C/EBP Homology Protein (CHOP) Interacts with Activating Transcription Factor 4 (ATF4) and Negatively Regulates the Stress-dependent Induction of the Asparagine Synthetase Gene

C/EBP homology protein (CHOP), a stress-induced transcription factor, is involved in transcriptional regulation, cell cycle, and apoptosis. The present studies identified CHOP as an interacting partner of activating transcription factor (ATF) 4 in a yeast two-hybrid screen and confirmed their interaction in HEK293T cells. CHOP protein levels rose modestly and transiently during amino acid deprivation, whereas endoplasmic reticulum stress caused a much higher and sustained expression of CHOP protein. Exogenous CHOP expression enhanced the TRB3 gene induction by amino acid deprivation. Conversely, CHOP suppressed the induction of the endogenous asparagine synthetase (ASNS) gene and inhibited transcription from a reporter gene driven by the ASNS promoter following activation by ATF4 or amino acid deprivation. Short interfering RNA-mediated knockdown of CHOP further enhanced the induction of ASNS by either amino acid deprivation or endoplasmic reticulum stress. The CHOP-dependent repression of the ASNS gene required the entire CHOP protein, arguing against the possibility of simple sequestration of ATF4 by the CHOP leucine zipper domain, and chromatin immunoprecipitation analysis showed association of CHOP with the ASNS and TRB3 promoters. Interestingly, chromatin immunoprecipitation also showed that CHOP was associated with the C/EBP-ATF composite site regions of the SNAT2, VEGF, and CAT-1 genes, despite no significant effect on their expression after exogenous CHOP overexpression. Collectively, the results document that CHOP is a member of the transcription factor network that controls the stress-induced regulation of specific C/EBP-ATF-containing genes, such as ASNS. C/EBP homology protein (CHOP), a stress-induced transcription factor, is involved in transcriptional regulation, cell cycle, and apoptosis. The present studies identified CHOP as an interacting partner of activating transcription factor (ATF) 4 in a yeast two-hybrid screen and confirmed their interaction in HEK293T cells. CHOP protein levels rose modestly and transiently during amino acid deprivation, whereas endoplasmic reticulum stress caused a much higher and sustained expression of CHOP protein. Exogenous CHOP expression enhanced the TRB3 gene induction by amino acid deprivation. Conversely, CHOP suppressed the induction of the endogenous asparagine synthetase (ASNS) gene and inhibited transcription from a reporter gene driven by the ASNS promoter following activation by ATF4 or amino acid deprivation. Short interfering RNA-mediated knockdown of CHOP further enhanced the induction of ASNS by either amino acid deprivation or endoplasmic reticulum stress. The CHOP-dependent repression of the ASNS gene required the entire CHOP protein, arguing against the possibility of simple sequestration of ATF4 by the CHOP leucine zipper domain, and chromatin immunoprecipitation analysis showed association of CHOP with the ASNS and TRB3 promoters. Interestingly, chromatin immunoprecipitation also showed that CHOP was associated with the C/EBP-ATF composite site regions of the SNAT2, VEGF, and CAT-1 genes, despite no significant effect on their expression after exogenous CHOP overexpression. Collectively, the results document that CHOP is a member of the transcription factor network that controls the stress-induced regulation of specific C/EBP-ATF-containing genes, such as ASNS. Mammalian cells have evolved an array of pathways that mediate the cellular response to changes in the environment, including UV irradiation, endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; AAR, amino acid response; AARE, amino acid response element; ASNS, asparagine synthetase; ATF, activating transcription factor; CAT-1, cationic amino acid transporter 1; C/EBP, CCAAT/enhancer-binding protein; CHOP, C/EBP homology protein; ChIP, chromatin immunoprecipitation; HisOH, histidinol; NSRE, nutrient sensing response element; qPCR, quantitative real time PCR; RT, reverse transcriptase; Tg, thapsigargin; TRB3, tribbles 3; SNAT2, system A transporter 2; VEGF, vascular endothelial growth factor; MEM, minimal essential medium; PERK, protein kinase-like endoplasmic reticulum kinase; aa, amino acids; siRNA, short interfering RNA; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UPR, unfolded protein response; IP, immunoprecipitation; co-IP, co-immunoprecipitation; GFP, green fluorescent protein; HA, hemagglutinin; bZIP, basic leucine zipper. stress, and deprivation of nutrients, such as amino acids and glucose (1.Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2381) Google Scholar). The cascade of events that is triggered by ER stress is referred to as the unfolded protein response (UPR), and the pathway initiated by amino acid limitation is called the amino acid response (AAR) pathway. These pathways initiate a wide array of adaptive mechanisms and ultimately, if necessary, programmed cell death (1.Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2381) Google Scholar, 2.Zinszner H. Kuroda M. Wang X.Z. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. Gene. Dev. 1998; 12: 982-995Crossref PubMed Scopus (1683) Google Scholar). Amino acid limitation leads to an increase in uncharged tRNA, which binds to and activates a kinase called GCN2 (general control nonderepressible 2) (3.Sood R. Porter A.C. Olsen D.A. Cavener D.R. Wek R.C. Genetics. 2000; 154: 787-801Crossref PubMed Google Scholar, 4.Zhang P. McGrath B.C. Reinert J. Olsen D.S. Lei L. Gill S. Wek S.A. Vattem K.M. Wek R.C. Kimball S.R. Jefferson L.S. Cavener D.R. Mol. Cell. Biol. 2002; 22: 6681-6688Crossref PubMed Scopus (348) Google Scholar, 5.Hinnebusch A.G. Annu. Rev. Microbiol. 2005; 59: 407-450Crossref PubMed Scopus (903) Google Scholar). ER stress, triggered by perturbation of calcium homeostasis, glucose deprivation, or other causes of misfolded protein accumulation in the ER lumen, activates a kinase called PERK (double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase) (6.Okada T. Yoshida H. Akazawa R. Negishi M. Mori K. Biochem. J. 2002; 366: 585-594Crossref PubMed Scopus (413) Google Scholar, 7.Harding H.P. Zhang Y. 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Full Text Full Text PDF PubMed Scopus Google as as C/EBP-ATF composite J.L. T. Biochem. J. PubMed Scopus Google Scholar, H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Wang C. I. Y. K. Wek R.C. M. Biochem. J. PubMed Scopus Google Scholar). the ATF4 asparagine synthetase (ASNS) and CCAAT/enhancer-binding protein homology protein (CHOP), also as growth and protein ASNS, which the gene the and of acid to is by ATF4 R. H. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The ASNS promoter and and that as an to mediate the transcriptional activation of the gene by either the or the pathway C. C. J. Biol. 2000; Full Text Full Text PDF PubMed Google Scholar). The is a C/EBP-ATF composite site that by and to ATF4 following of ATF4 following activation of either the or the H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, R. H. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). C/EBP-ATF composite of a C/EBP and a J.L. T. Biochem. J. PubMed Scopus Google Scholar, J.L. T. Mol. Cell. Biol. PubMed Scopus Google Scholar). CHOP was as a gene in response to I. S. A. PubMed Scopus Google and to by amino acid deprivation A. C. Wang Ron D. M. P. J. Biol. Full Text Full Text PDF PubMed Scopus Google and ER stress X.Z. B. H. A. R. Ron D. Mol. Cell. Biol. PubMed Scopus Google Scholar). The C/EBP protein a transcriptional and a which of a basic and a leucine zipper The basic of CHOP and in to essential to the interaction of with C/EBP D. Gene. Dev. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). was that CHOP and the of by their to D. Gene. Dev. PubMed Scopus Google Scholar). to C/EBP in and in CHOP the of other C/EBP to promoter D. Gene. Dev. PubMed Scopus Google Scholar). studies that a as a Kuroda M. J. Batchvarova N. R. P. H. Ron D. J. 1998; PubMed Scopus Google Scholar). TRB3 was also identified as a gene N. S. T. K. H. J. 2005; PubMed Scopus Google Scholar). CHOP an essential in the response to a wide of cell and cell and in response to ER stress H. Kuroda M. Wang X.Z. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. Gene. Dev. 1998; 12: 982-995Crossref PubMed Scopus (1683) Google Scholar). The expression of CHOP is in cell but expression is transcription D. Gene. Dev. PubMed Scopus Google Scholar, J. A. C. P. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). CHOP is of the in the network of transcription and the of CHOP is the mechanisms the cellular response to stress. the of on interaction of CHOP with transcription and the of is T. Mol. Cell. Biol. PubMed Scopus Google that CHOP by a to the effect on C/EBP N. S. T. K. H. J. 2005; PubMed Scopus Google showed that CHOP and ATF4 TRB3 expression during ER stress but or response required CHOP the of ATF4 and C/EBP-ATF composite but the of S. H. J. A.G. S. A. PubMed Scopus Google Scholar). The was from S. J. C. J. PubMed Scopus Google showed that CHOP binds to ATF4 in on and that in a reporter CHOP the of the interaction ATF4 and CHOP was in and the results also that CHOP transcriptional activation of the ASNS The effect of CHOP on the ASNS gene was either the or of the protein, C/EBP domain, CHOP binds to the ASNS which a C/EBP-ATF composite site that the response to amino acid deprivation or ER stress. the CHOP was during amino acid deprivation, the increase in CHOP protein was and the increase in CHOP protein during the was much and was sustained with the of CHOP amino acid deprivation cell whereas ER stress triggered the pathway and CHOP and from ATF4 was by ATF4 protein. and from The following expression of specific ATF4 was to the ATF4 expression driven by the promoter CHOP was by from a and which was used to the protein an or a that the and The reporter was by an ASNS promoter of the reporter gene the site of the ATF4 and green fluorescent protein used to the in HEK293T cells. and from A was as in the from cells with to or of ATF4 by the as and the The and yeast growth and as in the and identified cells or HEK293T cells in minimal essential or to amino 4 and in a and with or and to to that the cells in the Amino acid deprivation was by of cells to HisOH, which of the and deprivation and activation of the cascade C. Biochem. J. PubMed Scopus Google Scholar). ER stress was by of cells to which activates the pathway of of the ER M. C. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). and was from cells or HEK293T cells the including a the to to the the of ASNS quantitative analysis was a system and with I. the the as ASNS, and CHOP, and SNAT2, and VEGF, and TRB3, and CAT-1, and and glyceraldehyde-3-phosphate and the transcription from the ASNS from ASNS and used to as H. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar). chromatin CHOP was by in The was with and was to the immunoprecipitation ATF4 or was by qPCR, as The results as the to from in and the by the the the and used to the ASNS promoter to and and ASNS as a control and a control to the TRB3 promoter to CHOP N. S. T. K. H. J. 2005; PubMed Scopus Google also used in the The the TRB3 gene as and CHOP to the C/EBP-ATF regions of other amino acid was also as SNAT2, and VEGF, and and CAT-1, and The analysis was a and the with I. of used to a the of the and the the The to the by and by in the from to to that a was in the The results as the to from in and the by the CHOP and 4 from cells in a of in and was to the of of and a following cells with PBS, MEM, and The was and with control MEM, HisOH, or a or protein and by or cells in with or of or or a calcium was by the of cells to cells. after cells with PBS, by and by in and 4 with by immunoprecipitation of cell with with a 4 with The by with and the in of and immunoprecipitation of CHOP, cell with and protein 4 with The by and with and the with of to and to the of ATF4 in a yeast two-hybrid to A ATF4 was the and used as the ATF4 showed activation of the A analysis was and ATF4 the The ATF4 transcriptional activation and also showed The ATF4 activation was further amino but The ATF4 that have was the and was used as to screen a from cells that in transcription such as and ATF4 identified and factor to ATF4 P. B. D. J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, L. J.M. J. C. R. J. Biol. 2005; PubMed Scopus Google Scholar). the to the of the CHOP was the entire domain, and the leucine zipper CHOP with ATF4 in further the interaction of CHOP and ATF4 in the protein and CHOP either or in in HEK293T cells. cell to immunoprecipitation with and the of CHOP was by analysis CHOP was in the and CHOP but or CHOP was that the of CHOP with ATF4 was a cell from HEK293T cells CHOP and to with as a ATF4 was from the after with but ATF4 with that ATF4 and CHOP with other in and with the in by S. J. C. J. PubMed Scopus Google Scholar). CHOP from ASNS that the induction of the ASNS gene by amino acid limitation is by ATF4 H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, R. H. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google a CHOP was cells with the reporter and with a CHOP expression and in or ASNS promoter as by was by and of CHOP the induction in a CHOP ATF4 on the ASNS cells with the reporter and expression ATF4 and CHOP cells with of ATF4 expression ASNS promoter was to a with that by of CHOP and ATF4 a CHOP repression of the promoter by Exogenous expression of ATF4 and CHOP no significant effect on the ASNS promoter if the and that CHOP the transcription by the the ASNS of CHOP on CHOP the endogenous expression of ASNS and other amino genes, HEK293T cells with CHOP or green fluorescent protein expression of was as by the of fluorescent cells to cells exogenous CHOP protein by showed a expression from a of cells in the exogenous CHOP and the of endogenous CHOP expression was CHOP the of the endogenous ASNS but the induction of ASNS expression by was by the of the effect of CHOP on the endogenous ASNS gene is to other that by amino acid H. J. Biol. Full Text Full Text PDF PubMed Scopus Google 2002; Google CAT-1 J. Wang C. R. L. I. M. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google and CHOP A. C. Wang Ron D. M. P. J. Biol. Full Text Full Text PDF PubMed Scopus Google by CHOP the CHOP was in a with the regulation of the gene TRB3 was C. C. A.C. L. Y. C. R. P. A. P. J. Biol. Full Text Full Text PDF PubMed Scopus Google have that TRB3 expression is following amino acid and N. S. T. K. H. J. 2005; PubMed Scopus Google that the TRB3 gene that to activation by ATF4 and the TRB3 was modestly by but the increase was enhanced by of These results with TRB3 as a gene and in to the repression of ASNS gene of CHOP expression and following amino acid limitation and ER stress. cells in MEM, HisOH, or the was and by analysis CHOP or The the the with the a of protein and to analysis CHOP, and The is a and the ATF4 the of the cells in MEM, HisOH, or the protein and to analysis and and The is of of CHOP by the and induction of CHOP expression by amino acid deprivation and ER stress was in cells by in MEM, HisOH, or either or CHOP expression was and the time but the was the with the AAR, activation of the caused a induction of CHOP by the of CHOP protein was after or CHOP protein was in the of the protein of CHOP 4 and after during the protein of CHOP 4 a much higher that HisOH, and that the entire The and of CHOP protein expression following amino acid deprivation an of cellular in a the ATF4 protein was also and was of a the pathways with a and induction of ATF4 protein H.P. Novoa I. Zhang Y. Zeng H. Wek R. M. Ron D. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google that the pathway is the pathway the CHOP induction by ER stress. the of ATF4 with the of CHOP induction by amino acid deprivation or ER stress, the ATF4 from to the control and with the CHOP induction The induction of ATF4 by is higher that by the of stress the ATF4 protein was in the cells but in the cells. higher CHOP induction in the cells is with higher ATF4 levels during the of the response also to the CHOP induction the of as the induction of ATF4 was higher that by but the CHOP induction is These that during the or mechanisms that control the CHOP protein ER but Amino the and time of CHOP protein expression following activation of the and that CHOP is to an of cell and to pathways have in of and if with CHOP protein cell in MEM, HisOH, or cell specific and to analysis to of which is an of cell S. D. A. Mol. Biol. Google Scholar). in levels of to of whereas significant was after of of ASNS by CHOP CHOP induction by the and was used to knockdown the CHOP expression and the induction of ASNS by stress pathways was cells with either control or CHOP siRNA, and the cells in MEM, HisOH, or 4 by and and protein expression of CHOP the of CHOP the induction of ASNS transcriptional by either or was further enhanced with the that CHOP protein is during the of ASNS transcription during the was that during These further the that CHOP as a of ASNS transcription in response to the and stress of CHOP Both and of the and with mechanisms have CHOP of which the of CHOP and The from that CHOP with and from D. Gene. Dev. PubMed Scopus Google whereas the is on the that CHOP to specific N. S. T. K. H. J. 2005; PubMed Scopus Google Scholar). the that the leucine zipper of CHOP is to as by the yeast two-hybrid if CHOP ATF4 on the ASNS gene by from to the ASNS the to ATF4 