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* University of Geneva Medical School, Centre Médical Universitaire, Geneva, Switzerland;
NovImmune, Geneva, Switzerland; and
Swiss Institute for Experimental Cancer Research, Swiss Institute of Bioinformatics, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced cells, and induce broad domains of histone hyperacetylation. These results reveal a hitherto unexpected level of complexity involving long distance control of MHC-II expression by multiple distal regulatory elements. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and 
-chains of the classical MHC-II molecules HLA-DP, HLA-DQ, and HLA-DR. The intracellular transport and peptide loading of these molecules require the function of accessory genes, including those encoding the invariant chain (Ii) and the nonclassical MHC-II molecules HLA-DO and HLA-DM (1, 3). With the exception of the Ii gene, which is located on chromosome 5, all these genes are clustered in the class II region of the MHC locus on the short arm of chromosome 6 (4, 5).
Establishing a proper pattern of MHC-II-restricted Ag presentation is critical for the immune system. The MHC-II and accessory genes are consequently tightly coregulated and expressed in a precisely controlled fashion (6, 7). Constitutive expression is primarily restricted to specialized APCs (B cells, dendritic cells, and macrophages) and epithelial cells of the thymus. Many other cell types can be induced to coexpress these genes by exposure to IFN- or other stimuli (7). Coordinate expression of MHC-II and accessory genes is controlled at the level of transcription by a shared 59- to 68-bp regulatory module situated within 
300 bp upstream of the transcription initiation site of each gene (8). This promoter-proximal regulatory module is a composite motif consisting of four well-defined sequence elements, the S, X, X2, and Y boxes, present in a strictly constrained order, orientation, and spacing (Fig. 1A). This characteristic architecture is essential for function of the S-Y module (9, 10)
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RFX and CIITA are both essential and specific for activation of MHC-II promoters. This is underlined by the fact that mutations in CIITA or one of the three subunits of the RFX complex (RFXANK, RFX5, and RFXAP) are responsible for a severe hereditary immunodeficiency disease characterized by a highly specific defect in MHC-II expression (7, 11, 12, 13, 14, 15, 27, 28).
There is no doubt that the promoter-proximal S-Y modules are essential for the expression of MHC-II and accessory genes, and research in the field has thus concentrated on these regions (7, 25, 26). However, there is growing evidence that additional distal regulatory elements also play a key role. First, a locus control region (LCR) has been described upstream of the mouse H2-Ea gene (29, 30). Although a detailed dissection of this LCR is lacking, it is clear that one of its key elements is an inverted copy of the S-Y module (S'-Y') situated 1.3 kb upstream of the H2-Ea gene and 2.3 kb upstream of the orthologous human HLA-DRA gene. We have shown that this distal S'-Y' enhancer is bound in vivo by RFX and CIITA and exhibits functional features strongly reminiscent of the LCR of the -globin locus (31). Second, an enhancer resembling the S-Y module has been described in the first intron of the Ii gene (32).
