Article15 May 1997free access Expression of MHC class II molecules in different cellular and functional compartments is controlled by differential usage of multiple promoters of the transactivator CIITA Annick Muhlethaler-Mottet Annick Muhlethaler-Mottet L.Jeantet Laboratory of Molecular Genetics, Department of Genetics and Microbiology, University of Geneva Medical School, 1, rue Michel-Servet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Luc A. Otten Luc A. Otten L.Jeantet Laboratory of Molecular Genetics, Department of Genetics and Microbiology, University of Geneva Medical School, 1, rue Michel-Servet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Viktor Steimle Viktor Steimle Hans-Spemann-Laboratories, Max-Plank-Institut für Immunbiologie, Stübeweg 51, 79108 Freiburg, Germany Search for more papers by this author Bernard Mach Corresponding Author Bernard Mach L.Jeantet Laboratory of Molecular Genetics, Department of Genetics and Microbiology, University of Geneva Medical School, 1, rue Michel-Servet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Annick Muhlethaler-Mottet Annick Muhlethaler-Mottet L.Jeantet Laboratory of Molecular Genetics, Department of Genetics and Microbiology, University of Geneva Medical School, 1, rue Michel-Servet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Luc A. Otten Luc A. Otten L.Jeantet Laboratory of Molecular Genetics, Department of Genetics and Microbiology, University of Geneva Medical School, 1, rue Michel-Servet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Viktor Steimle Viktor Steimle Hans-Spemann-Laboratories, Max-Plank-Institut für Immunbiologie, Stübeweg 51, 79108 Freiburg, Germany Search for more papers by this author Bernard Mach Corresponding Author Bernard Mach L.Jeantet Laboratory of Molecular Genetics, Department of Genetics and Microbiology, University of Geneva Medical School, 1, rue Michel-Servet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Author Information Annick Muhlethaler-Mottet1, Luc A. Otten1, Viktor Steimle2 and Bernard Mach 1 1L.Jeantet Laboratory of Molecular Genetics, Department of Genetics and Microbiology, University of Geneva Medical School, 1, rue Michel-Servet, CH-1211 Geneva 4, Switzerland 2Hans-Spemann-Laboratories, Max-Plank-Institut für Immunbiologie, Stübeweg 51, 79108 Freiburg, Germany The EMBO Journal (1997)16:2851-2860https://doi.org/10.1093/emboj/16.10.2851 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The highly complex pattern of expression of major histocompatibility complex class II (MHC-II) molecules determines both the immune repertoire during development and subsequently the triggering and the control of immune responses. These distinct functions result from cell type-restricted expression, developmental control and either constitutive or inducible expression of MHC-II genes. Yet, in these various situations, MHC-II gene expression is always under the control of a unique transactivator, CIITA. Here we show that the CIITA gene is controlled by several distinct promoters, two of which direct specific constitutive expression in dendritic cells and B lymphocytes respectively, while another mediates γ-interferon-induced expression. Thus the cellular, temporal and functional diversity of MHC-II expression is ultimately controlled by differential activation of different promoters of a single transactivator gene. This provides novel experimental tools to dissect compartment-specific gain or loss of MHC-II function in vivo. Introduction The level of major histocompatibility class II (MHC-II) expression directly influences T-lymphocyte activation. The highly complex regulation of MHC-II expression thus controls both the generation of the immune repertoire and the triggering and maintenance of an immune response (Benacerraf, 1981; Janeway et al., 1984). It also represents an interesting example of a tight and complex control of a multi-gene family. Two main modes of MHC-II expression can be distinguished, constitutive or inducible. MHC–II genes are expressed constitutively in only a very restricted number of cell types, specialized in antigen presentation, such as dendritic cells and B lymphocytes. Within these cell lineages, MHC-II expression is also subject to strict developmental control. For example, in the murine B-lymphocyte compartment, early pro-B cells are MHC-II negative, expression in pre-B cells is inducible by interleukin-4 (IL-4), mature B-cells show constitutive MHC-II expression, while terminal differentiation into plasma cells is accompanied by the extinction of MHC-II expression (Glimcher and Kara, 1992). MHC-II expression can also be induced in a large variety of other cell types, in particular by γ-interferon (IFN-γ). In addition, modulation of MHC-II gene expression under a variety of other physiological and pathological conditions has been described (Glimcher and Kara, 