A Novel Element and a TEF-2-like Element Activate the Major Histocompatibility Complex Class II Transactivator in B-lymphocytes
1999; Elsevier BV; Volume: 274; Issue: 45 Linguagem: Inglês
10.1074/jbc.274.45.32342
ISSN1083-351X
AutoresNilanjan Ghosh, Janet F. Piskurich, Gabriëla Wright, Kevin Hassani, Jenny P.‐Y. Ting, Kenneth L. Wright,
Tópico(s)T-cell and B-cell Immunology
ResumoMajor histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly. Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly. interferon class II transactivator polymerase chain reaction dimethylsulfate activation response element nuclear factor-1 major histocompatibility complex Major histocompatibility complex (MHC)1 class II molecules play a fundamental role in presenting exogenous antigenic peptides to CD4+ helper T lymphocytes (1Benacerraf B. Science. 1981; 212: 1229-1238Crossref PubMed Scopus (472) Google Scholar). These activated CD4+ T cells release stimulatory cytokines that augment the response of CD8+ cytotoxic T lymphocytes (2Frasca L. Piazza C. Piccolella E. Crit. Rev. Immunol. 1998; 18: 569-594Crossref PubMed Google Scholar). Constitutive expression of MHC class II molecules is restricted to professional antigen-presenting cells, such as B lymphocytes and dendritic cells, and can be induced by interferon-γ (IFN-γ) in macrophages, endothelial cells, and fibroblasts (3Glimcher L.H. Kara C.J. Annu. Rev. Immunol. 1992; 10: 13-49Crossref PubMed Scopus (504) Google Scholar, 4Ting J.P.-Y. Baldwin A.S. Curr. Opin. Immunol. 1993; 5: 8-16Crossref PubMed Scopus (202) Google Scholar). The MHC class II family of genes are coordinately regulated at the level of transcription through a conserved trimeric promoter motif (3Glimcher L.H. Kara C.J. Annu. Rev. Immunol. 1992; 10: 13-49Crossref PubMed Scopus (504) Google Scholar, 5Vilen B.J. Penta J.F. Ting J.P.-Y. J. Biol. Chem. 1992; 267: 23728-23734Abstract Full Text PDF PubMed Google Scholar). A major component of this transcription complex was cloned by genetic complementation of anin vitro mutagenized MHC class II negative B cell line, RJ2.2.5. This component was named the MHC class II transactivator (CIITA) and restored MHC class II cell surface expression in one subgroup of bare lymphocyte syndrome patient cells (6Steimle V. Otten L.A. Zufferey M. Mach B. Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (752) Google Scholar). CIITA has since been shown to be a key regulatory molecule in the expression of all the known genes involved in the MHC class II antigen-presenting pathway (6Steimle V. Otten L.A. Zufferey M. Mach B. Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 7Steimle V. Siegrist C.A. Mottet A. Lisowska-Grospierre B. Mach B. Science. 1994; 265: 106-109Crossref PubMed Scopus (679) Google Scholar, 8Chin K.-C. Mao C. Skinner C. Riley J.L. Wright K.L. Moreno C.S. Stark G.R. Boss J.M. Ting J.P.-Y. Immunity. 1994; 1: 679-689Abstract Full Text PDF Scopus (130) Google Scholar, 9Chang C.-H. Fontes J.D. Peterlin M. Flavell R.A. J. Exp. Med. 1994; 180: 1367-1374Crossref PubMed Scopus (297) Google Scholar). CIITA is required for constitutive expression of MHC class II on B-lymphocytes (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar) and with a few exceptions, such as in respiratory epithelial cells and dendritic cells (11Gao J. De B.P. Banerjee A.K. J. Virol. 1999; 73: 1411-1418Crossref PubMed Google Scholar, 12Williams G.S. Malin M. Vremec D. Chang C.H. Boyd R. Benoist C. Mathis D. Int. Immunol. 1998; 10: 1957-1967Crossref PubMed Scopus (81) Google Scholar), is required for cytokine-induced expression in other cell types. Mice in which functional CIITA protein was ablated clearly demonstrated an absolute requirement for CIITA in B cell expression of MHC class II (13Chang C.H. Guerder S. Hong S.C. van Ewijk W. Flavell R.A. Immunity. 