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. 2015 Jun 19;290(25):15878-15891.
doi: 10.1074/jbc.M114.635144. Epub 2015 Apr 30.

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

Rebecca R Crawford  1 , Eugenia T Prescott  1 , Charity F Sylvester  1 , Ashlee N Higdon  1 , Jixiu Shan  2 , Michael S Kilberg  2 , Imran N Mungrue  3
Affiliations

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

Rebecca R Crawford et al. J Biol Chem. .

Abstract

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

Keywords: ATF3; ATF4; CEBPβ; CHAC1; DNA transcription; DNA-protein interaction; apoptosis; endoplasmic reticulum stress (ER stress); oxidative stress; unfolded protein response (UPR).

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Figures

FIGURE 1.
FIGURE 1.
Bioinformatic identification of CHAC1 promoter elements and the translation start site. A, using an online bioinformatics tool, the CHAC1 promoter sequence was probed for known transcription factor binding motifs. Those discovered are highlighted by boxes and overlaid onto the multiZ alignment of this locus available from the UCSC genome browser. CCAAT (R) refers to a CCAAT box found on the reverse strand. B, luciferase constructs containing 2.6 kb upstream of the CHAC1 transcriptional start and 133 bp of the 5′UTR (including the annotated translation start site) with (+ATG) or without (−ATG) the proposed CHAC1 start codon were cotransfected with 1/20× CMV-Renilla. Raw luciferase values were normalized to raw Renilla values (Student's t test; #, p < 0.001).
FIGURE 2.
FIGURE 2.
Robust ER stress induction of the human CHAC1 promoter requires the ATF/CRE in HEK293 cells. The sequence containing 2.6 kb upstream of the CHAC1 transcriptional start and 133 bp of the 5′UTR without the CHAC1 ATG start codon was cloned upstream of a luciferase reporter gene. Each construct was cotransfected with 1/20× CMV-Renilla. Raw luciferase values were normalized to raw Renilla values and then to −2600/+133 treated with control media, which was set equal to one. The locations of predicted regulatory sites are shown with open boxes. Treatments were thapsigargin (Th), tunicamycin (Tm), and histidinol (HisOH) for 24 h. *, p < 0.05; #, p < 0.001.
FIGURE 3.
FIGURE 3.
Robust ER stress induction of the human CHAC1 promoter requires the ATF/CRE in U2OS cells. Each construct was cotransfected with 1/20× CMV-Renilla. Raw luciferase values were normalized to raw Renilla values and then to −2600/+133 treated with control media, which was set equal to one. The locations of predicted regulatory sites are shown with open boxes. Treatments were Th, Tm, and HisOH for 24 h. *, p < 0.05; #, p < 0.001.
FIGURE 4.
FIGURE 4.
The ATF/CRE and ACM direct transcription of the human CHAC1 promoter in HEK293 cells. A–C, mutational analysis of the ATF/CRE (A), ACM (B), and CARE (C). Each construct was cotransfected with 1/20× CMV-Renilla. Raw luciferase values were normalized to raw Renilla values and then to the wild-type plasmid treated with control. Mutated residues are identified in boldface and underlined. Treatments were Th and Tm for 24 h. Scr, scrambled. *, p < 0.05; #, p < 0.001.
FIGURE 5.
FIGURE 5.
The ATF/CRE and ACM direct transcription of the human CHAC1 promoter in HEK293 cells. A–C, mutational analysis of the ATF/CRE (A), ACM (B), and CARE (C). Each construct was cotransfected with 1/20× CMV-Renilla. Raw luciferase values were normalized to raw Renilla values and then to the wild-type plasmid treated with control. Mutated residues are identified in boldface and underlined. Treatments were Th and Tm for 24 h. Scr, scrambled. *, p < 0.05; #, p < 0.001.
FIGURE 6.
FIGURE 6.
The ATF/CRE and ACM sites drive robust transcription in the absence of other regulatory sequences. A and B, internal deletions in the CHAC1 promoter were created using a Sac1 cloning site. Dashed lines encompass the deleted regions. Each construct was cotransfected with 1/20× CMV-Renilla in HEK cells (A) or U2OS cells (B). Raw luciferase values were normalized to raw Renilla values and then to the wild-type plasmid. Treatments were Th and Tm for 24 h. *, p < 0.05; #, p < 0.001.