1992; Mach et al., 1996). This complex cellular and temporal control of MHC-II genes has important functional consequences in terms of both normal and aberrant T lymphocyte activation. Our current understanding of the mechanisms of MHC–II gene expression results to a large extent from the study of various regulatory mutant cell lines, affected in certain essential MHC-II regulatory factors (Griscelli et al., 1989; Mach et al., 1994). In particular, analysis of cell lines from patients suffering from hereditary MHC-II deficiency (also called bare lymphocyte syndrome; BLS), a genetically heterogeneous disease of gene regulation, has led us to the discovery of three distinct, MHC-II-specific, transacting factors: RFX-5, RFX-AP and CIITA. Each of these factors is essential for the expression of all MHC–II genes and, in all three cases, mutations in the relevant regulatory genes could be identified in BLS patients. RFX–5 (Steimle et al., 1995) and RFX-AP (RFX-associated protein) (Durand et al., 1997) are components of the multi-subunit RFX complex (Reith et al., 1988) that binds specifically to the X box of all MHC-II promoters. The MHC-II transactivator CIITA (Steimle et al., 1993) has been found to act as a master-regulator of MHC-II expression (Reith et al., 1995; Mach et al., 1996). Whereas the different protein subunits of the RFX complex are expressed ubiquitously and are also present in MHC-II-negative cells, CIITA expression can only be detected in MHC-II-positive cell types and tissues (Steimle et al., 1993, 1994). CIITA and MHC-II expression are not only qualitatively but also quantitatively correlated (L.Otten, V.Steimle, S.Bontron and B.Mach, in preparation). In addition to its essential role in constitutive MHC-II expression, CIITA is also the obligatory mediator of IFN-γ-inducible MHC-II expression, since IFN-γ induces CIITA expression which in turn activates MHC-II transcription (Steimle et al., 1994). Furthermore, in plasmocytes, which are MHC-II negative, it was shown that CIITA expression is abolished and that re-expression of CIITA is sufficient to reactivate MHC-II expression in these cells (Silacci et al., 1994). In most cell types, introduction and expression of the CIITA gene is sufficient to activate expression of all MHC-II genes (Mach et al., 1996). These examples demonstrate that control of MHC-II expression is ultimately dependent on the control of expression of the CIITA gene itself. It was therefore an obvious challenge to explore the mechanisms that regulate CIITA expression in various cell types and biological situations and thus to understand how a single rate-limiting transactivator controls the highly complex pattern of regulation of MHC-II genes, including constitutive and inducible expression. We show here that expression of CIITA is controlled, in an unexpected manner, by four independent CIITA promoters leading to CIITA transcripts with four distinct first exons. The individual CIITA promoters are used in an alternative and tissue-specific manner. Distinct CIITA promoters control either MHC-II constitutive expression in dendritic cells (promoter I, PI) and in B lymphocytes (PIII), or IFN-γ-inducible expression (PIV) in a variety of MHC-II-negative cell types. We conclude that the cellular, temporal and functional diversity in MHC-II expression is not regulated at the level of the MHC-II genes themselves, but is ultimately under the control of several promoter sequences that activate differentially the same transactivator gene. Results Multiple 5′ ends of CIITA mRNA When the murine homologue of human CIITA (Steimle et al., 1993) was isolated from a mouse cDNA library by low stringency hybridization (L.Otten, V.Steimle, S.Bontron and B.Mach, in preparation), sequence comparison between human and mouse CIITA cDNAs showed excellent identity downstream of position +167 of the human CIITA cDNA, but totally divergent 5′ ends. To establish the structure of the 5′ region of CIITA mRNA, a systematic analysis of CIITA 5′ ends was performed by RACE–PCR both on murine and human RNAs isolated from different tissues, using primers from the homologous region (Figure 1). These assays led to the isolation of four different CIITA cDNA 5′ sequences in human and three in mouse. Sequence analysis revealed that all these different cDNA clones had identical 3′ ends but different 5′ ends. In each case, the boundary between the diverging 5′ ends and the common 3′ ends was located, in human and mouse, 21 nucleotides upstream of the second ATG codon of the CIITA cDNA sequence described previously (Steimle et al., 1993) (Figure 1). The different CIITA 5′ ends were numbered I–IV according to their relative position in the genomic map of the 5′-flanking region of the CIITA gene (see below). CIITA type I, III and IV 5′ sequences were identified in human and mouse genomic DNA, while the CIITA type II 5′ end