With this in mind, we designed a computational approach relying on the unique architecture of the composite S-Y regulatory module to identify novel target sites of RFX and CIITA. This led to the unequivocal identification of six novel S-Y like (S'-Y') modules. These include four sites placed at strategic positions within the MHC-II locus and two intronic enhancers in the Ii gene. Formation of the MHC-II enhanceosome and recruitment of CIITA to these S'-Y' modules mediates long-range chromatin remodeling, as indicated by the induction of global histone hyperacetylation over large domains. These findings reveal that the regulation of MHC-II expression exhibits a previously unsuspected level of complexity, involving a combination of promoter-proximal and -distal regulatory elements.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The sequence alignment used to create the search profile was constructed using promoter sequences from the following genes and alleles: DRA (X83114), DRB1*0101 (M81172), DRB1*0102 (X64441), DRB1*0302 (X64440), DRB1*0405 (L07840), DRB1*0802 (X64442), DRB1*0803 (X64439), DRB1*1201 (X64438), DRB1*1301 (X65565), DRB1*1302 (X65564), DRB1*1401 (X65563), DRB1*1402 (X65562), DRB1*03011 (Z84489), DRB1*0802 (X64442), DRB1*0901 (L07839), DRB2 (S57469), DRB3 (S57471), DRB3*0101 (X65558), DRB3*0201 (X65559), DRB4 (S57473), DRB4*0101 (L07841), DRB5 (S57475), DRB5*0101 (X64548), DRB5*02 (X64549), DRB-WS9009 (M81174), DRB-WS9010 (M81171), DRB-WS9011 (M81180), DMA (AJ249712), DMB (AJ249714), DQA1-DRw17-Dw3 (M97464), DQA1-DRw8-Dw8.3 (M97463), DQA1-DR4-DR5 (M97462), DQA1-DR4-Dw4 (M97461), DQA1-DRw8-Dw8.1 (M97459), DQA1-DR9-Dw23 (M97458), DQA1-DR7-DB1 (M97457), DQA1-DRw15-Dw2 (M97455), DQA1-DR1-Dw1 (M97454), DQB1*02 (U49059), DQB1*0201 (X74230), DQB1*0402 (Z80898), DQB1-Dw4-DR4 (K01499), DPA (X02228), DPB (X02228), DOA (Z81310), DOB (L29472), and Ii (NT_029289). A frequency table was established for the nucleotides at all positions, converted to a generalized profile (33), and modified to increase the weight of the core X and Y boxes. The final search profile was the following:
ID MHC_CLASS_2_PRM; MATRIX.
AC NS00001;
DT OCT-2001 (CREATED).
DE mammalian MHC class II promoter model
MA/GENERAL_SPEC: ALPHABET = ‘ACGT’; LENGTH = 68;
MA/DISJOINT: DEFINITION = PROTECT; N1 = 1; N2 = 68;
MA/NORMALIZATION: MODE = 1; FUNCTION = LINEAR; R1 = 0.0; R2 = 0.01;
MA TEXT = ‘ru’;
MA/CUT_OFF: LEVEL = 0; SCORE = 500; N_SCORE = 10.0; MODE = 1;
MA/DEFAULT: B0 = 0; B1 = *; E0 = 0; E1 = *; SY_M = ‘N'; II = *;
MA/I: B0 = 0; B1 = 0;
MA/M: SY = ‘G’; M = –10, –282, 50, –142;
MA/M: SY = ‘R’; M = 12, 0, 22, –142;
MA/M: SY = ‘R’; M = 28, 0, –16, –46;
MA/M: SY = ‘Y’; M = –282, 42, –282, 16;
MA/M: SY = ‘Y’; M = –46, 42, –46, –16;
MA/M: SY = ‘Y’; M = –46, 12, –142, 36;
MA/M: SY = ‘T’; M = –46, –142, –46, 52;
MA/M:/M:/M:/M:/M:/M:/M:/M:/M:
MA/I: MD = 0;/I: MD = 0;/I: MD = 0;/I: MD = 0;/I: DM = 0;
MA/M: SY = ‘Y’; M = –141, 16, –141, 16;
MA/M: SY = ‘Y’; M = –141, 16, –141, 16;
MA/M: SY = ‘Y’; M = –141, 16, –141, 16;
MA/M: SY = ‘Y’; M = –141, 16, –141, 16;
MA/M: SY = ‘R’; M = 16, –8, 16, –71;
MA/M: SY = ‘C’; M = –9, 26, –56, –71;
MA/M: SY = ‘C’; M = –71, 28, –56, –23;
MA/M: SY = ‘Y’; M = –23, 17, –71, 8;
MA/M: SY = ‘A’; M = 112, –282, –222, –282;
MA/M: SY = ‘G’; M = –282, –282, 118, –282;
MA/M: SY = ‘Y’; M = 24, 46, –282, 40;
MA/M: SY = ‘R’; M = 92, –282, 4, –282;
MA/M: SY = ‘A’; M = 110, –92, –282, –282;