1996; 4: 167-178Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). It has been shown that the CIITA gene has four distinct promoters each transcribing a unique first exon, which are located within approximately 13 kilobases of DNA (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar). The promoters are utilized in a tissue-specific manner. CIITA expression in dendritic cells is primarily from promoter I. Promoter III is predominantly used in B-lymphocytes, while promoter IV is used predominantly in IFN-γ-induced expression. The first exon associated with the B cell and dendritic cell promoters encodes distinct amino-terminal residues of 24 and 101 amino acids, respectively, while mRNA from promoters II and IV initiates translation from within the common exon 2 (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar). Promoter II has yet to be characterized. The IFN-γ-responsive control elements at CIITA promoter IV were recently characterized (14Piskurich J.F. Linhoff M.W. Wang Y. Ting J.P.Y. Mol. Cell. Biol. 1999; 19: 431-440Crossref PubMed Google Scholar, 15Muhlethaler-Mottet A. Di Berardino W. Otten L.A. Mach B. Immunity. 1998; 8: 157-166Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Both STAT-1 and IRF-1 are required at the proximal promoter for full response. Binding of the USF-1 transcription factor stabilizes STAT-1 binding (15Muhlethaler-Mottet A. Di Berardino W. Otten L.A. Mach B. Immunity. 1998; 8: 157-166Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Initial mapping of the CIITA promoter III revealed that sequences that lie between −545 and −113 base pairs relative to the transcription start site are required for constitutive expression in B-lymphocytes (16Piskurich J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar). While this region of the promoter is not responsive to IFN-γ, IFN-γ activation of CIITA promoter III can be observed when an additional 4000 base pairs of upstream DNA are present (16Piskurich J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar). However, nothing is known about the specific factors and promoter elements that control constitutive transcription from CIITA promoter III in B-lymphocytes. Tight regulation of the expression of MHC class II genes has been observed during B cell development. MHC class II gene transcription begins in the pre-B cell stage, is maintained until B-lymphocytes attain maturity (17Miki N. Hatano M. Wakita K. Imoto S. Nishikawa S. Nishikawa S. Tokuhisa T. J. Immunol. 1992; 149: 801-807PubMed Google Scholar, 18Tarlinton D. Int. Immunol. 1993; 5: 1629-1635Crossref PubMed Scopus (25) Google Scholar), and is down-regulated when B-lymphocytes terminally differentiate into plasma cells (19Dellabona P. Latron F. Maffei A. Scarpellino L. Accolla R.S. J. Immunol. 1989; 142: 2902-2910PubMed Google Scholar, 20Latron F. Jotterand-Bellomo M. Maffei A. Scarpellino L. Bernard M. Strominger J.L. Accolla R.S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2229-2233Crossref PubMed Scopus (44) Google Scholar). CIITA expression is also up-regulated in the pre-B cell stages and then lost upon terminal differentiation (21Silacci P. Mottet A. Steimle V. Reith W. Mach B. J. Exp. Med. 1994; 180: 1329-1336Crossref PubMed Google Scholar, 22Henderson A. Calame K. Annu. Rev. Immunol. 