was found only in human DNA. Figure 1.The four different 5′ ends of the human CIITA mRNAs. Coding regions are represented by wide boxes, 5′-untranslated regions by narrow boxes. The non-homologous regions are hatched differently. The positions of the two primers (P1, P2) used for RACE–PCR analysis are indicated by small arrows. The 3′ boundary of exon 2 has not been determined. Download figure Download PowerPoint Figure 2.Genomic organization of the 5′-flanking regions of the human and murine CIITA gene. The black boxes represent the different first exons, the small open boxes correspond to introns. The restriction sites for EcoRI (E), KpnI (K), NheI (N), SacI (S), XbaI (X) and XhoI (Xh) are indicated. The arrows represent the major initiation site of each first exon. Download figure Download PowerPoint Of the four different forms of CIITA transcripts which where thus identified, human CIITA type III corresponds to the previously described form of CIITA cDNA (Steimle et al., 1993). The four transcripts share a common open reading frame starting from the AUG which is located 21 bp downstream of the 5′ end of the common nucleotide sequence (Figure 1). For CIITA forms II and IV, this AUG, located in the context of a perfect Kozak consensus (Kozak, 1989), is the first initiation codon (Figures 1 and 3B and D). CIITA forms I and III display, in addition, upstream AUGs in favourable translation initiation contexts, which potentially lead to CIITA proteins with an additional 101 or 24 N-terminal amino acids respectively (Figures 1 and 3A and C). These features are shared between human and murine CIITA 5′ ends I, III and IV. The respective nucleotide conservation within these different CIITA 5′ ends is 75, 68 and 66%. For the sake of coherence and clarity, we propose to maintain the numbering of the amino acid sequence of the body of the CIITA sequence as published originally for CIITA type III (Steimle et al., 1993). Figure 3.Sequence comparison of the first exons of CIITA with their respective 5′-flanking regions in human and mouse. (A) CIITA type I. (B) CIITA type II. (C) CIITA type III. (D) CIITA type IV. For CIITA types I, III and IV, the upstream regions showing significant sequence homology between human and mouse are shown. Major mRNA initiation sites are indicated by horizontal arrows. The +1 positions of the murine sequences were defined as the homologous positions of the human +1 sites. For human CIITA type II, the +1 site is defined by RACE–PCR. Exonic regions are shaded. For CIITA types I and III, the predicted amino acid sequences starting from the upstream AUGs (white boxes) are shown in the one-letter code. The 3′ exon–intron boundaries of the different exons 1 are indicated by a vertical bar and the splice donor sites are shown in lower case letters. The different binding sites for known transcription factors are indicated. The white circle represents an AP1 site, the grey box a CCAAT box. Box1 is composed of either two NF-IL6 sites or two PEA3 sites in palindromic orientation. Download figure Download PowerPoint Since the nucleotide sequences of the 5′ region of all four different CIITA mRNAs differ, they could be generated either by alternative splicing of different 5′ exons in the CIITA gene or by a differential usage of four different promoters. Complete lack of sequence homology at the very 5′ ends of the divergent sequences favoured the second hypothesis. Genomic organization of the 5′-flanking region of the CIITA gene A human and a murine genomic λ phage library were screened using DNA fragments from the human type III and the murine type I and III 5′ ends of CIITA cDNAs, respectively. Three overlapping human genomic clones and five overlapping murine genomic clones covering 24 and 35 kb were obtained, and a detailed restriction map was established (Figure 2). Fragments hybridizing to the four different 5′ ends were identified, subcloned and sequenced. In all cases, the genomic sequence corresponded exactly to the sequences of the different 5′ ends of CIITA cDNA clones and thus represented distinct exons. Furthermore, the nucleotide sequences of exon–intron boundaries agreed with consensus splice donor sequences (Breathnach and Chambon, 1981). Screening of the murine genomic clones with a human CIITA type II 5′ end cDNA probe did not yield a positive signal. The four different 5′ exons (i.e. exon 1 of CIITA mRNA I, II, III and IV respectively) were mapped within the 5′-flanking region of the CIITA gene. The distance between exon 1 of CIITA type I and exon 1 of CIITA type IV is ∼13 kb in human and 10 kb in the mouse. The relative order and, in part, the genomic distances between homologous promoters are maintained between the two species, indicating a strongly conserved genomic organization (Figure 2). Multiple transcription initiation sites reveal four distinct CIITA promoters The transcription