MA/M: SY = ‘C’; M = –222, 110, –92, –282;
MA/M: SY = ‘W’; M = 11, –19, –71, 14;
MA/M: SY = ‘G’; M = –7, –71, 23, –23;
MA/M: SY = ‘A’; M = 32, –222, 32, –282;
MA/M: SY = ‘T’; M = –282, –282, –42, 104;
MA/M: SY = ‘G’; M = –10, –282, 94, –162;
MA/M: SY = ‘A’; M = 108, –74, –282, –282;
MA/M: SY = ‘Y’; M = –23, 0, –23, 18;
MA/M: SY = ‘G’; M = –19, –71, 23, –8;
MA/M: SY = ‘Y’; M = –23, 11, –71, 15;
MA/M: SY = ‘Y’; M = –8, 11, –23, 6;
MA/M: SY = ‘R’; M = 21, –23, –8, –23;
MA/M:/M:/M:/M:/M:/M:/M:/M:/M:/M:/M:
MA/I: MD = 0;/I: MD = 0;/I: MD = 0;/I: MD = 0;/I: DM = 0;
MA/M: SY = ‘C’; M = –71, 23, –23, –8;
MA/M: SY = ‘T’; M = –71, –8, –71, 26;
MA/M: SY = ‘G’; M = –11, –23, 24, –71;
MA/M: SY = ‘A’; M = 118, –282, –282, –282;
MA/M: SY = ‘T’; M = –282, –282, –282, 108;
MA/M: SY = ‘T’; M = –92, –282, –92, 102;
MA/M: SY = ‘G’; M = –220, –142, 106, –90;
MA/M: SY = ‘G’; M = –282, –282, 118, –282;
MA/I: E0 = 0; E1 = 0;//
Scans of the Genomic Reference Sequence of Homo sapiens, build 29 (International Human Genome Sequencing Consortium), were performed with the program pfscan from the pftools package (available at: ftp://ftp.isrec.isb-sib.ch/pub/software/unix/pftools/), which is a software implementation of the generalized profile method (33).
Cell lines
RJ2.2.5 is a CIITA-deficient mutant derived from the wild-type human Raji B cell line (27, 34). The RFXANK-deficient B cell line BLS1 was derived from a BLS patient with a null mutation in the RFXANK gene (13, 35). BLS1c is BLS1 complemented stably with a wild-type RFXANK cDNA (36). ME67.8 is a human melanoma cell line previously used to study IFN--induced CIITA and MHC-II expression (37). Cells were grown in RPMI 1640 and Glutamax medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% FCS and antibiotics. Me67.8 cells were induced with 200 U/ml human IFN- (Invitrogen Life Technologies).
Chromatin immunoprecipitation (ChIP) assays
ChIP assays were performed as previously described (31) using polyclonal rabbit anti-RFX and anti-CIITA antisera (11, 38); anti-histone H3 (ab1791; Abcam, Cambridge, UK), anti-acetylated histone H3, 8, and histone H4 Abs (06-599 and 06-866; Upstate Biotechnology, Lake Placid, NY); and a mixture of two anti-CREB Abs (sc-186 and sc-58; Santa Cruz Biotechnology, Santa Cruz, CA). Ten micrograms of chromatin (corresponding to 1.2 x 107 cells), fragmented by sonication to an average size of 400 bp, was used for each immunoprecipitation. All ChIP experiments were repeated at least twice with identical results. DNA sequences present in the immunoprecipitates were quantified by real-time PCR using the primers listed in Table I. To avoid cross-reactivity with different S-Y modules, at least one of the primers in each pair was placed at unique sequences situated outside of the conserved S, X, X2, and Y boxes (Fig. 2D). PCR was performed using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) and a SYBR Green-based kit for quantitative PCR (Eurogentec, Seraing, Belgium). The specificity of amplification was controlled by gel electrophoresis and dissociation curve analysis. The amounts of immunoprecipitated DNA were calculated by comparison with a standard curve generated with serial dilutions of input DNA. For global acetylation patterns, the signals were corrected for nucleosome density by dividing signals obtained with anti-acetylated histone Abs by signals obtained with Abs directed against unmodified histone H3.
