(Received for publication, October 13, 1994; and in revised form, December 19, 1994)
From the
We have previously described a mutant B lymphoblastoid cell line, Clone-13, that expresses HLA-DQ in the absence of HLA-DR and -DP. Several criteria indicated that the defect in this cell line influences the activity of an isotype-specific transcription factor. Indeed, transient transfection of HLA-DRA and DQB reporter constructs indicated that the affected factor operates via cis-elements located between -141 base pairs and the transcription initiation site. A series of hybrid DRA/DQB reporter constructs was generated to further map the relevant cis-elements in this system. Insertion of oligonucleotides spanning the DQB X-box (but not the DQB-W region or the DQB Y-box) upstream of -141 in a DRA reporter plasmid rescued expression to nearly wild-type levels. Substitution promoters were then generated where the entire X-box, or only the X1- or X2-boxes of HLA-DRA were replaced with the analogous regions of HLA-DQB. The DQB X2-box was able to restore expression to the silent DRA reporter construct. Moreover, replacement of the DQB X2-box with the DRA X2-box markedly diminished the activity of the DQB promoter in the mutant cell. None of the hybrid reporter constructs were defective when transfected into the wild-type, HLA-DR/-DQ positive parental cell line, Jijoye. These studies suggest that the divergent X2-box of the class II major histocompatibility complex promoters plays an important role in influencing differential expression of the human class II isotypes.
The class II molecules of the major histocompatibility complex
(MHC) ()present processed peptides derived from exogenous
antigen to CD4-positive helper T cells(1) . The human genome
encodes three functional isotypic forms of these molecules (HLA-DR,
-DQ, and -DP), each of which is a disulfide-linked heterodimer of two
transmembrane glycoproteins: an acidic
chain and a
chain.
The genes encoding these polypeptides are clustered in discrete loci in
the 1 megabase HLA-D region on the short arm of chromosome 6 (2) . Unlike the related class I MHC molecules, which bind and
present peptide antigen to cytotoxic T cells, class II MHC molecules
are expressed only on a limited number of cell types such as dendritic
cells, B cells, macrophages, and activated T cells, and the expression
on these cell types is developmentally regulated(3) . The
expression of the class II molecules can also be induced on normally
class II-negative cells by specific cytokines such as interferon-
or interleukin-4(4) . These induction pathways are probably
operating at sites of active immune response, where de novo expression of the class II molecules is frequently observed.
De novo expression of class II molecules is also observed at target sites in organ-specific autoimmune disease, and it has been hypothesized that this might trigger or exacerbate the disease by presentation of tissue-specific antigen to helper T cells(5) . In contrast, hereditary MHC class II deficiency, also called bare lymphocyte syndrome (BLS) results from the complete lack of expression of class II MHC molecules(6) . Patients afflicted with this autosomal recessive disease are prone to multiple infections and usually die during childhood. The potential association of these two types of disorders with aberrant class II MHC gene expression have prompted an intense analysis of the regulatory elements and transcription factors that control the proper expression of these genes(7) .
The regulation of the class II MHC genes is mainly transcriptional, and all of the class II proximal promoters studied to date contain highly conserved elements referred to as the X- and Y-boxes(3) . A number of DNA-binding proteins that interact with these and other less conserved sequences have been identified, and complementary DNAs encoding some of the factors have been molecularly cloned(4) . The regulatory roles of the individual recombinant proteins encoded by the cDNAs have been investigated via cotransfection or in vitro transcription and have been found to encode both activators and repressors of class II gene transcription(5, 6, 7, 8, 9, 10, 11, 12, 13) .