initiation sites of the different human CIITA mRNAs were mapped by RNase protection assays with fragments covering the different CIITA exons 1 and their respective 5′-flanking regions. For CIITA type I, three protected fragments were detected using mRNA from liver (data not shown). The major protected fragment corresponded to a major transcription initiation site located 380 bp upstream of the 3′ end of exon 1. It has been defined as nucleotide +1 of CIITA type I mRNA (Figure 3A). The two minor fragments indicate initiation start sites at positions −14 and +8 of CIITA type I. These transcription initiation sites fit well with those obtained by RACE–PCR in mouse. For CIITA type III, several protected fragments were obtained using mRNA from a B-lymphocyte cell line (data not shown). The major transcription initiation site located 183 bp upstream of the 3′ end of exon 1 defines position +1 of CIITA type III mRNA (Figure 3C). Two other main initiation start sites are located at positions −8 and −4, and several minor sites from positions −23 to +34. The longest CIITA type III cDNAs obtained by RACE–PCR fit with these initiation start sites. The CIITA cDNA sequence initially described (Steimle et al., 1993) begins at position +17. For CIITA type IV, multiple fragments were protected, using mRNA from an IFN-γ-induced melanoma cell line, with the major transcription initiation site located 75 bp upstream of the 3′ end of exon 1 (data not shown). This defines nucleotide +1 of CIITA type IV mRNA (Figure 3D). A second major and six minor transcription initiation sites were identified at position +17 and between positions −54 and +69 of exon 1 of CIITA type IV, respectively. The 5′ ends of many CIITA cDNAs obtained by RACE–PCR coincide with these initiation sites. No tissue or cell line could be identified so far in which the CIITA type II 5′ end is expressed at a significant level; the exact initiation site of this form of CIITA transcript was, therefore, not analysed further. Since all types of CIITA mRNAs were transcribed from multiple transcription initiation sites, we can conclude that these sites represent bona fide 5′ ends of transcription products from four distinct CIITA promoters (PI, PII, PIII and PIV) located upstream of four different CIITA exons 1 (of type I, II, III and IV respectively) (Figure 2). Analysis of promoter sequences Comparison of the sequences of the four different CIITA promoters showed no significant homology to each other, both in human and mouse. None of the regions upstream of the different CIITA first exons contains a consensus TATA box element or GC box. The absence of a TATA box is consistent with the finding that there are multiple initiation start sites (Breathnach and Chambon, 1981). In contrast, comparison of the homologous CIITA promoters between human and mouse showed significant sequence homology. For CIITA promoters I, III and IV, this homology extends over 120, 800 and 350 nucleotides respectively (Figure 3). More interestingly, within these promoter regions, small segments that are very well conserved within the two species could be identified (Figure 3). This sequence conservation suggests functional relevance and potential cis-acting elements. On the basis of this strong human–mouse conservation, as well as on the basis of sequence homology with known cis-acting elements, the following potential cis-acting elements of the different CIITA promoters can be pointed out. CIITA promoter I contains an NF-GMb site (Shannon et al., 1988), an NF-IL6 site (Akira and Kishimoto, 1992), two NF-IL6 inverted sites, a PEA3 site (Wasylyk et al., 1989), a PEA3 inverted site and an E2A site (Murre et al., 1989) in human and mouse respectively. In addition, human CIITA promoter I includes an AP1 site (Pollock and Treisman, 1990) and a CCAAT box (Dorn et al., 1987) (Figure 3A). In CIITA promoter III, one observes an E2A inverted box (Murre et al., 1989), an IRF1/2 site (Tanaka et al., 1993), an MYC inverted site (Agira et al., 1989) and an OCT inverted site (Rosales et al., 1987) in human and mouse, respectively, as well as two CCAAT boxes in mouse (Figure 3C). CIITA promoter IV, the IFN-γ-responsive promoter (see below), contains a NF-GMa site (Shannon et al., 1988), a GAS box (Pellegrini and Schindler, 1993), an adjacent E box (Blackwell et al., 1990) and an IRF1/2 site, both in human and mouse (Figure 3D). In addition, there is an NFκB site (Sen and Baltimore, 1986) in human and two AP1 sites in mouse. Although it is beyond the scope of this report, a systematic functional analysis of the role of these various potential cis-acting motifs is obviously required. Differential usage of the multiple CIITA promoters To study the pattern of expression of the different forms of CIITA mRNAs, and thus of the different CIITA promoters, RNase protection analyses using probes specific for each type of