DEK binding to class II MHC Y-box sequences is gene- and allele-specific
© Adams et al., licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. 2003
Received: 23 September 2002
Accepted: 29 April 2003
Published: 23 May 2003
Using electrophoretic mobility shift assays, we examined sequence-specific binding of DEK, a potential autoantigen in juvenile rheumatoid arthritis, to conserved Y-box regulatory sequences in class II MHC gene promoters. Nuclear extracts from several cell lines of different phenotypes contained sequence-specific binding activity recognizing DRA, DQA1*0101, and DQA1*0501 Y-box sequences. Participation of both DEK and NF-Y in the DQA1 Y-box binding complex was confirmed by 'supershifting' with anti-DEK and anti-NF-Y antibodies. Recombinant DEK also bound specifically to the DQA1*0101 Y box and to the polymorphic DQA1*0501 Y box, but not to the consensus DRA Y box. Measurement of the apparent dissociation constants demonstrated a two- to fivefold difference in DEK binding to the DQA1 Y-box sequence in comparison with other class II MHC Y-box sequences. Residues that are crucial for DEK binding to the DQA1*0101 Y box were identified by DNase I footprinting. The specific characteristics of DEK binding to these related sequences suggests a potential role for DEK in differential regulation of class II MHC expression, and thus in the pathogenesis of juvenile rheumatoid arthritis and other autoimmune diseases.
KeywordsDEK genetic polymorphism HLA-DQA1 HLA-DRA juvenile rheumatoid arthritis
Although juvenile rheumatoid arthritis (JRA) is the most common cause of disability in children, its etiology is unknown. Immune dysregulation appears to play a key pathogenic role, as circulating autoantibodies are common in patients with certain JRA clinical subtypes [1–7]. Two recent studies have shown a highly significant association between early-onset pauciarticular JRA and circulating antibodies to the 43-kDa nuclear protein DEK [8, 9]. Although circulating DEK antibodies have subsequently been found in children and adults with other autoimmune diseases [10, 11], these two studies did reveal that children with JRA are significantly more likely to have anti-DEK antibodies than are children without rheumatic disease. Children with pauciarticular-onset JRA were also significantly more likely to have anti-DEK antibodies than were children with polyarticular-onset or systemic-onset JRA or other rheumatic diseases. Among children with pauciarticular JRA, DEK autoantibodies were significantly more common in those with JRA-associated uveitis than in those without eye disease [8, 9]. DEK reactivity was also found to be strongly associated with onset of any JRA subtype before age 6 years .
DEK is a nuclear protein that is not structurally related to any known family of proteins [12, 13]. Although it may also participate in DNA replication and RNA processing [14, 15], we have identified DEK as a DNA-binding protein that recognizes the TG-rich peri-ets (pets) regulatory element in the human immunodeficiency virus type 2 (HIV-2) enhancer . The pets site is important in mediating HIV-2 enhancer stimulation in activated T cells and monocytes [17–19], suggesting that DEK may play an immunomodulatory role as it participates in transcriptional activation through this and related sites.
Materials and methods
Cell culture and preparation of nuclear extracts
Preparation of partially purified recombinant DEK protein
Construction of the poly-histidine-tagged DEK bacterial expression vector is described elsewhere . Full-length DEK or antisense DEK was prepared from cultures grown from individual colonies to log phase, induced with 1 mm isopropyl thiogalactose, and harvested by centrifugation after 4 hours. Recombinant protein was purified from bacterial lysates in accordance with the published method for the QIAexpress system (Qiagen, Valencia, CA, USA) with variations in Buffers B and D as noted in Additional file: 1. Procedures were carried out at 4°C; dialyzed recombinant DEK protein (rDEK) was stored at -80°C.
Preparation of FLAG-DEK
A FLAG-tagged DEK adenoviral vector constructed by the University of Michigan Vector Core was used to transduce T98G cells (ATCC) by incubation for 48 hours before harvesting for immunoprecipitation. FLAG-DEK was immunoprecipitated using anti-FLAG resin (Sigma-Aldrich, St Louis, MO, USA) in accordance with the manufacturer's instructions and was eluted by competition with peptide containing three FLAG recognition epitopes.