1998; 16: 163-200Crossref PubMed Scopus (111) Google Scholar). The mechanisms of activation and repression are unknown. CIITA promoter III does not contain any cis-acting sequences with significant homology to B cell activators except a putative octamer binding site. In this report, we now characterize the CIITA promoter III regulatory elements in B-lymphocytes and identify in vivo and in vitro five occupied sites. Importantly, two of the sites are absolutely required for CIITA transcription in B-lymphocytes. One is a TEF-2-like element, while the other represents a binding site for a novel transcription activator. The CIITA promoter III reporter constructs CIITAp3.1140-Luc and CIITAp3.545-Luc contain the human CIITA promoter III sequences from −1140 to +123 or −545 to +123 base pairs, respectively, relative to the transcription initiation site. These two constructs were originally carried in the pGL2-Basic vector and described previously as p1300CIITA.Luc and p668CIITA.Luc (16Piskurich J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar), but for this study they have been moved into the pGL3-Basic vector (Promega). Each of the other deletion constructs (namely CIITAp3.319-Luc, CIITAp3.195-Luc, CIITAp3.151-Luc, and CIITAp3.113-Luc) were constructed similarly and contain CIITA promoter III sequences from +123 up to the indicated number on the construct name. All of the mutations except as specifically noted were carried in the CIITAp3.545-Luc construct. Mutant constructs were derived by the unique site elimination method (CLONTECH) (23Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1078) Google Scholar). In the construct CIITAp3.545mtSiteA, the sequence 5′-TTGGCGGGCTCCCA-3′ was changed to 5′-TTGtCtaGaTCCCA-3′ (mutations in lowercase boldface type). In CIITAp3.545mtSiteB, 5′-TTCTTTGCATGT-3′ was changed to 5′-TTCgTcGacaGT-3′. In the construct CIITAp3.545mtARE-2, 5′-TGATGATCCCT-3′ was changed to 5′-TGATctagaCT-3′. In the construct CIITAp3.545mtARE-1, 5′-TTAAGGGAGTGTGGTAA-3′ was changed to 5′-TTAAGtctagaTGGTAA-3′. In the construct CIITAp3.545mtSiteC, 5′-GGAAGTGAAATT-3′ was changed to 5′-GGAgtcGAcgTT-3′. Three additional constructs with mutations in the activation response element-1 (ARE-1) sequence were developed in the pGL2-Basic vector backbone. The first (pGL2.545mtARE1–2) is in the context of the −545 base pair promoter, while the second (pGL2.151mtARE1–3) and third (pGL2.151mtARE1–4) constructs are in the context of the −151 base pair promoter. The specific base changes are shown in Table I. All mutations were confirmed by sequencing. The BSAP expression vector was cloned by reverse transcription PCR into pCDNA3.1 (InVitrogen) and confirmed by sequencing.Table IThe ARE-1 sequence element and four mutationsGAGGGCTTAA GGGAGTGTGG TAAAAWild type activityMutant activityRatioMt1———–tctaga——–120 ± 21.416.2 ± 4.60.13Mt2——–cta-a————120 ± 7.936.5 ± 2.30.30Mt3—————ctc——-101 ± 5.688.9 ± 7.40.88Mt4——–tt—————60.9 ± 10.451.9 ± 6.20.85Transcriptional activity is the average luciferase activity per μg of protein lysate from three independent experiments. Each mutation was tested independently with the appropriate wild type control, and the ratio is the value of activity of the mutant construct divided by the activity of the parent wild type construct. Mt1 and Mt2 are in the context of the 545-base pair promoter while Mt3 and Mt4 are in the context of the 151-base pair promoter. Partial sequence homologies are as follows: Myc/Myb (bold), TEF-2 (underline), and AP3 (italic). Open table in a new tab Transcriptional activity is the average luciferase activity per μg of protein lysate from three independent experiments. Each mutation was tested independently with the appropriate wild type control, and the ratio is the value of activity of the mutant construct divided by the activity of the parent wild type construct. Mt1 and Mt2 are in the context of the 545-base pair promoter while Mt3 and Mt4 are in the context of the 151-base pair promoter. Partial sequence homologies are as follows: Myc/Myb (bold), TEF-2 (underline), and AP3 (italic). Raji is a human Epstein-Barr virus-transformed Burkitt's lymphoma cell line that constitutively expresses MHC class II antigens (5Vilen B.J. Penta J.F. Ting J.P.