CIITA mRNA were performed (Figure 4A). A panel of different tissues and cell lines expressing the CIITA gene either constitutively or following IFN-γ induction were analysed. The results were quantified by PhosphorImager. Interestingly, there is a striking difference in the specific usage of each of the different CIITA promoters. CIITA type I mRNA, resulting from the use of promoter I, is expressed at a remarkably high level in dendritic cells (Figures 4B and 5). It is barely detectable in spleen and thymus, and no other tissue or cell line could be identified in which CIITA type I was expressed. Figure 4.Differential expression of CIITA types I, III and IV mRNAs. (A) Schematic representation of the probes used for analysis of expression pattern of the multiple CIITA mRNAs by RNase protection assays. The different specific probes are indicated with their sizes before and after RNase digestion. Each probe covers a part of exon 1 and 226 nucleotides. For example, the CIITA type I-specific probe protects a 333 nucleotide type I-specific fragment and protection of the 226 nucleotide fragment is due to hybridization with all other types of CIITA mRNAs (non-type I). The same applies to probes specific for CIITA type III and type IV. The RNAs were hybridized with the specific CIITA probe, an internal CIITA probe (‘internal’) and a probe for TBP (‘control’). (B) Analysis of CIITA type I expression. (C) Analysis of CIITA type III (upper panel) and CIITA type IV (lower panel). The positions of the protected fragments are indicated. Download figure Download PowerPoint Figure 5.Schematic representation of the differential expression of the four types of CIITA transcripts. The amount of the different types of CIITA mRNAs is given as a percentage of the total amount of CIITA mRNA expression as measured by the internal CIITA probe after quantification by PhosphorImager of the protected fragments obtained by RNase protection analysis. Download figure Download PowerPoint The presence of CIITA type III mRNA is detected at a high level in different B-lymphocyte cell lines, as well as in tissues rich in B lymphocytes such as spleen and tonsils, and also in the thymus (Figure 4C, upper panel, and Figure 5). In contrast, type III mRNA was detected only at a low level in dendritic cells and even less so in a variety of IFN-γ-induced cell lines (Figures 4C and 5). CIITA type IV mRNA is the major form expressed following induction by IFN-γ. This was observed in different IFN-γ-inducible cell lines, such as melanoma (Me67.1), monocytes (THP1), endothelial cells (HUVEC) and fibroblasts (PP2) (Figure 4C, lower panel, and Figure 5). In contrast, only a low level of type IV CIITA transcript was detectable in B lymphocytes or in dendritic cells (Figure 4C, upper panel, and Figure 5). No cell line or tissue could be identified to date in which a significant amount of CIITA type II mRNA is detectable by RNase protection assay (data not shown). The quantification of the differential expression of the different CIITA transcripts, from their respective endogenous promoters, is represented schematically in Figure 5, and the percentages of relative promoter usage are indicated in Table I. Table 1. Percentage of the different types of CIITA mRNAs observed in different tissues and cell lines (see Figures 4 and 5) Type I Type III Type IV Spleen 3.5 67 33 Tonsil 0 96 17 Thymus 6 60 33 Raji 0 86 2.5 Mann 0 72 17 Dendritic 74 39 2.7 Me67.1 + IFN-γ 0 2 88 THP1 + IFN-γ 0 14 62 HUVEC + IFN-γ ND 10 68 PP2 + IFN-γ ND 16 66 Differential CIITA expression is under transcriptional control To determine if the expression of the CIITA gene is controlled at the level of transcription, an alternative method to the classical run-on assay was carried out for the analysis of the transcription rate of CIITA RNA. Nascent RNA chains were isolated from nuclei by precipitation of a chromatin pellet containing DNA, histones and ternary transcription complexes (Wuarin and Schibler, 1994). Nascent RNA chains were then isolated from the chromatin pellet and analysed by RNase protection assays. The transcription rate of the CIITA gene was analysed in the monocyte cell line THP1 before and after induction by IFN-γ. Before induction, no CIITA nascent RNA could be detected (Figure 6, lane 3). After induction by IFN-γ, CIITA nascent RNA was detected at a high level (Figure 6, lane 5). As a control, nascent RNA from a B-lymphocyte cell line (Raji), which expresses CIITA constitutively, was analysed (Figure 6, lane 1). The level of nascent RNA was similar in the B-cell line and in IFN-γ-induced THP1 cells. These results show that the induction of the CIITA gene by IFN-γ is controlled mainly at the level of transcription. Moreover, no nascent RNA could be detected in a T-lymphoma cell line (CEM) constitutively negative for CIITA expression (data not shown). The constitutive expression of