Epigenetic Changes and Suppression of the Nuclear Factor of Activated T Cell 1 (NFATC1) Promoter in Human Lymphomas with Defects in Immunoreceptor Signaling
2007; Elsevier BV; Volume: 172; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2008.070294
ISSN1525-2191
AutoresAskar M. Akimzhanov, László Krenács, Timm Schlegel, Stefan Klein‐Hessling, Enikő Bagdi, Éva Stelkovics, Eisei Kondo, Sergei Chuvpilo, Philipp Wilke, Andris Avots, Stefan Gattenlöhner, Hans-Konrad Müller-Hermelink, Alois Palmetshofer, Edgar Serfling,
Tópico(s)Nuclear Receptors and Signaling
ResumoThe nuclear factor of activated T cell 1 (Nfatc1) locus is a common insertion site for murine tumorigenic retroviruses, suggesting a role of transcription factor NFATc1 in lymphomagenesis. Although NFATc1 is expressed in most human primary lymphocytes and mature human T- and B-cell neoplasms, we show by histochemical stainings that NFATc1 expression is suppressed in anaplastic large cell lymphomas and classical Hodgkin's lymphomas (HLs). In HL cell lines, NFATc1 silencing correlated with a decrease in histone H3 acetylation, H3-K4 trimethylation, and Sp1 factor binding but with an increase in HP1 binding to the NFATC1 P1 promoter. Together with DNA hypermethylation of the NFATC1 P1 promoter, which we detected in all anaplastic large cell lymphoma and many HL lines, these observations reflect typical signs of transcriptional silencing. In several lymphoma lines, methylation of NFATC1 promoter DNA resulted in a "window of hypomethylation," which is flanked by Sp1-binding sites. Together with the under-representation of Sp1 at the NFATC1 P1 promoter in HL cells, this suggests that Sp1 factors can protect P1 DNA methylation in a directional manner. Blocking immunoreceptor signaling led to NFATC1 P1 promoter silencing and to a decrease in H3 acetylation and H3-K4 methylation but not DNA methylation. This shows that histone modifications precede the DNA methylation in NFATC1 promoter silencing. The nuclear factor of activated T cell 1 (Nfatc1) locus is a common insertion site for murine tumorigenic retroviruses, suggesting a role of transcription factor NFATc1 in lymphomagenesis. Although NFATc1 is expressed in most human primary lymphocytes and mature human T- and B-cell neoplasms, we show by histochemical stainings that NFATc1 expression is suppressed in anaplastic large cell lymphomas and classical Hodgkin's lymphomas (HLs). In HL cell lines, NFATc1 silencing correlated with a decrease in histone H3 acetylation, H3-K4 trimethylation, and Sp1 factor binding but with an increase in HP1 binding to the NFATC1 P1 promoter. Together with DNA hypermethylation of the NFATC1 P1 promoter, which we detected in all anaplastic large cell lymphoma and many HL lines, these observations reflect typical signs of transcriptional silencing. In several lymphoma lines, methylation of NFATC1 promoter DNA resulted in a "window of hypomethylation," which is flanked by Sp1-binding sites. Together with the under-representation of Sp1 at the NFATC1 P1 promoter in HL cells, this suggests that Sp1 factors can protect P1 DNA methylation in a directional manner. Blocking immunoreceptor signaling led to NFATC1 P1 promoter silencing and to a decrease in H3 acetylation and H3-K4 methylation but not DNA methylation. This shows that histone modifications precede the DNA methylation in NFATC1 promoter silencing. In lymphoid cells, the three nuclear factor of activated T-cell (NFATc) proteins NFATc1, -c2, and -c3 are expressed to regulate the transcription of numerous genes that control lymphocyte activation, differentiation, and apoptosis.1Serfling E Berberich-Siebelt F Chuvpilo S Jankevics E Klein-Hessling S Twardzik T Avots A The role of NF-AT transcription factors in T cell activation and differentiation.Biochim Biophys Acta. 2000; 1498: 1-18Crossref PubMed Scopus (170) Google Scholar, 2Hogan PG Chen L Nardone J Rao A Transcriptional regulation by calcium, calcineurin, and NFAT.Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1528) Google Scholar, 3Macian F NFAT proteins: key regulators of T-cell development and function.Nat Rev Immunol. 2005; 5: 472-484Crossref PubMed Scopus (1093) Google Scholar The activity of NFATc factors is controlled by immunoreceptor signals that, in addition to other signaling events, lead to a rise in intracellular free Ca2+ and the subsequent activation of the Ca2+/calmodulin-dependent Ser/Thr-specific phosphatase calcineurin. Calcineurin binds to the regulatory region within the N-terminal half of NFATc proteins and removes most of numerous phospho-residues, thereby promoting the nuclear translocation, DNA binding, and transactivation of NFATc. In lymphocytes, NFATc factors control the transcription of numerous lymphokine genes, such as interleukin-2, interleukin-4, and interferon-γ genes, but also of genes that regulate proliferation and apoptosis. Examples of such genes are the CDK4 gene and the Fas ligand gene, whose promoters are bound and controlled by NFATc factors.4Baksh S Widlund HR Frazer-Abel AA Du J Fosmire S Fisher DE DeCaprio JA Modiano JF Burakoff SJ NFATc2-mediated repression of cyclin-dependent kinase 4 expression.Mol Cell. 2002; 10: 1071-1081Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 5Latinis KM Norian LA Eliason SL Koretzky GA Two NFAT transcription factor binding sites participate in the regulation of CD95 (Fas) ligand expression in activated human T cells.J Biol Chem. 1997; 272: 31427-31434Crossref PubMed Scopus (199) Google Scholar, 6Xiao S Matsui K Fine A Zhu B Marshak-Rothstein A Widom RL Ju ST FasL promoter activation by IL-2 through SP1 and NFAT but not Egr-2 and Egr-3.Eur J Immunol. 1999; 29: 3456-3465Crossref PubMed Scopus (56) Google Scholar, 7Li-Weber M Laur O Krammer PH Novel Egr/NF-AT composite sites mediate activation of the CD95 (APO-1/Fas) ligand promoter in response to T cell stimulation.Eur J Immunol. 1999; 29: 3017-3027Crossref PubMed Scopus (64) Google Scholar, 8Lee MO Kang HJ Kim YM Oum JH Park J Repression of FasL expression by retinoic acid involves a novel mechanism of inhibition of transactivation function of the nuclear factors of activated T-cells.Eur J Biochem. 2002; 269: 1162-1170Crossref PubMed Scopus (19) Google Scholar, 9Rengarajan J Mittelstadt PR Mages HW Gerth AJ Kroczek RA Ashwell JD Glimcher LH Sequential involvement of NFAT and Egr transcription factors in FasL regulation.Immunity. 2000; 12: 293-300Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar These and other experimental data suggested that, in addition to their role in normal lymphocyte development, NFATc factors, if aberrantly expressed, might also be involved in the generation of lymphoid tumors (see also10Viola JP Carvalho LD Fonseca BP Teixeira LK NFAT transcription factors: from cell cycle to tumor development.Braz J Med Biol Res. 2005; 38: 335-344Crossref PubMed Scopus (67) Google Scholar).Experimental evidence for this conclusion can be derived from retroviral tagging experiments in which the murine Nfatc1 locus has been identified as a common insertion site for oncogenic viruses (see also the Mouse Retrovirus Tagged Cancer Gene Database at http://RTCGD.ncifcrf.gov).11Akagi K Suzuki T Stephens RM Jenkins NA Copeland NG RTCGD: retroviral tagged cancer gene database.Nucleic Acids Res. 2004; 32: D523-D527Crossref PubMed Google Scholar In these studies, all retroviral insertions were found to be located close to either the two Nfatc1 promoters or polyadenylation sites,12Serfling E Chuvpilo S Liu J Hofer T Palmetshofer A NFATc1 autoregulation: a crucial step for cell-fate determination.Trends Immunol. 