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Plasmids and reporter gene assays
The pDRAprox contains an HLA-DRA promoter fragment (from –151 to +10) inserted upstream of a firefly luciferase reporter gene in the pGL3-Basic vector (Promega, Madison, WI) (31). The pDRAmin contains only the HLA-DRA core promoter (from –60 to +10) in the same reporter plasmid (31). Plasmids containing the 4, 5, 8, and Ii S''-Y'' sequences were created by replacing the MluI-BglII fragment spanning the S-Y region of pDRAprox with the corresponding 4, 5, 8, and S''-Y'' motifs amplified by PCR from genomic DNA. Primers used to generate these constructs are listed in Table II. Raji, RJ2.2.5, BLS1c, and BLS1 cells were cotransfected (in a 10:1 ratio) with the firefly reporter plasmids containing the S-Y sequences and a control Renilla luciferase plasmid (pRL-TK). Transfections were performed by electroporation (950 µF, 0.21–0.25 V in 4-mm cuvettes). Dual luciferase reporter gene assays were performed according to the manufacturer’s instructions (Promega).
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Promoter pull-down assays with whole cell extracts prepared from BLS1 (RFXANK–/–) and BLS1c (wild-type) cell lines were performed as described previously (18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A sequence alignment of the S-Y modules from all classical and nonclassical MHC-II genes, including allelic variants, was used to calculate nucleotide frequencies at all positions in the S, X, X2, and Y boxes. These frequencies together with the S-X and X2-Y spacing constraints were used to create a generalized profile, which is represented graphically by the sequence logo in Fig. 1A.
The MHC-II profile was used to scan the entire human genome using the pfscan program (33). This program assigns scores proportional to the similarity between the hits and the search profile. The hits with scores >10 are plotted in the human genome in Fig. 1B. As expected, the search identified the promoters of the Ii gene on chromosome 5 and all classical and nonclassical MHC-II genes on chromosome 6 (Fig. 1B). These sequences were assigned scores ranging from 12.5–17.3 (with the exception of HLA-DQA, where the score was 7.6). In addition, the search found a background of low scoring sequences (scores 10–13) spread throughout the genome at an average density of roughly one per million base pairs. Subsequent ChIP experiments demonstrated that three randomly chosen hits on chromosomes 3, 5, and 11 are not bound by RFX or CIITA despite the fact that they were assigned scores of 13.0, 10.2, and 11.2, respectively (Fig. 2, A and B). Moreover, a control scan performed with a meaningless profile consisting of an inverted MHC-II profile containing G/C and A/T transversions resulted in a similar number of hits spread throughout the genome (Fig. 1C). Therefore, the vast majority of these background hits probably represent fortuitous similarities to the MHC-II profile (see Discussion).
Interestingly, the density of hits within the MHC-II locus was found to be >10-fold higher than elsewhere in the genome, even after eliminating the distal HLA-DRA enhancer and the promoter regions of all known MHC-II genes and pseudogenes (Fig. 1D). This is due to the presence of nine potential S-Y modules (Fig. 1B; hits 1–9) found in two clusters situated in the DQ region and adjacent to the DP region. These sequences are henceforth referred to as S'-Y' modules.
The scan with the MHC-II profile failed to pick up any of the S-Y motifs found in the promoters of MHC-I genes, which were shown previously to be targets of RFX and CIITA (39). This was expected, because the MHC-II and MHC-I promoter consensus sequences differ at several critical positions within the conserved S, X, X2, and Y boxes. A separate search based on the MHC-I profile correctly identified all classical MHC-I genes, but did not find any new candidate sequences (data not shown).
