and PCT expressions, as well as RET mutation, were significantly associated with a 5-year recurrence, while the ATF4 expression was significantly related to better 5-year survival. ATF4 and PCT expressions combined with RET mutation are related to the clinical prognosis of SMTC and can predict SMTC staging.
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Lassot, Irina, Emmanuel Ségéral, Clarisse Berlioz-Torrent та ін. "ATF4 Degradation Relies on a Phosphorylation-Dependent Interaction with the SCFβTrCPUbiquitin Ligase". Molecular and Cellular Biology 21, № 6 (2001): 2192–202. http://dx.doi.org/10.1128/mcb.21.6.2192-2202.2001.
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ABSTRACT The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein βTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IκBα and β-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of βTrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of βTrCP. The F-box-deleted βTrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCFβTrCP complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.
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Proshkin, Sergey A., Elena K. Shematorova та George V. Shpakovski. "The Human Isoform of RNA Polymerase II Subunit hRPB11bα Specifically Interacts with Transcription Factor ATF4". International Journal of Molecular Sciences 21, № 1 (2019): 135. http://dx.doi.org/10.3390/ijms21010135.
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Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial α subunit and forms a complex with Rpb3 subunit analogous to prokaryotic α2 homodimer, which is involved in RNA polymerase assembly and promoter recognition. In humans, a POLR2J gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown. To further characterize the minor human specific isoform of RNA polymerase II subunit hRPB11bα, the only one from hRPB11 (POLR2J) homologues that can replace its yeast counterpart in vivo, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library. By this analysis and subsequent co-purification assay in vitro, we identified transcription factor ATF4 as a prominent partner of the minor RNA polymerase II (RNAP II) subunit hRPB11bα. We demonstrated that the hRPB11bα interacts with leucine b-Zip domain located on the C-terminal part of ATF4. Overexpression of ATF4 activated the reporter more than 10-fold whereas co-transfection of hRPB11bα resulted in a 2.5-fold enhancement of ATF4 activation. Our data indicate that the mode of interaction of human RNAP II main (containing major for of hRPB11 subunit) and minor (containing hRPB11bα isoform of POLR2J subunit) transcription enzymes with ATF4 is certainly different in the two complexes involving hRPB3–ATF4 (not hRPB11a–ATF4) and hRpb11bα–ATF4 platforms in the first and the second case, respectively. The interaction of hRPB11bα and ATF4 appears to be necessary for the activation of RNA polymerase II containing the minor isoform of the hRPB11 subunit (POLR2J) on gene promoters regulated by this transcription factor. ATF4 activates transcription by directly contacting RNA polymerase II in the region of the heterodimer of α-like subunits (Rpb3–Rpb11) without involving a Mediator, which provides fast and highly effective activation of transcription of the desired genes. In RNA polymerase II of Homo sapiens that contains plural isoforms of the subunit hRPB11 (POLR2J), the strength of the hRPB11–ATF4 interaction appeared to be isoform-specific, providing the first functional distinction between the previously discovered human forms of the Rpb11 subunit.
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Ross, Joseph, Kamiko Bressler, and Nehal Thakor. "Eukaryotic Initiation Factor 5B (eIF5B) Cooperates with eIF1A and eIF5 to Facilitate uORF2-Mediated Repression of ATF4 Translation." International Journal of Molecular Sciences 19, no. 12 (2018): 4032. http://dx.doi.org/10.3390/ijms19124032.
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A variety of cellular stresses lead to global translation attenuation due to phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2), which decreases the availability of the eIF2-GTP-Met-tRNAi ternary complex. However, a subset of mRNAs continues to be translated by non-canonical mechanisms under these conditions. In fact, although translation initiation of activating transcription factor 4 (ATF4) is normally repressed by an upstream open reading frame (uORF), a decreased availability of ternary complex leads to increased translation of the main ATF4-coding ORF. We show here that siRNA-mediated depletion of eIF5B—which can substitute for eIF2 in delivering Met-tRNAi—leads to increased levels of ATF4 protein in mammalian cells. This de-repression is not due to phosphorylation of eIF2α under conditions of eIF5B depletion. Although eIF5B depletion leads to a modest increase in the steady-state levels of ATF4 mRNA, we show by polysome profiling that the depletion of eIF5B enhances ATF4 expression primarily at the level of translation. Moreover, eIF5B silencing increases the expression of an ATF4-luciferase translational reporter by a mechanism requiring the repressive uORF2. Further experiments suggest that eIF5B cooperates with eIF1A and eIF5, but not eIF2A, to facilitate the uORF2-mediated repression of ATF4 translation.
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Zhao, Yunze, Jie Zhou, Dan Liu, et al. "ATF4 plays a pivotal role in the development of functional hematopoietic stem cells in mouse fetal liver." Blood 126, no. 21 (2015): 2383–91. http://dx.doi.org/10.1182/blood-2015-03-633354.