2006; 27: 461-469Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar suggesting that retroviral insertions modulate NFATc1 expression. At similar positions, retroviral insertion sites have also been identified within the murine Nfatc3 gene, and proviral insertion resulted in a loss or decrease of NFATc3 expression. Because infections of newborn mice with the T-cell lymphomagenic retrovirus SL3-3 MLV led to an acceleration of lymphoma generation in NFATc3-deficient mice compared with wild-type mice, we concluded that NFATc3 exerts a tumor suppressor-like activity for the generation of viral-induced lymphoid tumors.13Glud SZ Sorensen AB Andrulis M Wang B Kondo E Jessen R Krenacs L Stelkovics E Wabl M Serfling E Palmetshofer A Pedersen FS A tumor-suppressor function for NFATc3 in T-cell lymphomagenesis by murine leukemia virus.Blood. 2005; 106: 3546-3552Crossref PubMed Scopus (39) Google Scholar Investigations of NFATc2-deficient mice that develop chondrocytic neoplasms led to a similar conclusion, ie, NFATc2 can act like a tumor suppressor for the generation of chondrocytic malignancies.14Ranger AM Gerstenfeld LC Wang J Kon T Bae H Gravallese EM Glimcher MJ Glimcher LH The nuclear factor of activated T cells (NFAT) transcription factor NFATp (NFATc2) is a repressor of chondrogenesis.J Exp Med. 2000; 191: 9-22Crossref PubMed Scopus (163) Google ScholarHowever, in other cell types and under other experimental settings, NFAT factors can also exert oncogenic activities. Transformation of pre-adipocytes by a constitutively active version of NFATc1/A showed that this NFAT protein acts as an oncogene for the development of human adipocytic and other tumors,15Neal JW Clipstone NA A constitutively active NFATc1 mutant induces a transformed phenotype in 3T3–L1 fibroblasts.J Biol Chem. 2003; 278: 17246-17254Crossref PubMed Scopus (117) Google Scholar whereas in human classical Hodgkin's lymphoma (cHL), NFATc1 was found to be suppressed.16Marafioti T Pozzobon M Hansmann ML Ventura R Pileri SA Roberton H Gesk S Gaulard P Barth TF Du MQ Leoncini L Moller P Natkunam Y Siebert R Mason DY The NFATc1 transcription factor is widely expressed in white cells and translocates from the cytoplasm to the nucleus in a subset of human lymphomas.Br J Haematol. 2005; 128: 333-342Crossref PubMed Scopus (65) Google Scholar In a subset of large B-cell lymphomas, NFATc1 and c-Rel were detected to be expressed constitutively, to interact with each other, and to control synergistically the expression of CD40 ligand/CD154, thereby maintaining the survival of these aggressive lymphomas.17Pham LV Tamayo AT Yoshimura LC Lin-Lee YC Ford RJ Constitutive NF-kappaB and NFAT activation in aggressive B-cell lymphomas synergistically activates the CD154 gene and maintains lymphoma cell survival.Blood. 2005; 106: 3940-3947Crossref PubMed Scopus (96) Google Scholar In a similar way, NFATc1 and NFATc2 contribute to the constitutive expression of B-lymphocyte stimulator in large B-cell lymphomas and mantle cell lymphomas and support the survival and proliferation of these malignant B-cell lymphomas.18Kawano S Otsu K Kuruma A Shoji S Yanagida E Muto Y Yoshikawa F Hirayama Y Mikoshiba K Furuichi T ATP autocrine/paracrine signaling induces calcium oscillations and NFAT activation in human mesenchymal stem cells.Cell Calcium. 