While the different class II isotypes HLA-DR, -DQ, and -DP are
usually coordinately expressed, there is now abundant evidence that
differential regulation of the class II genes occurs in particular cell
types and in response to various
stimuli(4, 14, 15) . Such differential
regulation could result either from the use of different transcription
factors or from mechanisms that would selectively inhibit the
interaction of shared transcription factors with a subset of the class
II gene promoters. Apriori, both mechanisms appear
to be feasible. The genes encoding the and
chain
polypeptides of each isotype are located in distinct physically
separate loci (approximately 350 kilobases separate the DP and DQ loci
and approximately 100 kilobases separate the DQ and DR loci), which
could easily accommodate distinct chromatin structures at each locus,
resulting in differential accessibility of shared transcription
factors. On the other hand, although the different class II genes share
highly conserved X- and Y-box elements, the nucleotide sequence of
intervening and surrounding regions of the proximal promoter diverge
significantly. Therefore, there is ample opportunity for differential
binding of specific transcription factors to the various class II
promoters. Moreover, these two potential control mechanisms for
differential expression are not necessarily mutually exclusive.
We have previously described a mutant cell line, Clone-13, that expresses HLA-DQ in the absence of HLA-DR and -DP(16) . Using a variety of criteria, the differential expression of isotypes in this cell line was shown to result from a defect in (or operating through) a trans-acting factor since the transcriptionally silent genes in this cell line could be reactivated upon interspecific fusion with a MHC class II positive murine cell line. Transient transfection assays also indicated that the defective factor operated through the proximal promoter (between -141 base pairs and the transcription initiation site). To further study the molecular basis of defective transcription of a subset of the human class II MHC genes in this cell line, we have more precisely mapped the cis-elements that mediate this defect using a series of hybrid promoter constructs between the transcriptionally competent HLA-DQB gene and the transcriptionally silent HLA-DRA gene. The data indicate that the divergent X2-box found immediately 3` of the consensus X1-box mediates the isotype-specific defect in this cell line and provides further evidence that this element may play a major role in determining the isotype-specific expression observed in vivo.
The template for the
amplification was plasmid BSDRA300 containing nucleotides -260 to
+20 from the DRA gene inserted between the HindIII and EcoRI sites of the pBluescrpt cloning vector. ()The
insert for this plasmid was derived from the pp34-RI fragment
originally isolated by the laboratory of S. Weissman, Yale
University(22) . These primers incorporate unique HindIII and XbaI sites into the amplification
product. The PCR product was then purified by affinity to silica
fragments (Bio 101), sequentially digested with HindIII and XbaI restriction enzymes, subjected to a second round of
purification by silica affinity, and subcloned into the pCAT basic
plasmid between the HindIII and XbaI sites (Promega
Corp., Madison, WI). Construct 3 was generated by amplification of the
DNA fragment containing nucleotides -173 to -42 from the
DQB gene promoter using purified genomic DNA from ATCC CCL86 as a
template. The following oligonucleotides
(5`-GGGGAAGCTTAATTTGAAGACGTCACAGTGC-3` and
5`-GGGGAAGCTTTGGTAGGATTGGATGGTCCTT-3`) were employed and introduce HindIII sites on both ends of the amplification product. After
purification and restriction with HindIII, this DQB proximal
promoter fragment (lacking its own transcription initiation site) was
inserted into HindIII cleaved/calf intestinal alkaline
phosphatase-treated construct 1. Constructs 4-6 were generated by
first producing double-stranded oligonucleotides containing the
following strands and their complements by annealing via sequential
incubation in decreasing temperatures: DQB W,
5`-TCCAGTGCAGGCACTGGATTCAGAACCTTCACAAAAAAAAAA-3`; DQB X,
5`-CAAAAAAAAAATCTGCCCAGAGACAGATGAGGTCCTTCAG-3`; DQB Y,
5`-GTCCTTCAGCTCCAGTGCTGATTGGTTCCTTTCCAAGG-3`.
