Identification of E2F1 as an Important Transcription Factor for the Regulation of Tapasin Expression
2010; Elsevier BV; Volume: 285; Issue: 40 Linguagem: Inglês
10.1074/jbc.m109.094284
ISSN1083-351X
AutoresJuergen Bukur, F Herrmann, Diana Handke, Christian V. Recktenwald, Barbara Seliger,
Tópico(s)Cell Adhesion Molecules Research
ResumoHER-2/neu overexpression in tumor cells caused abnormalities of MHC class I surface expression due to impaired expression of components of the antigen-processing machinery (APM) including the low molecular weight proteins, the transporter associated with antigen processing (TAP), and the chaperone tapasin, whereas the expression of MHC class I heavy chain as well as β2-microglobulin was only marginally affected. This oncogene-mediated deficient APM component expression could be reverted by interferon-γ treatment, suggesting a deregulation rather than structural alterations as underlying molecular mechanisms. To determine the level of regulation, the transcriptional activity of APM components was analyzed in HER-2/neu− and HER-2/neu+ cells. All major APM components were transcriptionally down-regulated in HER-2/neu+ when compared with HER-2/neu− cells, which was accompanied by a reduced binding of RNA polymerase II to the APM promoters. Site-directed mutagenesis of the p300- and E2F-binding sites in the APM promoters did not reconstitute the oncogene-mediated decreased transcription rate with the exception of tapasin, which was restored in HER-2/neu+ cells to levels of wild type tapasin promoter activity in HER-2/neu− fibroblasts. The E2F-directed control of tapasin expression was further confirmed by chromatin immunoprecipitation analyses showing that E2F1 and p300 bind to the tapasin and APM promoters in both cell lines. Moreover, siRNA-mediated silencing of E2F1 was associated with an increased tapasin expression, whereas transient overexpression of E2F1 launch a reduced tapasin transcription, suggesting that E2F1 is an essential transcription factor for tapasin. HER-2/neu overexpression in tumor cells caused abnormalities of MHC class I surface expression due to impaired expression of components of the antigen-processing machinery (APM) including the low molecular weight proteins, the transporter associated with antigen processing (TAP), and the chaperone tapasin, whereas the expression of MHC class I heavy chain as well as β2-microglobulin was only marginally affected. This oncogene-mediated deficient APM component expression could be reverted by interferon-γ treatment, suggesting a deregulation rather than structural alterations as underlying molecular mechanisms. To determine the level of regulation, the transcriptional activity of APM components was analyzed in HER-2/neu− and HER-2/neu+ cells. All major APM components were transcriptionally down-regulated in HER-2/neu+ when compared with HER-2/neu− cells, which was accompanied by a reduced binding of RNA polymerase II to the APM promoters. Site-directed mutagenesis of the p300- and E2F-binding sites in the APM promoters did not reconstitute the oncogene-mediated decreased transcription rate with the exception of tapasin, which was restored in HER-2/neu+ cells to levels of wild type tapasin promoter activity in HER-2/neu− fibroblasts. The E2F-directed control of tapasin expression was further confirmed by chromatin immunoprecipitation analyses showing that E2F1 and p300 bind to the tapasin and APM promoters in both cell lines. Moreover, siRNA-mediated silencing of E2F1 was associated with an increased tapasin expression, whereas transient overexpression of E2F1 launch a reduced tapasin transcription, suggesting that E2F1 is an essential transcription factor for tapasin. IntroductionThe expression of multiple components of the antigen-processing machinery (APM) 2The abbreviations used are: APMantigen-processing machineryβ2-mβ2-microglobulinHCMHC class I heavy chainLMPlow molecular weight proteinlucluciferasemutmutantRNA pol IIRNA polymerase IITAPtransporter associated with antigen processingTFtranscription factorTFBStranscription factor-binding siteCREBcAMP-response element-binding proteinCBPCREB-binding proteinIRFinterferon regulatory transcription factorqRT-PCRquantitative RT-PCRmutmutant. is a prerequisite for constitutive MHC class I surface expression and necessary for recognition of non-self antigens by CD8+ cytotoxic T lymphocytes (1Garbi N. Tanaka S. van den Broek M. Momburg F. Hämmerling G.J. Immunol. Rev. 2005; 207: 77-88Crossref PubMed Scopus (33) Google Scholar, 2Jensen P.E. Nat. Immunol. 