2006; 39: 313-324Crossref PubMed Scopus (142) Google Scholar NFATc1 was found to be overexpressed in human pancreatic tumors and to enhance c-myc expression, and inhibition of calcineurin/NFATc1 signaling resulted in attenuation of cell proliferation and anchorage-independent growth.19Buchholz M Schatz A Wagner M Michl P Linhart T Adler G Gress TM Ellenrieder V Overexpression of c-myc in pancreatic cancer caused by ectopic activation of NFATc1 and the Ca2+/calcineurin signaling pathway.EMBO J. 2006; 25: 3714-3724Crossref PubMed Scopus (213) Google Scholar Similar results on a constitutive activation of calcineurin have recently been reported for T-cell acute lymphoblastic leukemias.20Medyouf H Alcalde H Berthier C Guillemin MC Dos Santos NR Janin A Decaudin D de The H Ghysdael J Targeting calcineurin activation as a therapeutic strategy for T-cell acute lymphoblastic leukemia.Nat Med. 2007; 13: 736-741Crossref PubMed Scopus (119) Google Scholar Induced by integrin signals, NFATc2 (and NFAT5) promotes the invasion of human breast cancer cells21Jauliac S Lopez-Rodriguez C Shaw LM Brown LF Rao A Toker A The role of NFAT transcription factors in integrin-mediated carcinoma invasion.Nat Cell Biol. 2002; 4: 540-544Crossref PubMed Scopus (347) Google Scholar by the induction of cyclooxygenase 2, which controls prostaglandin E2 synthesis.22Yiu GK Toker A NFAT induces breast cancer cell invasion by promoting the induction of cyclooxygenase-2.J Biol Chem. 2006; 281: 12210-12217Crossref PubMed Scopus (130) Google Scholar The NFATc2-induced motility and invasion of breast cancer cells can be blocked by Akt/PKB signals,23Yoeli-Lerner M Yiu GK Rabinovitz I Erhardt P Jauliac S Toker A Akt blocks breast cancer cell motility and invasion through the transcription factor NFAT.Mol Cell. 2005; 20: 539-550Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar which were shown to suppress the activation of NFATcs in thymocytes and peripheral T cells.24Patra AK Na SY Bommhardt U Active protein kinase B regulates TCR responsiveness by modulating cytoplasmic-nuclear localization of NFAT and NF-kappa B proteins.J Immunol. 2004; 172: 4812-4820Crossref PubMed Scopus (31) Google Scholar, 25Patra AK Drewes T Engelmann S Chuvpilo S Kishi H Hunig T Serfling E Bommhardt UH PKB rescues calcineurin/NFAT-induced arrest of Rag expression and pre-T cell differentiation.J Immunol. 2006; 177: 4567-4576Crossref PubMed Scopus (30) Google Scholar Taken together, these data indicate that alterations in calcineurin/NFATc signaling play an important role in human cancerogenesis, and because of their immunoreceptor-dependent activation, aberrant NFATc activity affects particularly the generation and maintenance of human lymphomas.In this study, we have investigated the expression of NFATc1 (and NFATc2) in human lymphomas. By immunohistochemical staining, we found that NFATc1 expression is suppressed in human anaplastic large cell lymphomas (ALCLs) and cHLs. Both ALCL and cHL are lymphoma entities with a defective immunoreceptor signaling.26Bonzheim I Geissinger E Roth S Zettl A Marx A Rosenwald A Muller-Hermelink HK Rudiger T Anaplastic large cell lymphomas lack the expression of T-cell receptor molecules or molecules of proximal T-cell receptor signaling.Blood. 2004; 104: 3358-3360Crossref PubMed Scopus (150) Google Scholar, 27Schwering I Brauninger A Klein U Jungnickel B Tinguely M Diehl V Hansmann ML Dalla-Favera R Rajewsky K Kuppers R Loss of the B-lineage-specific gene expression program in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma.Blood. 2003; 101: 1505-1512Crossref PubMed Scopus (319) Google Scholar, 28Marafioti T Pozzobon M Hansmann ML Gaulard P Barth TF Copie-Bergman C Roberton H Ventura R Martin-Subero JI Gascoyne RD Pileri SA Siebert R Hsi ED Natkunam Y Moller P Mason DY Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma.Leukemia. 