These blunt ended
double-stranded oligonucleotides were then ligated, and dimers were
purified by elution from polyacrylamide gels. HindIII linkers
were then attached to the purifed dimers, the ligation products were
cleaved with HindIII, and the purified dimers were ligated
into the HindIII site of construct 1. The orientation and
number of oligonucleotides inserted into the HindIII site was
determined using the DRA primer (5`-TCTTGCAAAGGGTCCAGGACA-3`), which
primes synthesis of the ``bottom'' strand of the DRA proximal
promoter oriented 5` to 3` toward the upstream direction. Constructs
containing two oligonucleotides ligated in the wild-type orientation
were identified and propagated. Construct 7 was generated by first
ligating the 564-base pair HindIII fragment of bacteriophage
DNA to the DQBX-box dimer isolated from construct 5, followed by
gel purification of the dimer/564-base pair ligation products. This was
then ligated into the HindIII site of construct 1. Plasmids
containing the DQBX-box dimer ligated in the wild-type orientation were
identified by dideoxy sequence analysis using a primer that initiates
DNA synthesis through the HindIII insertion site from the
plasmid backbone. Construct 8 was generated by first ligating the
2,322-base pair bacteriophage
HindIII fragment to the
DQBX-box dimer, followed by gel purification of the dimer/2,322
ligation products. This was then inserted into the HindIII
site of construct 1. Construct 9 was generated by ligating BamHI linkers onto the DQBX-box dimer, cleaving with BamHI, and insertion of the dimer into the unique BamHI site downstream of the chloramphenicol acetyltransferase
gene in the pCAT basic plasmid. Constructs 10-14 were generated
by overlap PCR of two or three PCR subfragments to generate the final
chimeric class II promoters. The nucleotide sequences of the
recombination sites are shown in the appropriate figures.
Figure 1: Insertion of two DQB X-box oligonucleotides upstream of -141 in a DRACAT reporter plasmid restores expression in Clone-13 cells. A, scheme for construction of hybrid constructs from a -173 to -42 DQB amplification product and dimerized DQB W(S), X- and Y-box oligonucleotides. As described under ``Materials and Methods,'' the PCR product and oligonucleotides were inserted upstream of nucleotide -141 in the DRA141CAT reporter plasmid. The numbers refer to the construct designations referred to in the text, and the oligonucleotide sequences are shown at the bottom of this panel. The consensus binding sites for the conserved elements are boxed in solidblackboxes over the sequences. A representative CAT assay from transfection of Clone-13 cells with these constructs is shown. B, results of quantitation of transfections of the constructs in 1A into Clone-13 cells using three independent plasmid preparations and and an n = 5 for each plasmid preparation. The results are relative CAT activities using the mean DQBCAT activity as a 100% maximum activity. The p values for 2, 3, and 5 are <0.005 relative to 1, and p < 0.05 for 7 relative to 1.
Figure 2: Replacement of DRA X2 with DQBX2 restores expression to the DRA reporter plasmid. A, scheme for construction of substitution plasmids. The exact nucleotide sequences that are replaced are indicated under ``Results.'' The numbers at the left of the panel refer to the construct number used in the text. These replacement plasmids were generated by overlap PCR. B, results of transient transfections of the constructs in A into Clone-13 cells. Three independent plasmid preparations and an n = 5 for each preparation were performed. The mean values and S.E. are indicated by errorbars. The p values of 2, 10, and 12 are < 0.005. C, a representative CAT assay from B.
Each of these replacement constructs were then transfected transiently into Clone-13 cells, and the resulting CAT activities were quantitated (Fig. 2B). Construct 10, containing a complete replacement of both X1 and X2-elements of DRA with the analogous DQB elements had a transcriptional activity approximately 85% of the wild-type DQB reporter construct. Construct 11, containing a DQB substitution only at the X1-element, had a variable transcriptional activity (with a mean of 7% of DQB and a maximum activity of 17% of DQB; Fig. 2C). Construct 12, containing a DQB substitution only at the X2-element, had a mean transcriptional activity of 88% of DQB. These data demonstrate that the DQB X2-element can functionally replace the DRA X2-element in the context of the DRA promoter and indicate that the defective transcription factor in Clone-13 ``interacts'' directly with or requires interaction with a factor bound to the DRA X2-element.