-Y. J. Biol. Chem. 1992; 267: 23728-23734Abstract Full Text PDF PubMed Google Scholar). NCI-H929 and U266 are human plasmacytoma cell lines and were grown in 10% heat-inactivated fetal bovine serum, 2 mml-glutamine, and 100 units/ml penicillin and streptomycin. NCI-H929 cells were also supplemented with 0.05 mm 2-mercaptoethanol. Cells were transfected by electroporation using the Bio-Rad Gene Pulser II apparatus. Cells (1 × 107) were pulsed with 200 V at a capacitance of 1070 microfarads, and cells were harvested after 42 h for luciferase assay. All cell lysates were normalized to recovered protein concentration with the Bradford protein assay (Bio-Rad). In vivo methylation of cells with dimethylsulfate and DNA preparation were as described previously (24Pfeifer G.P. Tanguay R.L. Steigerwald S.D. Riggs A.D. Genes Dev. 1990; 4: 1277-1287Crossref PubMed Scopus (199) Google Scholar). In vivo UV irradiation footprinting was done essentially as described by Pfeifer and Tornaletti (25Pfeifer G.P. Tornaletti S. Methods. 1997; 11: 189-196Crossref PubMed Scopus (18) Google Scholar). Briefly, cells were collected by centrifugation at 4 °C and resuspended at 1 × 107 cells/ml in Dulbecco's phosphate-buffered saline. Cells (1 ml) were spread on a 100-mm plastic dish and exposed to 500–2000 J/m2 UV light (UV Crosslinker; Fisher). After exposure (30–120 s), the cells were immediately harvested by the addition of an equal volume of 2× lysis buffer (200 mmEDTA, 120 mm Tris-HCl (pH7.5), 0.2% SDS, 1 mg/ml proteinase K). The DNA was isolated and digested withHindIII as described for the dimethylsulfate-treated samples. The DNA (20 μg) was cleaved at cyclobutane pyrimidine dimers as described previously using 20 units T4 endonuclease V (Epicenter Technologies) and 5 μg of Escherichia coli photolyase generously provided by A. Sancar (University of North Carolina). Control in vitro UV-irradiated DNA samples were obtained by first isolating and purifying the genomic DNA from untreated cells, dissolving 20 μg of DNA in 100 μl of buffer (10 mmTris-HCl (pH 7.5), 0.1 mm EDTA) and spotting the DNA as 5-μl aliquots on parafilm. UV treatment and processing was as described for the in vivo treated samples. The ligation-mediated, polymerase chain reaction-amplified in vivo genomic footprinting was as originally described (26Mueller P.R. Wold B. Science. 1989; 246: 780-786Crossref PubMed Scopus (787) Google Scholar) and modified by Wright and Ting (27Wright K.L. Ting J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7601-7605Crossref PubMed Scopus (60) Google Scholar). The upper strand was revealed by using two different primer sets, C2E and C2F. The sequence of the primers for C2E are primer 1 (5′-TCAGCTTCCCCAAGGATG-3′), primer 2 (5′-AGGATGCCTTCGGATGCCCAGCTC-3′), and primer 3 (5′-TGCCTTCGGATGCCCAGCTCAGAAGCAC-3′). The sequence of the primers for C2F are primer 1 (5′-AAACTCTCCCTGCAAGGT-3′), primer 2 (5′-TCTCCCTGCAAGGTGGCCCCAAGC-3′), and primer 3 (5′-CCTGCAAGGTGGCCCCAAGCGGTCAGATTTC-3′). The lower stand was revealed by two primer sets, C2C and C2G. The sequence of the primers for C2C are primer 1 (5′-AAGAAGTCCCCAGCAGAG-3′), primer 2 (5′-TCTGGGCGGAGGGCTATGATACTGGC-3′), and primer 3 (5′-CGGAGGGCTATGATACTGGCCCCATCCTGC-3′). The sequence of the primers for C2G are primer 1 (5′-CACCAAATTCAGTCCACAG-3′), primer 2 (5′-AGAGGTGTAGGGAGGGCTTAAGGGAG-3′), and primer 3 (5′-AGAGGTGTAGGGAGGGCTTAAGGGAGTGTG-3′). Nuclear extracts were prepared according to Dignamet al. (28Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1488Crossref PubMed Scopus (9132) Google Scholar). Gel shift analysis was performed as described previously (29Wright K.L. Dell'Orco R.T. van Wijnen A.J. Stein J.L. Stein G.S. Biochemistry. 