2005; 19: 856-861Crossref PubMed Scopus (21) Google Scholar The silencing of the NFATC1 locus is correlated with low levels of histone H3 acetylation, H3-K4 trimethylation, and Sp1 factor binding in its P1 promoter region, which is part of a CpG island and controlled by numerous inducible transcription factors.29Chuvpilo S Jankevics E Tyrsin D Akimzhanov A Moroz D Jha MK Schulze-Luehrmann J Santner-Nanan B Feoktistova E Konig T Avots A Schmitt E Berberich-Siebelt F Schimpl A Serfling E Autoregulation of NFATc1/A expression facilitates effector T cells to escape from rapid apoptosis.Immunity. 2002; 16: 881-895Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar We also show here that hypermethylation of P1 promoter DNA correlates with the suppression of NFATc1 expression that, in part, can be released by inhibition of de novo DNA methylation by 5-aza-2′-deoxycytidine (5-aza-dC). On interruption of immunoreceptor signaling by cyclosporin A (CsA) in EL-4 thymoma cells that results in the suppression of NFATc1, but not NFATc2, we observed distinct changes in histone modification, whereas DNA methylation remained unaffected. These results suggest that defects in immunoreceptor signaling in ALCL and HL cells lead to a cascade of epigenetic changes that, finally, contribute to the stable silencing of the NFATC1 promoter(s) in these human lymphomas.Materials and MethodsTissue Samples and CellsFormalin-fixed, paraffin-embedded samples from 3 hyperplastic tonsils, 2 lymph nodes with nonspecific hyperplasia, and 226 malignant lymphoma cases were selected from the histopathology files of the Institute of Pathology, University of Wuerzburg; the Laboratory of Tumor Pathology and Molecular Diagnostics, Institute of Biotechnology, Bay Zoltán Foundation for Applied Research Szeged; and the Department of Pathology, Okayama University. All samples from patients were used following permission and informed consent by the patients. The list of malignant lymphoma cases that were investigated is given in Table 1. Murine EL-4 T lymphoma cells; human Jurkat leukemic cells; the human ALCL lines SR-786, SUP-M2, and KARPAS 299; and the human HL cell lines L428, L540, L1236, and KM-H2 were grown in RPMI containing 10% fetal calf serum to a density of 2 × 105 cells per milliliter and used in all biochemical assays.Table 1NFATc1 Expression in Malignant Lymphoma Cases as Detected by ImmohistochemistryNFATc1 expression [n (%)]Cases studied (n)−±+Overall low (− and ±) [n (%)]Systemic ALCL (22)17 (77)5 (23)022 (100) ALK+ (10)8 (80)2 (20)010 (100) ALK− (12)9 (75)3 (25)012 (100)Cutaneous ALCL (11)1 (10)5 (45)5 (45)6 (55)Other PTCL (49)2 (4)5 (10)42 (86)7 (14) Unspecified (28)2 (7)3 (11)23 (82)5 (18) AILT (10)0010 (100)0 ETL (11)02 (18)9 (82)2 (18)Mature B-cell neoplasms (93)11 (12)3 (3)79 (85)14 (15) B-CLL (6)006 (100)0 MCL (20)0020 (100)0 FL (5)005 (100)0 MZL (9)009 (100)0 DLBCL (13)0013 (100)0 Mediastinal (3)003 (100)0 Burkitt (18)0018 (100)0 Plasmacytoma/myeloma (19)11 (33)3 (9)5 (15)14 (43)Classical HL (38)33 (87)4 (10)1 (3)37 (97)NLP HL (13)01 (8)12 (92)1 (8)AILT, angioimmunoblastic T cell lymphoma; ALK, anaplastic lymphoma kinase; B-CLL, B-cell chronic lymphoid leukemia; DLBCL, diffuse large B-cell lymphoma; ETL, enteropathy-type T-cell lymphoma; FL, follicular lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; NLP HL, nodular lymphocyte predominance HL; PTCL, peripheral T-cell lymphoma. Open table in a new tab Immunohistochemistry and Western BlotsFor detection of NFATc1 expression, a mouse IgG1 monoclonal antibody was used recognizing amino acids 197 to 304 of human NFATc1 (clone 7A6; sc-7294; Santa Cruz Biotechnology, Santa Cruz, CA), common to all major NFATc1 isoforms.30Northrop JP Ho SN Chen L Thomas DJ Timmerman LA Nolan GP Admon A Crabtree GR NF-AT components define a family of transcription factors targeted in T-cell activation.Nature. 