2007; 8: 1041-1048Crossref PubMed Scopus (250) Google Scholar, 3Raghavan M. Del Cid N. Rizvi S.M. Peters L.R. 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We here show for the first time an important role for E2F1 in the transcriptional regulation of tapasin in untransformed and HER-2/neu-transformed fibroblasts.DISCUSSIONA detailed analysis of the APM components in HER-2/neu− versus HER-2/neu+ cells revealed a strong coordinated down-regulation of the expression of several APM components, with the exception of β2-m and HC (63Herrmann F. Lehr H.A. Drexler I. Sutter G. Hengstler J. Wollscheid U. Seliger B. Cancer Res. 2004; 64: 215-220Crossref PubMed Scopus (100) Google Scholar, 67Choudhury A. Charo J. Parapuram S.K. Hunt R.C. Hunt D.M. Seliger B. Kiessling R. Int. J. Cancer. 2004; 108: 71-77Crossref PubMed Scopus (131) Google Scholar). However, the molecular mechanisms involved in the impaired APM component expression in the HER-2/neu+ cells have not yet been identified.By investigating the TAP1, LMP2, TAP2, and tapasin promoter activity, a transcriptional down-regulation of the respective APM promoters was found in HER-2/neu-overexpressing fibroblasts when compared with non-transformed cells varying from 38% (TAP2) to 84% (LMP2). These data were further confirmed by qRT-PCR analyses of HER-2/neu− versus HER-2/neu+ fibroblasts. Based on these results, we postulated that the mRNA levels of APM components could be modulated by (i) an instability of transcription initiation due to the absence of the enhancing transcription factor CBP/p300, which might be limited in oncogene-transformed cells (8Setiadi A.F. David M.D. Seipp R.P. Hartikainen J.A. Gopaul R. Jefferies W.A. Mol. Cell Biol. 2007; 27: 7886-7894Crossref PubMed Scopus (53) Google Scholar), or the presence of negatively acting suppressors; (ii) a chromatin-mediated physical barrier in HER-2/neu+ cells, thereby reducing the access of the RNA pol II complex to the tapasin promoter; or (iii) the absence of transcription elongation factors, which might control the constitutive transcription level in a gene- or locus-specific manner (70Ares Jr., M. Proudfoot N.J. Cell. 2005; 120: 163-166Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 71Brès V. Yoh S.M. Jones K.A. Curr. Opin. Cell Biol. 2008; 20: 334-340Crossref PubMed Scopus (168) Google Scholar, 72Fujita T. Piuz I. Schlegel W. FEBS Lett. 2009; 583: 2893-2898Crossref PubMed Scopus (16) Google Scholar).To address these questions, the role of TFBS controlling the TAP1/LMP2, TAP2, and tapasin promoter activity was determined using TFBS KO mutants generated by site-directed mutagenesis (62Herrmann F. Trowsdale J. Huber C. Seliger B. Immunogenetics. 2003; 55: 379-388Crossref PubMed Scopus (17) Google Scholar). Mutations of the E2F and p300 TFBS caused a reconstitution of the tapasin promoter activity in HER-2/neu+ cells, suggesting the involvement of E2F1 and p300 in the transcriptional down-regulation of tapasin in oncogenic transformants. In contrast, E2F is not crucial for the HER-2/neu-mediated down-regulation of TAP1 transcription, whereas the E2F-binding site at position −85 plays an important role for TAP2 transcription. These data were further validated by investigating the access of RNA pol II complexes to the APM promoters using ChIP. The decreased binding of RNA pol II to the APM promoters in HER-2/neu+ cells mirrored the decreased transcription level of tapasin, TAP1, LMP2, and TAP2 in these cells. These results are in accordance with a recent report by Jefferies and co-workers (8Setiadi A.F. David M.D. Seipp R.P. Hartikainen J.A. Gopaul R. Jefferies W.A. Mol. Cell Biol. 2007; 27: 7886-7894Crossref PubMed Scopus (53) Google Scholar) demonstrating an association of a weak TAP1 promoter activity with reduced recruitment of CBP to the TAP1 promoter in TAP1-deficient cells.To clarify the role of the histone-acetylating protein p300, a slightly reduced attachment of p300 to tapasin and LMP2 but not to the TAP2 promoter in HER-2/neu+ cells when compared with untransformed cells was found, suggesting a correlation between RNA pol II and p300 access to these promoters. Because the binding of E2F1–3 to promoters is associated with the acquisition of the histone H3 and H4 acetylation (73Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816PubMed Google Scholar, 74Taubert S. Gorrini C. Frank S.R. Parisi T. Fuchs M. Chan H.M. Livingston D.M. Amati B. Mol. 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