FIGURE 7.
FIGURE 7.
ATF4 and CEBPβ transfection activates the CHAC1 promoter. A, cotransfection of CHAC1 promoter reporter plasmids with expression plasmids containing the control plasmid (pMAX-GFP) or various transcription factors and 1/20× CMV-Renilla. Raw luciferase values were normalized to raw Renilla values and then to −267/+133 treated with the control. *, p < 0.05; #, p < 0.001. Mutated sequences are shown with an X. scr, scrambled. B, transcription factor expression was verified using immunoblotting. NRF2 expression was verified using GFP fluorescence prior to harvesting the cells.
FIGURE 8.
FIGURE 8.
The CHAC1 promoter luciferase reporter is more responsive to ATF4 than the CHOP and ASNS promoters. Luciferase reporter plasmids driven by CHAC1, CHOP, and ASNS promoters with all of the required elements for full activity were cotransfected with varying amounts of ATF4 (1× = 0.15 μg) and 1/20× CMV-Renilla. Total DNA was kept constant with a GFP expression plasmid. Raw luciferase values were normalized to raw Renilla values and then to the 1× GFP average of each plasmid. #, p < 0.001 versus ASNS.
FIGURE 9.
FIGURE 9.
ATF4 binds to both the ATF/CRE and ACM sites, whereas ATF3 binds to the ACM. A, short oligonucleotide probes containing wild-type or mutated ATF/CRE and ACM sequences were incubated with nuclear extract cells treated with 3 μm Th versus control for 4 h. B and C, proteins were separated on a native gels to identify ATF4 (B) or ATF3 (C) to determine transcription factor binding. Reactions without oligonucleotides (none) were included to detect free transcription factors.
FIGURE 10.
FIGURE 10.
ATF4, ATF3, and CEBPβ are enriched at the CHAC1 promoter. HEK293 cells were treated with 0 or 3 μm Th for 4 h. A—C, ChIP experiments were performed using ATF4 (A), ATF3 (B), and CEBPβ (C) antibodies and primers specific to the region of interest adjacent to the ATF/CRE and ACM (0 kb) or 1 and 3 kb distal. Data are presented as the bound/input ratio. No antibody samples were used as negative controls and showed little or no binding. One-way analysis of variance, Dunnett's post test; #, p < 0.001 versus 0 μm Th; ♦, p <0.05 versus no antibody. D, immunoblots and densitometry to determine transcription factor expression. Student's t test; *, p < 0.05.
FIGURE 11.
FIGURE 11.
CHAC1 overexpression depletes glutathione. HEK293 cells were transfected with control (pMAX-GFP), CHAC1, or CHAC1-E115Q, a catalytic site mutation, plasmids. Total glutathione was measured by Tietze recycling assay and normalized to total protein. One-way analysis of variance; Bonferroni post test; #, p < 0.001.
FIGURE 12.
FIGURE 12.
A model for the transcriptional regulation of CHAC1. A, under basal conditions, ATF3 with an unknown binding partner regulates CHAC1 transcription from the ATF/CRE. B, induction of the ER stress response leads to ATF4 binding to the ATF/CRE site with ATF3 and to the ACM with an unknown binding partner. CEBPβ is also recruited to the CHAC1 promoter, increasing transcription.
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References

    1. Schröder M., Kaufman R. J. (2005) The mammalian unfolded protein response. Annu. Rev. Biochem. 74, 739–789 - PubMed
    1. Tabas I., Ron D. (2011) Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat. Cell Biol. 13, 184–190 - PMC - PubMed
    1. Bernales S., Papa F. R., Walter P. (2006) Intracellular signaling by the unfolded protein response. Annu. Rev. Cell Dev. Biol. 22, 487–508 - PubMed
    1. Harding H. P., Novoa I., Zhang Y., Zeng H., Wek R., Schapira M., Ron D. (2000) Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099–1108 - PubMed
    1. Yoshizawa T., Hinoi E., Jung D. Y., Kajimura D., Ferron M., Seo J., Graff J. M., Kim J. K., Karsenty G. (2009) The transcription factor ATF4 regulates glucose metabolism in mice through its expression in osteoblasts. J. Clin. Invest. 119, 2807–2817 - PMC - PubMed

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