1994; 369: 497-502Crossref PubMed Scopus (522) Google Scholar, 31Chuvpilo S Avots A Berberich-Siebelt F Glockner J Fischer C Kerstan A Escheqjr C Inashkina I Hlubek F Jankevics E Brabletz T Serfling E Multiple NF-ATc isoforms with individual transcriptional properties are synthesized in T lymphocytes.J Immunol. 1999; 162: 7294-7301PubMed Google Scholar Immunohistochemical reactions were performed using a streptavidin-biotin-horseradish peroxidase complex method, following heat-induced antigen retrieval performed in 0.5 mol/L Tris buffer (pH 6.0) and pressure cooker. An immunoreaction was classified positive if proposed neoplastic cells revealed moderate to marked homogeneous (cytosolic) staining for NFATc1. In some cases, moderate to marked NFATc1 immunostaining occurred in a proportion of the tumor cells, and those that showed staining in at least one-half of these cells were scored to be positive. For the exact immunolocalization of NFATc1 in plasma cells with higher sensitivity, NFATc1 staining was combined with an Ig light chain antibody (Ab) cocktail, using double immunofluorescent stains and confocal laser scanning microscopy in formalin-fixed, paraffin-embedded sections of hyperplastic tonsils.Western blots were performed by fractionation of whole protein extracts from human and murine cell lines on 10% polyacrylamide gels followed by immunodetection using polyclonal Abs raised against the N-terminal halves of NFATc1 or c2, respectively (ImmunoGlobe, Groβostheim, Germany), or against Sp1.32Hagen G Muller S Beato M Suske G Sp1-mediated transcriptional activation is repressed by Sp3.EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar Equal protein loading of gels was determined by staining of filters with Poinceau red.DNA Methylation AssaysCpG methylation studies of the human NFATC1 promoter region were performed using the PCR-based sodium-bisulfite DNA modification procedure.33Frommer M McDonald LE Millar DS Collis CM Watt F Grigg GW Molloy PL Paul CL A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.Proc Natl Acad Sci USA. 1992; 89: 1827-1831Crossref PubMed Scopus (2480) Google Scholar The PCR products were directly sequenced, and semiquantitative data on methylation degree (Figure 2B) were derived from the sequencing peaks. The sequences of the primers used in PCR amplifications for sequencing of bisulfite-modified DNA were as follows. P1 promoter, distal block of homology: forward, 5′-AACAAATAAACRCRTCCCCRAACCTCCCCAC-3′ (for primary, outer amplification), and reverse, 5′-GAAYGGGTTAGAYGGGAYGTTTGAGTTTAY-3′ (for primary, outer amplification); forward, 5′-AAACRCRTCCCCRAACCTCCCCACRCCRACC-3′ (nested), and reverse, 5′-GAYGGGAYGTTTGAGTTTAYGYGGGTGTTYGG-3′ (nested). P1 promoter, proximal block of homology: forward, 5′-CCRCRACCCTAAAACCTACRCRATAAC (for primary, outer amplification), and reverse, 5′-GTTTTTAGGYGAGYGGTTGTYGYGGYG-3′ (for primary, outer amplification); forward, 5′-CRCRATAACTCCRAACCCTACCCRC-3′ (nested), and reverse, 5′-GGGYGTTYGGYGATTGTTTTYGGG-3′ (nested).Determination of gross DNA methylation in 5-aza-dC-treated HL cell lines (Supplemental Figure S1 at http://ajp.amjpathol.org) were performed by combined high-performance liquid chromatography/tandem mass spectroscopy assays.34Fink K Brink A Vienken J Heidland A Stopper H Homocysteine exerts genotoxic and antioxidative effects in vitro.Toxicol In Vitro. 2007; 21: 1402-1408Crossref PubMed Scopus (10) Google ScholarElectrophoretic Mobility Shift Assays and Chromatin Immunoprecipitation AssaysNuclear proteins from Jurkat T and HL cell lines were prepared and used in electrophoretic mobility shift assays (EMSAs) as described previously.29Chuvpilo S Jankevics E Tyrsin D Akimzhanov A Moroz D Jha MK Schulze-Luehrmann J Santner-Nanan B Feoktistova E Konig T Avots A Schmitt E Berberich-Siebelt F Schimpl A Serfling E Autoregulation of NFATc1/A expression facilitates effector T cells to escape from rapid apoptosis.Immunity. 