1992; 31: 2812-2818Crossref PubMed Scopus (36) Google Scholar) using synthetic oligonucleotides and 500 ng of the nonspecific competitor poly(dI:dC). The ARE-1 oligonucleotide spans from −151 to −121 base pairs of the promoter and is 5′-GAGGGCTTAAGGGAGTGTGGTAAAATTAGAGG-3′. The mutant ARE-1 oligonucleotide contains the same mutation as in construct CIITAp3.545mtARE-1. The ARE-2 oligonucleotide spans from −66 to −51 of the promoter and is 5′-GATCCTTGATGATCCCTCACTAGATC-3′. The mutant ARE-2 oligonucleotide contains the same mutation as in construct CIITAp3.545mtARE-2. The site A oligonucleotide spans from −34 to +1 base pairs of the promoter and is 5′-GGCTTAGCTTGGCGGGCTCCCAACTGGTGACTGG-3′. The site B oligonucleotide spans from −55 to −24 of the promoter and is 5′-TCACTTGTTTCTTTGCATGTTGGCTTAGCTTG-3′. The mutSiteA and mutSiteB oligonucleotides contain the same mutation as in constructs CIITAp3.545mtSiteA and CIITAp3.545mtSiteB, respectively. The site C oligonucleotide spanned from −190 to −157 base pairs of the promoter relative to the transcription initiation site and is 5′-GTCCACAGTAAGGAAGTGAAATTAATTTCAGAG-3′. The consensus NF-1 oligonucleotide is 5′-TTTTGGATTGAAGCCAATATGATAA-3′ (30Leegwater P.A. van Driel W. van der Vliet P.C. EMBO J. 1985; 4: 1515-1521Crossref PubMed Scopus (84) Google Scholar); the consensus PU.1 oligonucleotide is 5′-GGGCTGCTTGAGGAAAGTATAAGAAT-3′ (31Eichbaum Q.G. Iyer R. Raveh D.P. Mathieu C. Ezekowitz R.A. J. Exp. Med. 1994; 179: 1985-1996Crossref PubMed Scopus (101) Google Scholar); the consensus BSAP oligonucleotide (BSAP.CD19) is 5′-CCGCAGACACCCATGGTTGAGTGCCCTCCAGGCCC-3′ (32Kozmik Z. Wang S. Dorfler P. Adams B. Busslinger M. Mol. Cell. Biol. 1992; 12: 2662-2672Crossref PubMed Scopus (289) Google Scholar). The consensus TEF-2 oligonucleotide is 5′-GATCCTTAGGGTGTGGACCA-3′ (33Crossley M. Whitelaw E. Perkins A. Williams G. Fujiwara Y. Orkin S.H. Mol. Cell. Biol. 1996; 16: 1695-1705Crossref PubMed Scopus (209) Google Scholar). Unlabeled oligonucleotide competitors were used in a 50–100-fold molar excess. Cytoplasmic RNA was prepared from 1–5 × 107 cells using the RNeasy System (Qiagen). RNA (500 ng) was reverse transcribed using the cDNA Cycle kit (InVitrogen) and resuspended in 20 μl of H2O. The number of PCR cycles that produced a linear phase of amplification for each product was empirically determined. Semiquantitative PCR was performed in the linear phase of amplification using 0.5, 1.0, and 2.0 μl of the cDNA for each primer pair. Each reaction was done in the presence of 2.5 mmMgCl2, 0.5 mm each dNTP, 1 μmeach primer, 2 units of Taq DNA polymerase (Life Technologies), and 2 μCi of [α-32P]dCTP in a total volume of 20 μl. The CIITA promoter III has been shown to be constitutively active in B-lymphocytes (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar). To define the functional regions of the promoter, a series of progressive 5′ deletions of CIITA promoter III fused to the luciferase reporter gene were constructed and transiently transfected into the B cell line Raji. Constructs containing 1140, 545, or 319 base pairs of the promoter including the first 123 base pairs of 5′-untranslated sequences exhibit similar high levels of luciferase activity (Fig.1). Additional upstream sequences spanning 7000 base pairs of promoter III do not further enhance transcriptional activity (16Piskurich J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar). This indicates that the first 319 base pairs of CIITA promoter III are sufficient for transcriptional activation in B-lymphocytes. Deletion of the region from base pair −319 to −195 leads to a 40% decrease in transcriptional activity, which suggests the presence of a positive acting element(s) in this 124-base pair region. Deletion of base pairs −195 to −151 does not alter activity. However, deletion of the region between base pairs −151 and −113 greatly diminishes activity, to only 20% of that observed with the full-length promoter. This suggests the presence of a second