CIITA RNA is thus also controlled at the level of transcription. Comparison of lanes 5 and 6 shows that the relative amount of CIITA RNA versus TATA box-binding protein (TBP) RNA is completely different between nascent RNA and nuclear RNA. This excludes any contamination of the nascent RNA chains by nuclear RNA. Figure 6.Analysis of transcription from CIITA promoters III and IV. Nascent RNAs (lanes 1, 3 and 5) and free nuclear transcripts (lanes 2, 4 and 6) were analysed by RNase protection assays in B lymphocytes (Raji), and in monocytic cells (THP1) before (−) and after (+) induction by IFN-γ. The RNAs were hybridized with the internal CIITA probe, the TBP probe and the CIITA type III or type IV probe for Raji and THP1, respectively. Download figure Download PowerPoint The promoter-proximal regions control alternative usage of different CIITA promoters The functional activity and tissue specificity of CIITA promoters III (PIII) and IV (PIV) were analysed by transient transfection assays of promoter–reporter gene constructs. Since CIITA type III mRNA is the major form expressed in B lymphocytes and CIITA type IV mRNA is expressed preferentially in IFN-γ-induced cells, the B–lymphocyte cell line Raji and the melanoma cell line Me67.8 were chosen for these functional assays. Because of the very low level of expression of the CIITA gene (Steimle et al., 1993), it was necessary to use a highly sensitive reporter gene assay, such as quantitative RT–PCR transcription assay (Sperisen et al., 1992). For both CIITA promoters PIII and PIV, two different fragment sizes were analysed and compared with the promoterless plasmid pGβG(+) as a negative control. Transfections of pIII-974 and pIII-322 in B lymphocytes revealed a high activity of CIITA promoter PIII, whereas in Me67.8 cells, the same promoter PIII was inactive, before and after induction by IFN-γ (Figure 7A). In B lymphocytes, the activity of the short pIII-322 promoter was higher than that of the longer promoter pIII-974 (signal ratios of 8.2 and 2.6 respectively). The control pGβG(+) was negative in both cell types. Figure 7.Functional analysis of CIITA promoters III and IV. (A) Transient transfections of Raji and Me67.8 cells with the plasmids pIII-974, pIII-322 or the promoterless plasmid pGβG(+) and the reference plasmid. The input ratios between CIITA constructs and the reference plasmid were 4:1 for Raji and 9:1 for Me67.8. c, RT–PCR signals from CIITA 5′-flanking region–β-globin reporter gene construct; r, RT–PCR signals from reference plasmid. The signal ratio is determined by normalizing the mRNA signal derived from the CIITA construct to that obtained with the reference plasmid (see Materials and methods). (B) Transient transfections of Raji and Me67.8 with the plasmids pIV-950, pIV-461 or pGβG(+) and the reference plasmid, with the same ratio as in (A). Download figure Download PowerPoint In contrast, the CIITA promoter IV (pIV-950 and pIV-461) showed only a baseline activity in B lymphocytes (Raji) (signal ratios of 0.4 and 1.1 respectively), whereas the same CIITA promoter IV was highly induced by IFN–γ in Me67.8 cells (Figure 7B), as well as in other IFN–γ-inducible cell lines (HeLa, 2FTGH, data not shown). Signal ratios of promoters pIV-950 and pIV-461 rose from 0.13 and 0.18 before induction to 7.9 and 29.6, respectively, after IFN-γ induction. These results provide functional evidence for the existence of distinct CIITA promoter regions, located immediately upstream of the alternative 5′ exons type III and IV. They also showed that the use of these two distinct CIITA promoters is indeed controlled in a tissue-specific manner, either constitutively or following induction, as observed earlier for the transcription of the endogenous CIITA gene into distinct CIITA mRNA. Discussion The very complex and tight regulation of expression of the MHC-II gene family has direct implications for T–lymphocyte activation and thus for the control of the immune response. In addition, significant pathophysiological consequences such as immunodeficiency or aberrant T-cell activation may arise from the disregulation of MHC–II gene expression. The unsuspected findings reported here allow us to attribute the tissue-specific and temporal control of expression of MHC-II molecules, and hence of T-cell activation, to the selective usage of distinct alternative promoters of the regulatory gene CIITA. This indicates that the complex pattern of regulation that determines the diverse functions of MHC-II molecules, in positive and negative selection for the generation of the immune repertoire, as well as in the induction of an immune response by antigen-presenting cells (APCs), is controlled entirely by the differential activation of multiple promoters of a single transactivator gene. Expression of MHC-II molecules can be modulated by a number of different stimuli including