Electrophoretic mobility shift assays (EMSAs)
EMSAs were carried out as previously described , using 0.1–0.25 ng of radiolabeled oligonucleotide probe (2.5 × 104 counts per minute) per 15 μl binding reaction and 5 μg of nuclear extract (except as noted) or <1 μg of rDEK. For competition EMSAs, unlabeled double-stranded oligonucleotide was added to reaction mixtures before the radiolabeled probe. For antibody supershift of binding complexes, 1 μl anti-NF-YA antibody (gift of JP-Y Ting) or 1 μl high-titer anti-DEK human serum (gift of W Szer ) or 2–3 μl control human serum was added to the binding reaction, and the mixture was incubated on ice for 2 hours before the probe was added.
Sequence of oligonucleotide probes and competitors
See Fig. 1.
Measurement of apparent dissociation constants using EMSA
The 32P end-labeled oligonucleide probe (5 nM) was incubated with immunoprecipitation-purified FLAG-rDEK in a range of concentrations from one tenth to 10 times the estimated Kd[app] (apparent dissociation constant) as described elsewhere . Protein-bound DNA was separated from free probe as for EMSA in 1 × TBE (Tris-borate-EDTA buffer: Tris 89 mM, borate 89 mM, EDTA 2 mM). The dried gel was exposed to a phosphor screen overnight, and the bands were quantified using a Molecular Dynamics Storm 840 Phosphorimager with Image-Quant Software. The data were fit via nonlinear least-squares regression to the single-site binding isotherm:
% free DNA = Kd[app]/(Kd[app] + [protein]).
From this equation, the apparent Kd corresponds to the protein concentration at which half of the DNA is bound .
DNAse I protection assay
The 148-bp probe included DQA1*0101 promoter sequence from -53 to -200. PCR primer (200 ng) for probe sequence was end-labeled with 32Pγ-ATP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA), and was column purified. Labeled antisense primer (200 ng) and 200 ng unlabeled sense strand primer (or vice versa) were used in each 50.5-μl PCR reaction, with 0.2 mM dNTP, 1.5 mM MgCl2, 5 μl 10X PCR Buffer (Invitrogen, Carlsbad, CA, USA), and 1 μg Namalwa genomic DNA. Taq polymerase (Invitrogen) was added at 80°C after a 94°C 'hot start,' initiating 35 cycles of PCR: 94°C for 45 s, 55°C for 30 s, and 72°C for 90 s, with final extension at 72°C for 10 min. PCR products were purified with a High Pure PCR Product Purification Kit (Roche Applied Science, Indianapolis, IN, USA) and then used at 2.5 × 104 CPM/2 μl. DNase I digestion reaction and footprinting gel followed published techniques .
Y-box binding activity in nuclear extracts is gene- and allele-specific
The consensus (HLA-DRA) Y-box sequence differs from the HLA-DQA1*0101 Y box by a single nucleotide immediately 5' of the reverse CCAAT pentamer; this single-nucleotide polymorphism significantly changes the electrophoretic pattern (Fig. 3b) in comparison with that seen with the HLA-DQA1*0101 probe. HLA-DQA1*0101 oligonucleotide also competes poorly with the consensus Y-box probe for binding (Fig. 3b, lanes 5–6). Divergence of the HLA-DQA1*0501 Y-box sequence at one position within the required NF-Y binding site reduces its ability to compete with the DRA Y-box probe (Fig. 3b, lanes 7–8) as much as does mutation of all five nucleotides within the CCAAT sequence (Fig. 3b, lanes 9–10).
Both DEK and NF-Y participate in the HLA-DQA1 Y-box binding complex
Recombinant DEK protein binds in a sequence-specific manner to the DQA1 Y box but not to the DRA (consensus) Y box
Further characterization of rDEK binding to the polymorphic DQA1*0501 Y-box sequence and to the consensus (HLA-DRA) Y-box sequence establishes a relative hierarchy of DEK binding activity (Fig. 5b). With the DQA1*0501 probe, unlabelled DQA1*0101 sequence (Fig. 5b, lane 3) competed less well than did unlabelled probe. DRA Y box sequence (Fig. 5b, lane 4) and CCAAT mutant sequence (not shown) did not compete for binding. Inability of the HLA-DRA Y-box sequence to compete with the DQA1*0501 probe again indicates the relative importance of the adenine residue immediately 5' to the reverse CCAAT for DEK binding. With the DRA Y-box sequence as probe (Fig. 5b, lanes 6–10), the unlabelled probe sequence competes less well than do any of the other Y-box sequences, suggesting that rDEK alone binds to this sequence nonspecifically and with low affinity.