positive element in this 38-base pair region. Similar results were obtained by transient transfections into the Namalwa B cell line (data not shown). In vivo genomic footprint analysis of promoters has been invaluable in the visualization of protein/DNA interactions within the physiologically relevant setting of the intact cell. This technique was exploited to directly identify occupied DNA elements present in the CIITA promoter. In Raji B cells both strands of the promoter were analyzed from +69 to −319 base pairs using dimethylsulfate (DMS) as the modifying agent (Fig. 2 A, summarized in Fig.3). Close association of transcription factors in vivo with DNA can inhibit or enhance DMS methylation of guanine residues. The resulting pattern of methylation is resolved and compared with the pattern observed when the proteins are removed prior to DMS treatment. Analysis of the upper strand reveals seven very strong protections clustered between base pairs −142 and −133 (Fig. 2 A, lanes 2 and 4 compared with lanes 1 and3). These contacts map within the strong activation domain functionally defined in Fig. 1 between base pairs −151 and −113. For ease of discussion, this element will be defined as ARE-1. ARE-1 does not contain guanine residues on the lower strand; thus, DMS cannot reveal interactions on the lower strand (Fig. 2 B, lanes 5 and 6). A second region ofin vivo protein/DNA interaction is observed between base pairs −64 and −56, and is designated activation response element-2 (ARE-2). A single strong enhancement and a partial protection are detected on the upper strand (lanes 1 and2), while the lower strand displays three very strong protections (lanes 7 and 8). In addition to these two obvious domains of in vivo binding, three weaker sites of interaction were detectable. A cluster of five contacts designated site A is found between −18 and −27, corresponding to a sequence with homology to the NF-1 transcription factor binding site. Two partial protections are observed on the upper strand (lanes 1 and 2), while the lower strand displays two weak enhancements and a partial protection (lanes 7 and 8). A second cluster of contacts, site B, is located at a putative octamer-binding site homology (base pairs −45 to −38) and is visualized as a single protected residue on the upper strand flanked by a partial protection and weak enhancement on the lower stand. Finally, a single protection is observed at position −181 on the upper strand (lanes 3 and 4) and is designated site C. Protections were not detected on either strand upstream of base pair −195, and only two isolated weak enhancements were observed at base pairs −226 and −291.Figure 3Schematic of the sequence of CIITA promoter III and in vivo protein/DNA interactions in B-lymphocytes. Open head arrows(protections) and closed head arrows(enhancements) indicate in vivo protein/DNA interactions observed after DMS treatment of cells. Sites of ultraviolet light-induced enhancements of in vivo pyrimidine dimer formation are denoted by V. The 5′-end of each deletion construct tested in Fig. 1 is indicated by a vertical bar and deletion number. The transcription initiation site is indicated by +1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4DNA/protein interactions at the CIITA promoter III correlates with CIITA and MHC class II expression in myeloma cell lines. A, semiquantitative reverse transcription PCR demonstrates that NCI-H929 does not express CIITA or MHC class II mRNA, while U266 expresses low levels of both. B cell lines, Raji and IM9, are shown as positive controls. To ensure the reactions are in the linear range, each group of three lanes represents PCR done with 0.5, 1.0, or 2.0 μg of starting cDNA material. GAPDH was also amplified independently to control for the cDNA concentration and quality (data