Novel target sites of RFX and CIITA in the MHC-II locus
We performed ChIP experiments to determine whether the new S'-Y' modules in the MHC-II locus represent true targets of RFX and CIITA. Binding specificity was controlled by comparing wild-type B cells with mutant B cells lacking CIITA (RJ2.2.5 cells) or the RFXANK subunit of RFX (BLS1 cells). As a reference we used the promoter-proximal region of the HLA-DRA gene, which is the most well-studied target of RFX and CIITA (36, 40). Four of the S'-Y' modules (2, 4, 6, and 8; scores 10.1, 10.1, 11.3, and 11.9, respectively) are bound efficiently by RFX and CIITA in vivo in wild-type cells (Fig. 2). Binding of RFX at these sites ranges from 50–100% of that observed at the HLA-DRA promoter. Binding of CIITA ranges from 20–100%. This is highly significant because it, in fact, exceeds that observed at the drb6 pseudogene. The specificity of RFX and CIITA association with the new S'-Y' motifs is emphasized by their lack of binding to randomly chosen sequences situated outside the MHC locus (Fig. 2). Therefore, S'-Y' sequences 2, 4, 6, and 8 can be unambiguously defined as new RFX and CIITA target sites in vivo. The remaining candidates (1, 3, 5, 7, and 9; scores 9.9, 9.7, 14.1, 9.2, and 9.4, respectively) do not show significant binding and can be classified as false positives (Fig. 2). No obvious defects were pinpointed in candidate sequences that are not bound in vivo by RFX, CREB, or CIITA. The failure of these sequences to be bound could be due either to an accumulation of several minor deviations from the permissive consensus sequence or to chromatin interference at the loci containing these sites.
Binding of RFX and assembly of the MHC-II enhanceosome at the HLA-DRA promoter in B cells are independent of CIITA (36). Binding of RFX5 at the HLA-DRA promoter is thus normal in CIITA-deficient cells (Fig. 2A). In contrast, mutations in RFXANK abrogate formation of the RFX complex, which eliminates both enhanceosome assembly and CIITA recruitment at the HLA-DRA promoter (36). Occupation of the HLA-DRA promoter by both RFX5 and CIITA is thus lost in RFXANK-deficient cells (Fig. 2, A and B). A very similar pattern is observed at the S'-Y' sites (Fig. 2). It should be noted, however, that two of the sites exhibit minor, but notable, differences. First, at sequence 8, binding of RFX5 is partially dependent on CIITA, indicating that CIITA association has a stabilizing effect on the enhanceosome complex. This is reminiscent of the situation in IFN--induced cells, where full MHC-II promoter occupancy requires CIITA (see below). Secondly, at sequence 2, significant levels of RFX5, CREB, and CIITA association are detected in the BLS1 mutant, indicating that a partial enhanceosome complex can form in the absence of RFXANK. A similar observation has been made for certain other target sites, such as the promoter of the Ii gene (see below) (36). To confirm that partial enhanceosomes can form in the absence of RFXANK, we performed promoter pull-down assays in vitro. DNA fragments containing HLA-DRA promoter region were incubated with extracts obtained from BLS1 and BLS1c cells, and DNA-bound complexes were eluted and analyzed by Western blotting. As shown in Fig. 2E, a partial complex containing RFX5 and RFXAP can indeed assemble on the promoter in the absence of RFXANK. It should also be noted that the formation of a dimeric complex between RFX5 and RFXAP has recently been reported in vivo (41).
CREB binds to the X2 box of the HLA-DRA gene (16). However, it has not been shown that CREB also associates with other S-Y modules. We therefore performed ChIP experiments to examine whether the enhanceosome complexes formed at the S'-Y' motifs contain CREB. At the HLA-DRA promoter, CREB binding is, as expected, reduced 10-fold in the RFXANK mutant (Fig. 2C). A strong reduction in CREB association is also observed at the new target sites (Fig. 2C). At sequences 4, 6, and 8, this reduction is of the same order of magnitude as at the HLA-DRA promoter. As observed for RFX5, binding of CREB at sequence 2 is lost only partially in the BLS1 cells, confirming that a partial enhanceosome complex can form at this sequence in the absence of RFXANK. As expected, sequences that are not occupied by RFX or CIITA (such as sequence 3) are also not bound by CREB. Taken together, these results confirm that binding of CREB to the X2 box of the S'-Y' modules is strictly dependent on the cooperative binding interactions that mediate stable MHC-II enhanceosome assembly.