2002; 16: 881-895Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar For the detection of Sp1 binding to the P1 promoter, the following (double-stranded) oligonucleotides were incubated as radioactively labeled probes together with 5 μg of nuclear protein: −825 site, (−836) 5′-GGCCTCCCCACGCCGGCCCCTGCCA-3′ (−811); −585 site, (−594) 5′-CCCCCGGCCCCCGCCCCCCGCCCCT-3′ (−572); −210 site, (−220) 5′-CGCGGGGAGGGGCGGGCGCTCGGCG-3′ (−196); −15 site, (−30) 5′-TCCGAACTCGCCGGCGGAGTCG-3′ (−9). The nucleotides in bold correspond to the Sp1 core binding sequence; the underlined nucleotides correspond to the Sp1 consensus binding site.35Kadonaga JT Tjian R Affinity purification of sequence-specific DNA binding proteins.Proc Natl Acad Sci USA. 1986; 83: 5889-5893Crossref PubMed Scopus (715) Google ScholarFor competition, a consensus Sp1 site oligonucleotide (cSp1/3) and a mutated version (mSp1/3) (sc-2502 and sc-2503, respectively) were purchased from Santa Cruz Biotechnology. In addition, a Sp1/3−210 site methylated in all CpG residues (methyl-Sp1/3; Figure 4C) and a tandemly arranged Sp1/Sp3 site (Sp1/3tn; Figure 4B) from the NFATC1 P2 promoter were used. In supershift EMSAs, 1 μl of polyclonal Ab raised against Sp1 or Sp3 (sc-644x; Santa Cruz Biotechnology) was added to the incubations.Figure 4Binding sites of transcription factors Sp1 and Sp3 mark the borders of DNA methylation within the P1 NFATC1 promoter. A: Factor binding to the two Sp1/Sp3 sites around the positions −585 and −825 that flank a "window of hypomethylation" (see Figure 2B) within the distal block of homology of the P1 promoter. In EMSAs, the binding of Sp1 and Sp3 in nuclear protein extracts from Jurkat cells is demonstrated by competition with a 50-fold excess of an unlabeled consensus Sp1-binding site (lanes 2 and 6) and by supershifts with Abs specific for Sp1 (lanes 3 and 7, labeled by arrows) and Sp3 (see lanes 4 and 8). In lanes 9 to 16, EMSAs were performed with nuclear protein from uninduced cells (−) or from Jurkat, KM-H2, L428, and L540 cells induced by T+I for 4 hours (+). B: Sp1/Sp3 binding to the −15 and −210 sites of P1 promoter in the proximal block of homology. Nuclear proteins from induced Jurkat cells were incubated with an oligonucleotide probe of site −15 (lanes 1 to 10), of site −210 (lanes 21 to 29) or a Sp1 consensus site (lanes 11 to 20). In supershift assays, Sp1- and Sp3-specific Abs (αSp1 and αSp3) were added as indicated. For competition, a 5- or 50-fold excess of unlabeled oligonucleotides of a tandemly arranged Sp1/3 site from the P2 promoter (Sp1/2tn), a consensus Sp1/Sp3 site (cSp1/3), a mutated Sp1/3 consensus site (mSp1/3), or a Sp1/3−15 site were added to the incubations as indicated. C: DNA methylation does not impair Sp1/Sp3 binding. A chemically synthesized −210 oligonucleotide probe methylated at all C residues (methyl-Sp1/3) was incubated with nuclear protein from Jurkat cells and assayed as in A and B. For competition, a 5- or 50-fold molar excess was added of cSp1/3, a consensus Sp1/Sp3 site, mSp1/3, a mutated Sp1/3 consensus site, and methyl-Sp1/3, a Sp1/3−210 site that was methylated at all Cs. D: Detection of Sp1 expression in Jurkat, KM-H2, L428, and L540 cells by Western blot immunoassays. Nuclear proteins were analyzed from uninduced cells (lanes 1, 3, 5, and 7) or from cell induced by T+I for 4 hours (lanes 2, 4, 6, and 8). Comparable protein loading of lanes of the gel was detected by staining of filters with Poinceau red. E: Detection of Sp1 binding to the P1 and DHFR promoters in vivo by ChIP assays. Nuclear proteins were analyzed from uninduced cells (lanes 1, 3, 5, and 7) or from cells induced as in D (lanes 2, 4, 6, and 8).