Figure 3: Replacement of the DQB X2-element with the DRA X2-element abolishes transcription from the DQB promoter. A, diagram of the substitution reporter plasmids 13 and 14. The color coding is as designated in Fig. 2. The exact nucleotides substituted are indicated under ``Results.'' Substitution plasmids were synthesized by overlap PCR using complementary oligonucleotide containing substitution sequences at their 5` termini. B, results of transient transfection of plasmids in 3A into Clone-13 cells. Statistical analysis was by analysis of variance. Constructs 2 and 14 had p values of < 0.005 relative to 1. C, a representative CAT assay from B.
Finn and co-workers (27) have previously reported that a low mobility complex (complex A), which forms on extended class II MHC probes correlates with the class II positive phenotype in B cells and cytokine-induced nonlymphoid cells(27) . This complex was reported to be missing in the class II negative mutant cell line RJ2.2.5 and restored in HLA class II positive interspecific hybrids with a murine class II positive cell(28) . We therefore performed a similar analysis with extracts from a panel of human class II positive and negative cell lines, including Clone-13 (Fig. 4). In our EMSA analysis using a DRA probe containing nucleotides -141 to -43 (containing the S, X, Y, and octamer elements), we are able to distinguish six complexes in EMSAs using extracts from the class II positive cell line, Raji (lane1). The two highest mobility bands, B5 and B6 result from the presence of the octamer motif (which was absent in the probes used by Finn) as determined by competition analyses (not shown). Complex B6 is due to the B lymphoid specific factor Oct-2 (note that it is missing in HeLa and Jurkat cells, lanes8 and 9, respectively), and complex B5 is due to Oct-1 binding. Complex B4 is present in all cells and is competed by any single element (not shown) and is, therefore, probably a multiprotein complex containing S, X1-, X2- and Y-box binding factors. Complex B3 is only present in B cells and is therefore probably the multiprotein complex B3 containing Oct-2. Finally, complexes B1 and B2 probably correspond to Finn's complexes A and B, respectively. Complex B2, like complex B, is a low mobility complex present in all cells, and complex B1, like complex A, is both the lowest mobility complex and only formed in extracts from certain cells. Complex B1 (probably complex A) in our hands is absent in 6.1.6, BLS-1, TF, HeLa, Jurkat, and SJO nuclear extracts (this group contains both B and non-B cells, which are all class II negative), but is present in Raji, RJ2.2.5, Clone-13, and JY cells (this group contains both class II positive and negative B cells).
Figure 4:
Electrophoretic mobility shift analysis of
complex formation on DRA nucleotides -141 to -43 using
nuclear extracts from a panel of class II positive and negative cell
lines, including Clone-13. Six complexes that form on this probe are
indicated on the left of the gel and the source of the nuclear
extracts used in each lane is indicated above the gel. Lane2 received 100 cold competitor of the
probe. Complex B1 is probably the same as complex A as described in
Finn et al.(27, 28) . Complex B2 is probably
complex B using the Finn terminology. Complex B5 is due to Oct-1, and
complex B6 is due to Oct-2. The reasoning behind these designations is
described under ``Results.''
These data are in agreement with Finn's previous reports of a low mobility complex that forms preferentially in class II positive cells (note its absence in lanes4, 5, and 7-10: all class II negative cells). However, the formation of complex B1 in extracts from RJ2.2.5 is at variance with the previous findings. Moreover, the formation of complex B1 in extracts from Clone-13 cells (HLA-DR and -DP negative) also suggest that the correlation between complex B1 formation and class II positivity is not one-to-one. These data do show that multiprotein complexes can form on an extended DRA probe in EMSAs in both the RJ2.2.5 and Clone-13 mutant cell lines. This similar phenotype is consistent with the studies of Benichou and Strominger (18) that reported that these two cell lines fall into complementation group II. All of the cell lines that did not form complex B1 in this study fall outside complementation group II: BLS-1 (group I), 6.1.6 (group III), SJO, and TF (group IV).