The new S'-Y' motifs in the MHC-II locus function as enhancers
To evaluate whether the S'-Y' motifs can function as transcription control elements, we performed luciferase reporter gene assays. As done previously for the distal S'-Y' enhancers situated upstream of the HLA-DRA and E genes (31), we determined whether the new S'-Y' motifs could replace the S-Y module of the HLA-DRA promoter (Fig. 3). For this analysis we chose two representative sequences, sequence 4 from the DQ region and sequence 8 from the DP region. As negative control we chose sequence 5, which is not occupied significantly by RFX and CIITA. We found that sequences 4 and 8 can substitute very efficiently for the HLA-DRA S-Y module in the wild-type B cell lines (Raji and BLS1c; Fig. 3). Transactivation by the new S'-Y' motifs is fully dependent on both CIITA and RFX, because it is completely abolished in the mutant cell lines lacking CIITA (RJ2.2.5) and RFXANK (BLS1). This confirms that activation by the new S'-Y' modules involves the same transcriptional machinery controlling the classical S-Y elements of MHC-II genes.
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Expression of MHC-II genes can be induced in MHC-II-negative cell lines by stimulation with IFN-. This is mediated by the activation of CIITA expression and its recruitment to the enhanceosome assembled on MHC-II promoters (23, 40, 42). We therefore investigated whether occupation of the new S'-Y' modules by the enhanceosome and CIITA is also induced by IFN- (Fig. 4).
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10 to 100% of that observed at the control HLA-DRA promoter.
Binding of RFX is also enhanced by IFN-, achieving levels varying from 10 to 50% of that observed at the HLA-DRA promoter (Fig. 4A). At sequences 2, 4, and 8, this induction (ratio of induced to noninduced) is similar to (sequence 2) or even greater than (sequences 4 and 8) that observed at the HLA-DRA promoter.
Sequence 6 differs from the others in that occupation by RFX is not induced by IFN-, although a clear (albeit weak) increase in occupation by CIITA is observed. At this point we have no explanation for this difference between sequence 6 and the others. We also do not know why a high background level of RFX binding is observed at the negative control sequence 3 in the cells used for the IFN- induction. This high background was not observed in B cells (Fig. 2).
S'-Y' modules induce global histone acetylation
Assembly of the enhanceosome complex and the recruitment of CIITA enhances histone acetylation at the promoter proximal S-Y modules of MHC-II genes (36, 40). We therefore examined whether the newly identified S'-Y' modules also induce histone hyperacetylation in an RFX- and CIITA-dependent manner. ChIP experiments were first performed with Abs directed against pan-acetylated histone H3. At all four new S'-Y' modules, H3 acetylation is significantly greater in wild-type cells than in RFXANK-deficient cells (Fig. 5A). This is mainly due to the recruitment of CIITA, because a very similar reduction in H3 acetylation is observed in CIITA-deficient cells (see Fig. 5B).
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25 kb upstream of the gene (Fig. 5B) (31). We therefore determined whether occupation of the newly identified S'-Y' modules by RFX and CIITA is also accompanied by the spreading of hyperacetylation to adjacent regions. For this analysis we chose two S'-Y' modules: site 4 located upstream of the HLA-DQB gene, and the isolated enhancer 8 situated >30 kb away from the DP region. We examined H3 and H4 acetylation in wild-type and mutant cell lines lacking RFXANK or CIITA (Fig. 5B). As observed in the HLA-DRA upstream region, RFX- and CIITA-dependent histone hyperacetylation at the HLA-DQB locus is not restricted to the proximal S-Y and distal S'-Y' modules, but is evident over the entire 5-kb upstream domain examined. At sequence 8, the hyperacetylation extends in both directions, spanning a region of at least 5 kb. These results demonstrate that RFX and CIITA induce broad patterns of histone hyperacetylation not only in the vicinity of the upstream S'-Y' modules in the HLA-DRA and HLA-DQB genes, but also at the isolated motif 8. The latter is an important observation because it demonstrates that a single S'-Y' motif not closely associated with a promoter proximal S-Y module can induce hyperacetylation over a broad domain. The establishment of large open chromatin domains thus appears to be a general mechanism by which RFX and CIITA regulate genes in the MHC-II locus. Novel target sites of RFX and CIITA in the invariant chain gene
The promoter-proximal region of the Ii gene contains a typical S-Y module that is bound in vivo by RFX and CIITA (36). Our search with the MHC-II profile consequently picked up the Ii gene (Fig. 1B). Interestingly, we also identified two S-Y motifs, referred to here as S'-Y' and S''-Y'', in the first intron of the Ii gene (Fig. 6). Both motifs are conserved at equivalent positions in the mouse and rat Ii genes (Fig. 1E).
