Res. Immunol. i99i, i42, 393-399

Transcriptional regulation of HLA-DRA gene B.M. Peterlin Howard Hughes Medical Institute, University o f California, San Francisco, 3rd and Parnassus A venues, San Francisco, CA 94143 (USA)

SUMMARY Transcriptional regulation of class II genes is complex. DRA, for example, is expressed in both tissue-specific and iymphokine-inducible fashion. High levels of expression in B and activated T cells depend upon a lymphoid-specific transcriptional enhancer located in the first intron and on the upstream and downstream promoter elements. Conserved upstream promoter Z and X sequences (CUS) are the main determinants of this complex regulation. At least four distinct trans-acting factors that might be differentially expressed in various cell types bind to these Z and X boxes. These are members of JUN, helix-loop-helix {HLH), C/EBP and ETS families of proteins.

Key-words: MHC, Lymphocyte, HLA-DRA, Immunoregulation; Class II genes, Transcription, Bare Ivmphocyte syndrom.


Protein products of class II genes determine the qualitative and quantitative nature of the immune response (Sullivan et al., 1987; Kappes and Strominger, 1988). They function as restriction elements in presentation of antigenic peptides to T helper cells and they determine interactions between T and B cells that lead to the synthesis of specific antibodies (Sullivan et ai., 1987; Kappes and Strominger, 1988). Inappropriate expression of class II determinants has been correlated with autoimmunity (Botazzo et al., 1986) and their congenital absence leads to severe combined immunodeficiency and agammaglobulinaemia (bare lymphocyte syndrome, bls II) (de Preval et al., 1988; Kappes and Strominger, 1988; Griscelli et al., 1989). Besides having obvious immunological importance, the study of transcriptional regulation of class II genes is interesting from the standpoint of developmental, tissue-specific and lymphokineinducible expression of eukaryotic genes (Sullivan et al., 1987; Benoist and Mathis, 1990; Peterlin et aL, 1990). Furthermore, the class II cluster of genes contains several aberrantly- or non-expressed genes that

have normal coding sequences (Benoist and Mathis, 1990; Peterlin et al., 1990). Mutations in their promoters can be correlated with the normal expression of other members of this gene family. Finally, the study of transcriptional regulation of class II genes should lead to an understanding of his II which results in congenital absence of class II expression in o cells (de Preval et al., 1988; Griscelli et al., 1989). In this report, the regulation of the DRA gene will be dissected in detail. Correlations with expression of other class II 0t and [3 genes will be drawn. First, DRA gene will be analysed with respect to its state of DNA methylation and chromatin structure. Second, important cis-acting elements will be delineated. Third, the isolation, cloning, characterization, and expression of trans-acting factors will be described. Finally, progress in the studies of bls II will be summarized. Genetic structure

DRA is composed of five exons and four introns of which the first intron is the largest, spanning almost two kilobases of DNA (fig. IA and IB)

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_n U _ n LI •m [] m......- - O - - - - L ~ B2 A2 B1 A1






B1 B2 B3 B4




A2 B1 A1







$ B chain




TM Cyt

3' UT














D~ M











Fig. 1. Structure of the DRA gene. A) On the short arm of human chromosome 6, the DRA gene is located telomeric to other class II genes. Furthermore, the class II locus is situated between glyoxalase-I (GLO) and complement (class l i d and B, C and A (class I) genetic loci. Of sixteen genes depicted, seven are coordinately expressed (filled boxes), one is expressed at very low levels (DOB) and eight are pseudogenes (open


= chloramphenicol acetyltransferase. = conserved upstream sequence.


= thymidine kinase.

TRANSCRIPTIONAL REGULA TION OF HLA-DRA GENE (Peterlin et al., 1990). The promoter is located just 5' to the first exon (fig. 1C). The entire coding region contains a n u m b e r of CpG dinucleotides (fig. I D) (Wang and Peterlin, 1986). These are susceptible to cytosine methylation in eukaryotic cells. In the D R A promoter, a CpG-rich island which contains an abnormally high n u m b e r of CpG dinucleotides was observed (Wang and Peterlin, 1986). Such CpG-rich islands have been found near promoters o f active genes and at sites o f D N A recombination. Furthermore, the D R A CpG-rich island was demethylated in all cells (fig. 1D) (Wand and Peterlin, 1986). In contrast, the body o f the D R A gene was found to be hypermethylated irrespective of the state o f D R A transcription (Wang and Peterlin, 1986). Thus, D R A methylation does not correlate with D R A gene expression. However, the hypomethylated promoter suggests that it is always susceptible to interactions with D N A - b i n d i n g regulatory factors. Next, chromatin structure of the D R A gene was investigated (fig. ID) (Peterlin et al., 1987). In all ceils, a DNase I hypersensitive site was found at the p r o m o t e r . This hypersensitive site was more pronounced in B cells that express large amounts of class II determinants. Additionally, B cells were observed to contain two other DNase I hypersensitive sites in the first intron of the D R A gene (Peterlin et al., 1987). These intronic hypersensitive sites suggest that regulatory elements are also present in the body o f the D R A gene.