The tissue-specific expression of the class II genes of the MHC, like all other RNA polymerase II eukaryotic genes, requires the formation of a multiprotein complex on the proximal promoter, and appropriate interactions between the upstream activators and the basal transcriptional machinery(29) . In the class II genes, relatively conserved elements, designated S, X, and Y are found in all class II promoters sequenced to date, and a multiplicity of factors have been purified and/or molecularly cloned that exhibit varying degrees of sequence-specificity and/or preference for these and other elements (4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . With a few exceptions, most of the promoter-bound transcription factors that interact with the class II cis-elements are ubiquitously expressed and either do not or only weakly activate class II reporter constructs when overexpressed in transient assays. The newly identified factor, CIITA, is a dramatic exception, as its expression pattern correlates with class II positivity and has been shown to activate all of the class II genes upon transfection into several class II negative cells(30, 31) .
While it is likely that the different class II genes within a species are regulated by overlapping transcription factors (with CIITA being a central player), it is clear that this system is much more complex than originally thought. Multiple proteins can interact with each cis-element, and each of the cloned factors has turned out to have dramatically different affinities for their binding sites in the different class II genes(32) . Moreover, the field is only begining to uncover the protein-protein interactions that are necessary from transcription complex assembly, and additional proteins that participate in this process are probably yet to be discovered(33) .
In this report, we provide additional evidence of the complexity of this problem. It has become clear that the expression of the different class II genes in human cells is not always coordinate. In this study we have used the HLA-DQ positive, HLA-DR, and -DP negative cell line Clone-13, to investigate a molecular basis for this divergence in regulatory mechanisms. This particular model is attractive since the lack of DRA gene expression in this cell is not due to the influence of chromatin structure or distant elements but has been mapped to the immediate proximal promoter in transient assays.
Using a series of hybrid and substitution constructs, we show here that the differential expression of the DRA and DQB genes in this cell line is mediated by the divergent X2-box element (Fig. 1Fig. 2Fig. 3). The DQB X2-element can restore transcriptional activity to the DRA promoter (Fig. 2), and the DRA X2-element can abolish transcription from the DQB promoter (Fig. 3). These data strongly suggest that the X2 binding factors utilized by the two genes are distinct. These results further indicate that the factors that bind to the DQB X2-element can, however, functionally replace the factors that normally bind to the DRA X2-element. This in turn implies that the DQB X2 factors can participate in any of the required protein-protein interactions in which the DRA X2 factors normally participate. The data also indicate that the defect in the DRA X2-associated factor in Clone-13 is drastic enough to prohibit the formation of an active transcription complex with the other constituents of the normal DRA or DQB transcription complexes.
The concept that the DRA and DQB genes
bind different factors to their X2-elements is consistent with 1)
sequence divergence at this site between the two
promoters(4, 5, 6) , 2) multiple previous
EMSA experiments involving cross-competitions and using recombinant
X2-binding proteins (6, 34) , 3) the results of
antisense inhibition experiments where antisense hXBP-1 and c-fos RNAs specifically affected DR and DP antigen expression, and DRA
but not DQB promoter activity(5, 6) , and 4) our
studies on the steroid sensitivities of class II MHC genes that
indicate that DR and DP (but not DQ) antigen expression, and DRA but
not DQB promoter activity are inhibited by steroids. ()
The concept that the distinct X2-binding proteins can substitute for each other is consistent with the recent independent studies of Voliva and Reith(35, 36) . Voliva showed that substitution promoters (including core X-box replacements) can function despite clear evidence that both common and unique proteins interact with class II MHC cis-elements. Reith and co-workers showed that one member of the RFX family of proteins binds cooperatively with one of several potential Y-box binding proteins, NF-Y. This interaction occurred in vitro in the absence of the X2-element, indicating that RFX/NF-Y interaction does not require an X2BP. The fact that site-directed mutagenesis of the X2-element decreases class II promoter activity might suggest that each distinct X2BP that binds to the divergent X2-elements provides a common generic function (such as contributing to a multiprotein activation surface or assisting in the tethering of CIITA to the promoter).
The resolution of these issues requires the isolation of each component of the DRA and DQB transcription complexes, and a systematic analysis of protein/protein interactions between these factors.