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A deficiency in CIITA does not abolish enhanceosome assembly at the S'-Y' and S''-Y'' motifs, as revealed by normal binding of RFX and CREB in cells lacking CIITA (Figs. 6, A and B). In contrast, binding of RFX5, CREB, and CIITA is strongly reduced in cells lacking RFXANK (Fig. 6, A–C). The S'-Y' and S''-Y'' sequences are thus typical target sites for assembly of the MHC-II enhanceosome complex and recruitment of CIITA. However, as observed for the Ii promoter (36), a partial enhanceosome complex can form at the intronic sites in cells lacking RFXANK (Fig. 6, A and B). This is particularly evident at the S'-Y' sequence, where residual binding of RFX5 and CREB is clearly detected in the RFXANK-deficient cells.
To assess the functional importance of the new intronic S''-Y'' motif, we performed a luciferase reporter gene assay. The S''-Y'' module drives expression of the reporter gene with an efficiency attaining 50–80% that of the S-Y module of the HLA-DRA promoter (Fig. 7). This enhancer activity is strictly dependent on CIITA and RFX, because the construct is not active in the mutant cells lacking CIITA or RFXANK.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our search with the MHC-II profile proved to be quite stringent. The scan of the entire genome produced only a very low background (approximately one hit per million base pairs). Four lines of evidence suggest that most of these hits represent nonrelevant sequences. First, the majority are assigned relatively modest scores. Second, a similar background was obtained with a meaningless profile consisting of an inverted MHC-II profile containing G/C and A/T transversions at all positions (Fig. 1C). Third, several randomly chosen hits turned out to be false positives, as assessed by ChIP experiments (Fig. 2). Fourth, restricting the search to the upstream regions of known and predicted genes drastically reduces the number of hits (data not shown).
On the basis of the results obtained with the initial scan, we created a more discriminative profile that generates an improved correlation between the score and the binding data (Fig. 8). This emphasizes another strong aspect of our approach: as the knowledge of valid RFX and CIITA binding sites grows, we can reiterate searches with progressively improved profiles, thereby increasing the chances of identifying novel sites. If a sufficiently large set of binding sites can be identified, it may eventually become possible to incorporate parameters that have currently not been defined. Examples include preferential combinations of or incompatibilities between certain S, X, X2, and Y sequences; particular spacing requirements imposed by certain S, X, X2, or Y sequences; and influences of the sequences situated between the S and X or X2 and Y boxes. Taking such parameters into account would facilitate the identification of true target sites and reduce the frequency of false positive hits.
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-induced cells. These S'-Y' motifs function as CIITA- and RFX-dependent enhancers and induce RFX- and CIITA-dependent histone hyperacetylation in vivo. Together with the S'-Y' motif present upstream of the HLA-DRA gene, the human MHC-II locus thus contains at least five distal regulatory modules that are controlled by the same transcription machinery that is recruited to the proximal S-Y modules of MHC-II promoters. Interestingly, we also identified two S-Y-like motifs (S'-Y' and S''-Y'') in the first intron of the Ii gene, which is, for the moment, the only direct target gene of RFX and CIITA that is situated outside the MHC. Both intronic motifs are occupied by RFX, CREB, and CIITA in vivo and function as enhancers in B cells (Fig. 7) (32). Taken together, these results suggest that RFX and CIITA control their target genes via a combination of multiple homologous enhancer modules situated both near (S-Y) and far from (S'-Y') the promoters.