Cis-acting elements Following structural studies, an enhancer trap was constructed where fragments from the D R A gene were placed either 5' to the thymidine kinase (tk) promoter or 3' to the chloramphenicol acetyltransferase (CAT) reporter gene on plasmids that could





Fig. 2. Expression of the DRA gene in different cells. A) Lymphoid-specific transcriptional enhancer elements interact with an active DRA promoter in B and activated T cells. OCT-1/OCT-2 (star and circle) and C/EBP (striped circle) bind to the intronic transcriptional enhancer sequences. Z, X, Y and octamer promoter elements (see fig. 3) are active in these cells. B) In IFNy-induced cells, the transcriptional enhancer is inactive. However, the DRA promoter is active. OCT-1 binds to the octamer sequence in these cells (see fig. 4B). C) In uninduced cells, the intronic transcriptional enhancer and Z and X CUS are both inactive (see fig. 4C). Arrows denote start sites of transcription. Boxes 3' to the arrow represent exons.

boxes). Of these DOB, DQB2 and DQA2 have mutations in CUS. While DOB is expressed at low levels in B cells, it is not IFN-f-inducible. B) The DRA gene has five exons and four introns. In contrast to ~x-chain genes, which have a long first intron, [3-chain genes have a relative short first but long second introns. S, signal peptide; 1, 2, exons one and two ; TM, transmembrane peptide; Cyt, cytoplasmic domain ; 3'UT, 3' untranslated sequences. C) DRA promoter has the following CUS from the 5' to the 3' direction: Z (or W), X, Y, octamer (CCAAT in most other class II promoters) and TATA boxes. These conserved motifs were determined by comparing sequences of all murine and human class II promoters. D) DRA pro~aoter is contained within a CpG-rich island. Furthermore, eleven CpG sites can be studied by methylation-sensitive restriction endonucleases in the DRA gene. Whereas the CpG-rich island is demethylated (D), other CpG sites are methylated (M) in all cells. In the DRA gene, three DNase I hypersensitive sites (I, If, Jill) are present in B cells. Of these, only site I is present in uninduced and IFN-r-induced cells. Site Ill is contained within a lymphoid-specific transcriptional enhancer element.


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be transfected into human lymphoid and fibroblastoid cells (Wang et al., 1987). Activity of inserted fragments could be assayed by a simple enzymatic assay which measures the conversion of chloramphenicol to its mono- and diacetylated forms. Subsequent analyses revealed that sequences in the DRA promoter and in the first intron could function as lymphoid cell-specific transcriptional enhancers (fig. 2) (Wang et al., 1987). In particular, a region of 200 nucleotides in the first intron containing binding sites for C / E B P and OCT-1/OCT-2 was able to

confer lymphoid cell specificity upon the tk promoter (fig. 2) (Wang et al., 1987; Caiman and Peterlin, 1990). This activity was position-, orientation- and distance-independent and thus satisfied the criteria for a transcriptional enhancer (Wang et al., 1987). It is of interest that the stronger of the two intronic DNase I hypersensitive sites maps into this transcriptional enhancer element. The other DNase I hypersensitive site also maps into a region that was active in the enhancer trap experiments. Not only was the activity of this second site less than of its better




Z2-box -70







pyr -40


X-box -30


X2-box -20

-10 /





















Fig. 3. Sequence and consensus binding motifs of the DRA promoter. A) Sequences from positions - 150 to + 1 are required for B-cell-specific and IFN-/'-inducible expression of the DRA gene. Moreover, Z and X boxes function like tissue-specific and lymphokineinducible enhancer elements, such that 24 X box nucleotides and 56 nucleotides which additionally contain Z box sequences confer B-cell specificity and IFNy inducibility on heterologous promoters. Core box sequences are placed into open boxes and their functionally important extensions are represented by dashed lines. For example, the X box does not function without the pyrimidine (pyr) tract. The arrow denotes the start site of transcription. B) Consensus DNA-binding motifs in Z and X boxes of the DRA promoter. Two TPA-responsive (TRE) or cAMP-responsive (CRE) sites flank other sites. AP-1 binds to TRE, while CREBP and/or ATF bind to CRE. Core Z and X boxes are contained within two C/EBP sites. The C/EBP site in the X box is flanked by ETS- and HLH-binding sites. Ubiquituous and cell-type-specific or IFNyinducible factors that bind to these sequences have not been characterized completely.