Full textAbstract:
Abstract The fetal liver (FL) serves as a predominant site for expansion of functional hematopoietic stem cells (HSCs) during mouse embryogenesis. However, the mechanisms for HSC development in FL remain poorly understood. In this study, we demonstrate that deletion of activating transcription factor 4 (ATF4) significantly impaired hematopoietic development and reduced HSC self-renewal in FL. In contrast, generation of the first HSC population in the aorta-gonad-mesonephros region was not affected. The migration activity of ATF4−/− HSCs was moderately reduced. Interestingly, the HSC-supporting ability of both endothelial and stromal cells in FL was significantly compromised in the absence of ATF4. Gene profiling using RNA-seq revealed downregulated expression of a panel of cytokines in ATF4−/− stromal cells, including angiopoietin-like protein 3 (Angptl3) and vascular endothelial growth factor A (VEGFA). Addition of Angptl3, but not VEGFA, partially rescued the repopulating defect of ATF4−/− HSCs in the culture. Furthermore, chromatin immunoprecipitation assay in conjunction with silencing RNA-mediated silencing and complementary DNA overexpression showed transcriptional control of Angptl3 by ATF4. To summarize, ATF4 plays a pivotal role in functional expansion and repopulating efficiency of HSCs in developing FL, and it acts through upregulating transcription of cytokines such as Angptl3 in the microenvironment.
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Verginadis, Ioannis I., Harris Avgousti, Kyle Kim, et al. "Abstract 3178: A stromal integrated stress response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumor progression." Cancer Research 82, no. 12_Supplement (2022): 3178. http://dx.doi.org/10.1158/1538-7445.am2022-3178.
Full textAbstract:
Abstract Despite recent advances in prevention and treatment, including immune checkpoint inhibitors, malignant melanoma remains a particularly aggressive and deadly malignancy, which is partly attributed to its highly heterogeneous TME. However, malignant cells exhibit altered signaling pathways, which enables them to adapt to both cell-intrinsic and extrinsic stressors within TME. The activating transcription factor 4 (ATF4) is a master transcriptional effector of the Integrated Stress Response (ISR), a homeostatic mechanism coupling cell growth and survival to bioenergetic demands. We and others have established a critical tumor cell-intrinsic role of ATF4 which culminates in the promotion of primary tumor growth and in the establishment of micro- and macro-metastases in xenograft, allograft and transgenic models. However, the potential roles of the ISR and particularly of ATF4-mediated responses in host-dependent, tumor-related processes, have not been yet extensively investigated. Using novel conditional knockout ATF4 mouse models, we show that global loss of host ATF4 results in deficient tumor vascularization and a pronounced tumor growth delay in syngeneic melanoma and pancreatic tumor models. Immunofluorescence analysis revealed a severely impaired angiogenic phenotype in tumors grown in ATF4 KO mice which was accompanied by deficiencies in markers of CAF activation. Single-cell transcriptomic analysis of B16F10 melanoma tumors further localized this defect to a distinct CAF population, previously identified as vascular CAFs (vCAFs), and revealed a significant reduction in the expression of extracellular matrix components, primarily type I collagen, in tumors grown in ATF4 KO mice. Intriguingly, we identified a multifaceted impairment of the collagen biosynthetic pathway with the ATF4 to directly regulate the expression of the Col1a1 gene as well as the intracellular levels of glycine and proline, the major amino acids comprising collagen fibers. Moreover, we showed that the ATF4-deficient vCAFs secrete significantly lower levels of angiogenic factors (i.e., VEGF, SDF-1 etc.) in the perivascular area leading to an abnormal angiogenesis and significant attenuation of tumor growth. Specific deletion of ATF4 in the fibroblast compartment (Col1a1 promoter) produced a similar tumor growth delay as in the global ATF4 KO mice, and notably, co-injection of fibroblasts from ATF4-proficient mice led to significant recovery of tumor growth rates in ATF4-deficient mice. Finally, analysis of human melanoma and pancreatic tumor samples revealed a strong correlation between ATF4 and collagen levels and between an ISR gene signature and expression of collagen and CAF activation genes. Our findings uncover a novel role of stromal ATF4 in shaping CAF functionality, a key driver of disease progression, metastasis, and therapy resistance. Citation Format: Ioannis I. Verginadis, Harris Avgousti, Kyle Kim, Giorgos Skoufos, Frank Chinga, Nektaria Maria Leli, Ilias V. Karagounis, Brett I. Bell, Anastasia Velalopoulou, Victoria S. Wu, Yang Li, Jiangbin Ye, David A. Scott, Andrei L. Osterman, Arjun Sengupta, Aalim Weljie, Artemis G. Hatzigeorgiou, Sandra Ryeom, Alan J. Diehl, Serge Y. Fuchs, Ellen Puré, Constantinos Koumenis. A stromal integrated stress response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3178.