The S'-Y' motif of the H-2Ea gene lies at one end of a large region that has been shown to exhibit functions characteristic of a LCR in transgenic mouse experiments. These functions include cell type-specific, copy number-dependent, and position-independent transgene expression (29, 30). Moreover, binding of RFX and CIITA to the S'-Y' motif of the HLA-DRA gene establishes a broad hyperacetylated chromatin domain, promotes the recruitment of RNA polymerase II, and induces the synthesis of extragenic transcripts (31). These features are strongly reminiscent of the LCR of the -globin locus, where long-range chromatin remodeling associated with the synthesis of intergenic transcripts has also been documented (51, 52). Taken together, these features suggest that the distal S'-Y' motifs are likely to influence MHC-II gene expression by a mode of action akin to that of LCRs. This hypothesis is sustained by our analysis of the new S'-Y' motifs, which, like the HLA-DRA S'-Y' motif, induce RFX- and CIITA-dependent histone hyperacetylation over a broad domain that spreads upstream and downstream for several kilobases. It is therefore tempting to speculate that the S'-Y' motifs found in the MHC-II locus control the expression of MHC-II genes from a distance. Individual S'-Y' motifs could in this way regulate a specific gene or a defined subset of genes. This would be consistent with the strategic positions occupied by the S'-Y' sequences: one is found upstream of the HLA-DRA gene, three (sequences 2, 4, and 6) flank the central DQ region, and one (sequence 8) is present at the other end of the locus, near the DP region.
An alternative possibility is that the S'-Y' motifs exert a global influence over the whole MHC-II locus, for instance by generating a large (700 kb) open chromatin domain spanning all the coregulated MHC-II genes. Such broad domains have been documented in several systems, where they correlate with transcriptional competence and gene activation (43, 44, 45). The latter is an attractive model, because MHC-II genes have been maintained clustered together (5). Numerous rearrangements and duplications have occurred within the MHC-II locus to a much greater extent than in the flanking regions, yet in all species analyzed to date, except bony fishes, all MHC-II genes are present in the same region (5). This arrangement may have been maintained because there has been a selective pressure against rearrangements that translocate MHC-II genes outside of a globally regulated domain. In this context it should be noted that the MHC is indeed subjected to global regulatory events; the entire MHC locus undergoes changes in subnuclear localization in response to IFN- induction (53).
The DR-DQ region of the human MHC-II locus contains a particularly high density of S'-Y' motifs (Fig. 1). Moreover, as a consequence of the relative positions and orientations of the HLA-DRA (H-2E) and HLA-DQB (H-2A) genes, the DR-DQ (H-2E-H-2A) region is bracketed by two highly conserved S'-Y' enhancers (Fig. 1). This arrangement is particularly intriguing, because all vertebrate species have retained genes encoding the DR and DQ isotypes, whereas many have lost functional genes in the DP region (5). It is also striking that the DR-DQ region is separated from the DP region by a large domain containing the HLA-DO and HLA-DM genes, which are less tightly coregulated with the classical MHC-II genes, and genes that are not required for MHC-II-restricted Ag presentation. This prompts us to speculate that the S'-Y' enhancers in the DR-DQ region may define a more tightly coregulated subdomain within the MHC-II locus.
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Acknowledgments |
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Footnotes |
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1 This work was supported by grants from the Swiss National Science Foundation, the Comission pour la Technologie et de l’Innovation, and NovImmune. 
2 Address correspondence and reprint requests to Dr. Walter Reith, University of Geneva Medical School, Centre Médical Universitaire, 1 rue Michel-Servet, CH-1211, Geneva, Switzerland. E-mail address: walter.reith{at}medecine.unige.ch
3 Abbreviations used in this paper: MHC-II, MHC class II; ChIP, chromatin immunoprecipitation; Ii, invariant chain; LCR, locus control region; RFX, regulatory factor X complex.
Received for publication April 8, 2004. Accepted for publication August 17, 2004.
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-inducible expression of the HLA-DRA gene. J. Biol. Chem. 267:23728. mediated by the transactivator gene CIITA. Science 265:106.-globin gene cluster. Mol. Cell. Biol. 21:6507.-globin locus. Mol. Cell 5:377.[Medline]
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