characterized neighbor, but the two fragments together had only additive effects on the tk and DRA promoters (Wang et al., 1987; Caiman and Peterlin, 1990). Next, DRA promoter elements were investigated (fig. 3) (Tsang et al., 1988). Classical deletions from the 5' direction and placements of individual promoter elements upstream of the tk promoter revealed that sequences from positions - 150 to + 1 were sufficient to confer B-cell-specific and ~,interferon-(IFNy)-inducible regulation to the DRA promoter. Moreover, within this region, an oligonucleotide of 24 base pairs which contains the X box could confer B-cell specificity and a fragment of 56 nucleotides which contains both Z and X boxes additionally conferred IFN'r inducibility to the tk promoter (Tsang et ai., 1988). Thus, Z and X CUS (conserved upstream sequences) can be viewed as enhansons that together form an IFNT-inducible and B-cell-specific transcriptional enhancer element (Peteriin et al., 1990). Following this analysis, cluster point mutagenesis of the DRA promoter was performed (Tsang et al., 1990). Here, mutations in core Z, pyrimidine, X, Y and octamer sequences were deleterious to expression in B cells and in fibroblastoid cells following IFN'r administration. It is of interest that mutations in the octamer were deleterious only to expression in B cells, but not in IFNT-inducible cells (Tsang et al., 1990). Thus, the octamer in the DRA promoter has B-cell-specific activity. Further analysis revealed that while Z and X boxes regulate the quantity and quality of the transcription complexes, Y, octamer and TATA boxes position RNA po!ymerase !! at the start site of DRA transcription (Tsang et al., 1990).

Trans-acting factors As described above, the DRA promoter is composed of five regions which, from the ~' direction. are called TAP!A. octamer Y, X and Z boxes (fig. 3) (Sullivan et ai., 1987; Peterlin et ai., 1990). It is of some interest that the purified human TFIID does not bind to the TATA element from the DRA promoter in a DNase I footprinting assay (unpublished data). Both OCT-1 and OCT-2 bind to the octamer; however, OCT-2 is present only in B cells (Caiman and Peterlin, 1990). NF-YA and NF-YB bind to the Y box (Hooft van Huijsduijnen et al., 1990). Furthermore, NF-YA and NF-YB are human equivalents of HAP-2 and HAP-3 proteins in yeast (Hooft van Huijsduijnen et al., 1990). This heterodimer is present in all cells and is a potent transcriptional activator. Moving 5' to the X box a more complicated picture emerges. Proteins that bind to the X box represent a slow migrating complex called NF-Xc and a faster migrat-




ing band called NF-X2 (Tsang et al., 1990). Studies of cluster point mutations and DNA-protein interactions revealed that NF-X2 binds 3' of the X box, whereas NF-Xc binds to the pyrimidine and core X sequences (Tsang et al., 1990). Furthermore, NF-X2 was isolated by expression cDNA cloning and was revealed to be c-JUN (Andersson and Peterlin, 1990). Thus, NF-X2 is composed of a c-JUN and c-FOS heterodimer. It is of some interest that, although c-FOS and c-JUN are both expressed in most cells, they do not form a heterodimer with each other in B cells (Andersson and Peterlin, 1990). Either c-JUN preferentially binds to another protein in B cells or it is sterically hindered from binding c-FOS by some post-trans!atignal modification. In agreement with binding data, no activity of AP-1 sites in B cells could be demonstrated (Tsang et aL, 1990; Andersson and Peterlin, 1990). NF-Xc is slightly more complicated, since it appears to be a complex of proteins that have different DNA-binding properties (Peterlin et aL, 1990; Tsang et al., 1990). NF-Xc might be composed of as few as two and as many as three to five proteins. From the 3' to the 5' direction in the X box~ one can find consensus binding sites for H L H (Blackwell and Weintraub, 1990), C/EBP (McKnight et al., 1989), and ETS (Karin et al., 1990) proteins. By DNase I footprinting, AP-4 (Hu et al., 1990) binds to the X box (unpublished data) and by expression cDNA cloning, in addition to RF-X, members of the C/EBP family of proteins can bind to the core X sequenc~ (Reith et al., 1989). Finally, preliminary ~tudies suggest that ETS binds to the pyrimidine tract (unpublished data). The Z box is, in part, a duplication of the X box, since proteins that bind to the Z box can be competed for by the X box oligonucleotide (Tsang et al., 1990). However, the reverse is not true, i.e. the Z box cannot compete for the binding of proteins that bind to the X box (Tsang et al., 1990). Thus, Z box seQ , , ~ n o ~ ~ n n ~ r t o ,'ontain moat. but not all of the I)NA-bintllng sites in the X box. in particular, RFX or C / E B P might bind to the core and 3' end of the Z box, whereas members of J U N / C R E B / A T F family of proteins could bind to the Z2 box (fig. 3). Thus, the Z box appears to have binding sites for some but not all factors that bind to the X box. For example, there is no HLH binding site in the Z box (fig. 3). Complex interactions between these transcription factors are believed to mediate tissue-specific and IFN~-inducible regulation of the DRA gene (fig. 4). For example, proteins that contain leucine zippers like c-JUN, c-FOS, H L H and C/EBP could heterodimerize and interact in different combinations depending on cell type and expression of the particular protein. In addition to interactions when bound to DNA, these different complexes could attract distinct