possibility CHOP of ATF4 on the ASNS CHOP, the transcriptional or the of CHOP the expression and their expression was confirmed by HEK293T cells by analysis of the CHOP with the reporter was in with either or ATF4 in cells the CHOP protein the induction of ASNS promoter by or ATF4 overexpression. the and the of CHOP or no effect on the induction of transcription to CHOP to that CHOP of ATF4 the entire protein to the that is associated with CHOP on the ASNS CHOP to analysis was in cells in MEM, HisOH, or 4 The TRB3 promoter in that to CHOP N. S. T. K. H. J. 2005; PubMed Scopus Google Scholar). a control CHOP analysis of the TRB3 promoter was The of CHOP to the TRB3 promoter was following or activation of the and the association of CHOP with the ASNS promoter was also and the was in cells with that in the cells with the that CHOP protein to a higher a the association of CHOP with ASNS was minimal and after or the that the association of CHOP with the ASNS promoter is specific These in with the of that of CHOP to the ASNS promoter is a in the CHOP of ASNS the of CHOP to composite that have amino analysis was also on the SNAT2, VEGF, and CAT-1 and VEGF, was a of that was in response to or CAT-1, the CHOP association after the of CHOP association was CHOP and ATF4 to the a was in cells in MEM, HisOH, or 4 chromatin was with CHOP and to a immunoprecipitation with ATF4 or activation of the or the regions from the TRB3, ASNS, and in the chromatin with the control The of on gene that in the CHOP of on the was during but The of CHOP and ATF4 on the CAT-1 which is with the of CHOP on that a the association of with ASNS was minimal The results in document and the of CHOP on transcriptional activation of ASNS gene during amino acid deprivation or ER stress. The association of in was in cells by 2) CHOP transcriptional activation and the induction of endogenous ASNS expression by amino acid deprivation. CHOP is to during amino acid deprivation, protein induction is and 4 and the CHOP and protein is much and sustained during ER stress. CHOP knockdown results in enhanced induction of endogenous ASNS expression by either amino acid deprivation or ER stress. The effect of CHOP on ATF4 the entire protein, arguing against a by which CHOP from to analysis of ATF4 and CHOP to the ASNS promoter which the site The of triggered by ER stress or amino acid deprivation with the of CHOP protein The of cells to stress a array of cellular The transcription factor ATF4 is by stress such as amino acid deprivation H.P. Novoa I. Zhang Y. Zeng H. Wek R. M. Ron D. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google ER stress M. D. K. R. C. W. R. J. K. K. J.C. K. N. Cell. 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Biol. 2005; PubMed Scopus Google CHOP of either ATF4 or The present analysis that CHOP is associated with the ASNS promoter during amino acid deprivation and ER stress. a that and CHOP to the These results that the ASNS C/EBP-ATF as an to other C/EBP-ATF as quantitative of CHOP association genes, is to that the of the TRB3 which of the C/EBP-ATF was an of the the a is CHOP with ATF4 to the TRB3 whereas N. S. T. K. H. J. 2005; PubMed Scopus Google that the of the TRB3 or that to the of the a The stress response to of the TRB3 gene of of the a a and an AARE, which with the analysis showed that the site and the essential the TRB3 induction by ER stress, but the was also required N. S. T. K. H. J. 2005; PubMed Scopus Google Scholar). The ASNS promoter have the and is by CHOP to the possibility that the involved in the activation or repression by the a by (1.Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2381) Google identified to ATF4 their induction by ER stress. a by Yun S. Novoa I. Zhang Y. R. K. H.P. Ron D. Dev. PubMed Scopus Google identified to have induction following ER stress. Interestingly, of the of identified genes, the gene in studies to with the entire CHOP is also involved in the induction of cell and H. Kuroda M. Wang X.Z. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. Gene. Dev. 1998; 12: 982-995Crossref PubMed Scopus (1683) Google Scholar). the cells used in CHOP protein induction was during amino acid deprivation, and no was whereas CHOP protein was and a much higher during ER stress, and was Collectively, the results in document transcriptional of the mechanisms by which CHOP to the cellular stress pathways other of the and