Quantitative assessment of rDEK binding to related DQ-and DR- Y-box sequences
Apparent dissociation constant (Kd[app]) of rDEK binding to class II MHC Y-box sites
478 ± 103
582 ± 130
2480 ± 519
1977 ± 93
1266 ± 274
1041 ± 258
Localization of DEK binding within the Y box
All antigen-presenting cells upregulate MHC class II transcription in response to immune stimulation. We have previously shown that activation of promyeloid cells causes dephosphorylation of DEK and diminished DEK binding to the HIV-2 long terminal repeat [16, 17, 32]. In this study, we show that rDEK can bind to the DQA1 Y box, and that DEK in nuclear extracts participates in the DQA1 Y-box binding complex in vitro. Thus, we propose a model in which intracellular signaling modulates the ability of DEK to bind DNA, causing alteration of MHC class II transcription. Transient transfection experiments in cultured cell lines have not proved useful in examining this model, for DEK is highly expressed in most cells of hematopoietic lineage, and further overexpression has resulted in apparently nonspecific downregulation of transcriptional activity (as might be predicted from ). For this reason, we are currently pursuing other experimental approaches.
The DQA1*0501 promoter region (QAP 4.1), in which a single-base-pair polymorphism in the Y box significantly diminishes transcriptional activity , is a component of the so-called susceptibility haplotype for autoimmune disease. The DQA1*0501 allele is strongly associated with early-onset pauciarticular JRA in Northern European populations [23, 24] and with increased risk for juvenile dermatomyositis  and Sjögren's syndrome with high autoantibody production [26, 27]. In our proposed model, aberrant class II MHC regulation could result from altered DEK binding and/or interaction with NF-Y, and aberrant class II expression may alter or enhance reactivity against DEK-derived or other self peptides. The C-terminal region of DEK, which contains the putative DNA-binding domain, appears to be most antigenic  (and K Sitwala and DM Markovitz, unpublished observations), raising the possibility that altered DNA binding may expose other masked epitopes. Development of antibodies to DEK could even be a primary event in the pathogenesis of JRA, with disruption of nuclear events due to penetration of anti-DEK antibodies into living cells . It remains to be determined whether anti-DEK antibodies are directly involved in the pathogenesis of autoimmune disease, or if they result from generally enhanced immunoreactivity.
Three specific findings in this report support a potential role for DEK as a transcriptional modulator of MHC class II expression. One is that DEK binds to the HLA-DQA1 Y box in a sequence-specific manner. Another is that NF-Y and DEK both participate in the HLA-DQA*0101 Y-box binding complex, which coordinates DQ protein expression. Finally, DEK binds differentially to specific Y-box sequences found in HLA-DQA1(*0101 and *0501 alleles) and HLA-DRA, consistent with observations of DQA1 gene-specific cell-surface expression  and allele-specific promoter activity . The specificity of rDEK binding to these and other, related Y-box sequences (including HLA-DQB and HLA-DRB alleles associated with the DR4 haplotype) may correlate with a predisposition to autoim-mune disease seen with certain HLA haplotypes.
electrophoretic mobility shift assay
Five NH2-terminally deleted epilope
Major histocompatibility complex
human immunodeficiency virus
juvenile rheumatoid arthritis
apparent dissociation constant
polymerase chain reaction
recombinant DEK protein.
We thank W Szer for human anti-DEK serum, J Ting for anti-NF-YA antibody, and D Glass for multiple helpful discussions. This work was supported by grants from the Arthritis Foundation Michigan Chapter (BSA), the Arthritis Foundation (BSA, DMM), the American Cancer Society (DMM), and the National Institutes of Health (AI36685).
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