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Aberrantly or non-expressed class II genes










The cluster point mutagenesis of DRA promoter was performed in a cassette-forming fashion (Tsang et al., 1990). Elements of the promoter were separated from each other by new restriction endonuclease sites which were placed between Z, X, Y, octamer and TATA sequences. Thus, oligonucleotides corresponding to CUS could be mutated end replaced within these cassettes. This allowed for ~ e study of transcriptional motifs in the DRA promoter as well as those of other class II genes. By this approach, CUS from all class 1I e and ~ promoters could functionally substitute for the DRA Z and X boxes. The sole exceptions were the DOB and DQB2 which had defective Z sequences and QA2 which had defective Z and X elements. Once placed in the context of the DRA promoter, CUS from these promoters could not function and therefore resulted in low to undetect, ble expression in B cells and in the loss of IFN~, inducibility (unpublished data).


The bare lymphocyte syndrome

A) In B cells, C/EBP, ETS, and HLH proteins bind to Z and X boxes and are active. Presumably, these proteins interact with each other and with NF-YA and NFYB (NF-Y) and OCT-2 forming an active transcription complex. In B cells, no binding of FOS/JUN or ATF/CREBP can be detected. B) In IFNy-induced cells, additionally CREBP/ATF and FOS/JUN bind to X and Z box sequences. Even though C/EBP and ETS are still active, these new interactions with NF-Y and OCT-I result in the formation of a weaker transcription co.-nplex. C) In uninduced cells, although many of the same factors bind, they are not able to activate DRA transcription. Thl: could be clue to dilterences in post-translational modification of the same trans-acting factors or due to the presence of new and inactive proteins. Proteins are labelled in panel C. Filled circles or boxes represent active proteins, whereas white circles denote inactive proteins.

Careful analysis of cis-acting sequences and transacting factors that regulate the normal expression of the DRA gone has been invaluable for our studies of bls II (Caiman and Peterlin, 1987; Caiman and Peterlin, 1988; Caiman and Peterlin, 1990). There are four different complementation groups for class II non-expression in these cells since four different crosses (heterokaryons) between these ceils rescued expression. All four bls II lesions map into the X box sequences of class II promoters (unpublished data). Thus, if all described defects are genetic, four different complementation groups could be explained by four distinct proteins that bind to the X box. Conversely, genetic defects could be in factors that interact with but are not themselves DNA-binding proteins. Other scenarios involving yet more distal lesio:v~ can be envisioned. The biochemical isolation of proteins that bind to the DRA promoter should reveal these genetic defects.

adaptors or co-activators that would interact with downstream promoter elements to either increase or decrease the assembly of competent transcription complexes (Lewin, 1990). Complex models with permutations of this theme can be drawn. However, before individual factors are cloned and their protein-protein interactions characterized, definitive statements cannot be_ ..,au~." "4"

However, if biochemical studies and expression of cloned proteins fail to reveal trans-acting factors that are inactive or absent in bls II, a genetic approach will become useful. Here, either fragments of the human genome or expression cDNA libraries will be transfected into bls II cells and these cells monitored for reexpression of class II determinants. One of these approaches should reveal the gene(s) responsible for bls II. After characterization, these genetic 0robes will have great utility for population studies, for prenatal diagnosis and for possible gene therapy to reverse this severe combined immunodeficiency:

Fig. 4. Pictorial representation of DNA-binding proteins that interact with the DRA promoter in different cells.



Acknowledgements I would like to thank Michael Armanini for expert secretarial assistance and members of my laboratory for useful discussions and comments on the manuscript. This work was carried out in part by NIH grant (A129954).

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Transcriptional regulation of HLA-DRA gene.

Transcriptional regulation of class II genes is complex. DRA, for example, is expressed in both tissue-specific and lymphokine-inducible fashion. High...
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