not shown). B, DMS in vivo footprint analysis of CIITA promoter III in myeloma cell lines. This experiment is the same as in Fig. 2 A except lanes 1–4 are of the promoter in the U266 cells, and lanes 5–8 are of the promoter in the NCI-H929 cells. A nearly identical pattern of contacts is observed in the U266 line, while the NCI-H929 line is completely devoid of in vivo contacts. Lane markings are as described for Fig. 2 A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In vivo footprinting with DMS is limited to analysis of guanine residues, which may allow some interactions to escape detection. In order to expand the effective analysis of interactions on CIITA promoter III, we established in vivo genomic footprinting using UV light as the modifying agent in place of DMS. Ultraviolet light induces the formation of pyrimidine dimers that can be subsequently converted into double strand DNA breaks. Studies on thePKG1, JUN, PCNA, and FOSgene promoters have demonstrated suppression or enhancement of UV photoproduct formation associated specifically with known sites ofin vivo protein/DNA interaction (34Pfeifer G.P. Drouin R. Riggs A.D. Holmquist G.P. Mol. Cell. Biol. 1992; 12: 1798-1804Crossref PubMed Scopus (125) Google Scholar, 35Tornaletti S. Pfeifer G.P. J. Mol. Biol. 1995; 249: 714-728Crossref PubMed Scopus (102) Google Scholar). Analysis of CIITA promoter III demonstrates two strong photoproduct enhancements on the lower DNA strand at the ARE-1 element (Fig. 2 B, lane 6 versus lane 4). This is revealed by the more intense signal in vivo at these two pyrimidine pairs compared with the neighboring pyrimidine pairs at similar or lower doses of UV light. This confirms the strong in vivo protections that were detected by DMS on the upper strand and validates the usefulness of the technique. A second cluster of three very strong photoproduct enhancements is located between base pairs −183 and −172, corresponding to the single DMS enhancement detected on the upper strand at site C. This confirms that site C is a site of in vivo protein binding. In summary, the in vivo footprinting analyses identified five sites of protein/DNA interaction within the first 183 base pairs of the promoter (Fig. 3). Differentiation of B-lymphocytes into plasma cells is accompanied by suppression of MHC class II gene expression (36Halper J. Fu S.M. Wang C.-Y. Winchester R. Kunkel H.G. J. Immunol. 1978; 120: 1480-1484PubMed Google Scholar). In the single plasmacytoma cell line examined to date, CIITA expression was down-regulated, and introduction of CIITA rescued MHC class II expression (37Silacci P. Mottet A. Steimle V. Reith W. Mach B. J. Exp. Med. 1994; 180: 1329-1336Crossref PubMed Scopus (132) Google Scholar). However, studies of other plasmacytoma cell lines have identified some lines that weakly express MHC class II (38Yi Q. Dabadghao S. Osterborg A. Bergenbrant S. Holm G. Blood. 1997; 90: 1960-1967Crossref PubMed Google Scholar). This may be due to the state of differentiation of the transformed cell lines. Two different plasmacytoma cell lines were examined to determine the level of CIITA expression and if in vivo protein/DNA interactions at the CIITA promoter were altered. One cell line, NCI-H929, has no detectable MHC class II DRA or CIITA mRNA (Fig.4 A, lanes 7–9). In contrast, the U266 plasmacytoma cell line has low levels of both (lanes 10–12) compared with the B cell lines Raji and IM9 (lanes 1–6). When examined by DMS in vivo genomic footprinting, CIITA promoter III was completely bare of all in vivo protein/DNA interactions in the CIITA-negative NCI-H929 cell line (Fig.4 B). In contrast, in the low CIITA-expressing cell line U266, interactions were detected at the ARE-1, ARE-2, site A, and site B elements, which were
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