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Chromatin immunoprecipitation (ChIP) assays were performed as described previously.36Soutoglou E Talianidis I Coordination of PIC assembly and chromatin remodeling during differentiation-induced gene activation.Science. 2002; 295: 1901-1904Crossref PubMed Scopus (262) Google Scholar Cells were cross-linked using formaldehyde, nuclei were isolated and sonicated, and DNA-protein complexes were immunoprecipitated after pre-clearing with protein-A sepharose blocked with salmon sperm DNA using anti-acetylated histone H3 Ab (06-599; Upstate, Lake Placid, NY), anti-trimethylated H3-K4 Ab (8580; Abcam, Cambridge, UK), anti-dimethylated H3-K9 (05-768; Upstate), anti-trimethylated H3-K9 (8898; Abcam), anti-HP1 (sc-28735; Santa Cruz Biotechnology), and anti-Sp1.32Hagen G Muller S Beato M Suske G Sp1-mediated transcriptional activation is repressed by Sp3.EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar After washing, elution, and reversion of cross-links, the DNA was isolated and used in PCRs that were performed with the following primers to amplify human or murine NFATC1 promoters. Human NFATC1 P1 promoter, distal block of homology: forward, 5′-GAGACGTGAGAGAGGAAAGTGTGAGTGG-3′, and reverse, 5′-GAAAGCCCGGCATGCTGAAGTCATTATG-3′. Human DHFR promoter: forward, 5′-AACCTCAGCGCTTCACCCAA-3′, and reverse, 5′-CGCACGTAGTAGGTTCTGTC-3′.Murine Nfatc1 P1 promoter, distal block of homology: forward, 5′-GAAAAGGACTCCTGGGAA-3′, and reverse, 5′-CAAAGACCCAGAGGAGGA-3′. Murine Nfatc2 promoter: forward, 5′-GCTCTACCTTAGGGACCA-3′, and reverse, 5′-GGTTTGAATCCAGACTAGGA-3′.Real-Time PCR AssaysRNAs were extracted by TRIzol from cell lines treated with 5-aza-dC (1 to 5 μmol/L) for 4 days. RNAs were reverse transcribed using the iScript cDNA synthesis kit of Bio-Rad (Hercules, CA). SYBR Green real-time PCR assays were performed with an ABI PRISM 7700 sequence detection system according to the protocol of ABgene (Hamburg, Germany). For detecting NFATc1-, BOB.1/OBF1-, PLCγ2-, and Syk-specific RNAs, the following primers from Qiagen (Hilden, Germany) were used: Hs_NFATc1-1-SG (QT00094157); Hs_POU2AF1_1-SG (QT0.0001540), Hs_PLCg2 (QlT00050393), and Hs_SYK_1 (QT00050043). β-Microglobulin primers were used as an internal standard.ResultsNFATc1 Expression Is Suppressed in ALCLs and cHLsIn immunohistochemical stainings of human hyperplastic tonsils and lymph nodes using a NFATc1-specific monoclonal antibody raised against all NFATc1 isoforms (see Materials and Methods), we observed cytosolic staining of virtually all lymphoid T and B cells (with the exception of a faint or no nuclear staining in some germinal center B cells) (Supplemental Figure S2, A and B, at http://ajp. amjpathol.org). No staining was observed in endothelial cells and epithelial cells and, as shown in double stainings with monoclonal antibodies against Ig light chains, in plasma B cells (Supplemental Figure S2, C and D). When we used the same Ab for immunostaining of a large panel of human lymphoid tumors, the cytoplasm of the majority of tumor cells was also positively stained (Table 1). Thus, a strong cytosolic expression of NFATc1 was found in the majority of mature B-cell neoplasms: All cases of B-cell chronic lymphoid leukemia, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mediastinal large B-cell lymphoma, and Burkitt's lymphoma showed consistent NFATc1 staining, and in the majority of Burkitt's lymphoma cells, a nuclear appearance of NFATc1 was observed. In contrast to the loss of NFATc1 expression in normal plasma cells, neoplastic plasma cells of 8 of 19 plasmacytoma/myeloma cases revealed a positive NFATc1 immunostaining (
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