©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Human Leukocyte Antigen A2 Interferon-stimulated Response Element Consensus Sequence Binds a Nuclear Factor Required for Constitutive Expression (*)

Jeffrey F. Waring (1) (2) (3), James E. Radford (1), Linda J. Burns (1), Gordon D. Ginder (1) (2) (3)(§)

From the (1) Department of Medicine, Division of Medical Oncology, and the (2) Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455 and the (3) Genetics Ph.D. Program, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Both constitutive and interferon-inducible enhancer-like elements have been identified previously in the promoter of human leukocyte antigen (HLA) class I genes. One of these sites is termed the interferon-stimulated response element (ISRE). We have tested the function of an ISRE consensus sequence in the human HLA class I gene HLA-A2 and confirmed previous studies that showed that the HLA-A2 ISRE consensus sequence does not mediate a response to interferons. However, deletion of the ISRE consensus sequence caused a severalfold reduction in the constitutive expression of the HLA-A2 gene in K562 and Jurkat cells. Mobility shift assays performed with the HLA-A2 ISRE revealed the presence of a constitutive binding protein (ISRE/CBP). This protein binds specifically to the HLA-A2 ISRE sequence, and binding is not efficiently competed by the ISRE sequences of the HLA-B7 or ISG54 genes. Substitution of the HLA-B7 or ISG54 ISRE sequences for the HLA-A2 ISRE sequence caused a severalfold reduction in the constitutive expression of the HLA-A2 gene. Mass determinations showed the ISRE/CBP to be 105 kDa, different than any previously characterized ISRE binding proteins. We propose that ISRE/CBP is a novel positive transcriptional regulatory factor for the HLA-A2 gene that may contribute to the differential expression of HLA-A versus HLA-B genes.


INTRODUCTION

The MHC() class I genes encode for a highly polymorphic membrane-spanning 43-45-kDa polypeptide heavy chain that covalently associates on the cell surface with an invariant 12-kDa light chain -2 microglobulin (Hood et al., 1983). Most adult mammalian nucleated cells, with the exception of neurons, germ cells, and trophoblasts, express MHC class I antigens, also known in humans as HLA class I antigens (Singer and Maguire, 1990). The expression of HLA class I antigens is developmentally controlled (Burke et al., 1989; Koller and Orr, 1985) and can be up-regulated in certain cell types by type II () and type I ( and ) interferons (Ball et al., 1984; Basham et al., 1982; Chen et al., 1986; Friedman et al., 1984; Reis et al., 1992; Sanderson and Beverly, 1983). The classical MHC class I antigens, termed HLA-A, -B, and -C in humans and H-2 K, D, and L in mice, function by governing the interactions between cytotoxic T lymphocytes and their target cells and are the principle targets for cell-mediated lysis by cytotoxic T cells during the rejection of allogenic tissue transplants (Hood et al., 1983). Recent studies in transgenic mice lacking class I expression confirm the absolute requirement of class I expression for cytotoxic T lymphocyte function as well as a role in the development of natural killer cells (Zijlstra et al., 1990). Class I antigens have also been implicated in controlling the metastatic growth of tumors (Ball et al., 1984; Ackrill and Blair, 1988; Henseling et al., 1990; Kimura et al., 1986; Lenardo and Baltimore, 1989; Moller et al., 1987; Tanaka et al., 1985; Travers et al., 1982; Wallich et al., 1985).

There is emerging evidence for locus-specific expression of MHC class I genes in some cells such as human colorectal and melanoma cells and murine IC9 fibrosarcoma cells. (Soong et al., 1991; Marincola et al., 1994; Maschek et al., 1989). Studies of HLA class I locus-specific response to interferon also have shown that the different class I subtypes have varying levels of response to both type I and type II interferons (Sanderson and Beverly, 1983; Hakem et al., 1991).

Several cis-acting transcriptional regulatory sites have been identified in the 5` promoter region of most MHC class I genes. Two of these elements have been well characterized. The first is designated the class I regulatory element, or enhancer A. Studies of the H-2K and H-2L gene promoters have shown that this site acts as a constitutive enhancer for these genes in certain cell types; other investigators have shown that this element may play a role in the regulation of the HLA-B7 gene as well (Koller and Orr, 1985; Baldwin and Sharp, 1987; Chamberlain et al., 1991; Dey et al., 1992; Miyazaki et al., 1986). Numerous nuclear proteins have been shown to bind specifically to the enhancer A sequence, including H2TF1 (Baldwin and Sharp, 1987), KBF1 (Yano et al., 1987), NF-B (Levy et al., 1988; Sen and Baltimore, 1986), MBP-1 (Baldwin et al., 1990), and EBP-1 (Clark et al., 1988). To date, no example of a constitutive element involved in locus-specific expression of MHC class I genes has been reported.

A second regulatory site that has been identified in HLA class I genes, as well as in several other interferon responsive genes, is the interferon-stimulated response element (ISRE). This consensus sequence was first designated the interferon response sequence by virtue of its high sequence conservation in the promoters of a number of genes that are inducible by interferon (IFN-) (Friedman and Stark, 1985). In many cases, it has been shown that the ISRE is the major element mediating interferon , and in some cases, interferon responses in interferon inducible genes. However, it has been shown that ISRE consensus sequences in certain genes, such as in HLA-A2 and HLA-A3, do not mediate an interferon response and, as such, are not true ISREs (Hakem et al., 1991; Burns et al., 1993).

DNA-protein binding studies have shown that in some genes the ISRE binds several regulatory proteins or protein complexes, both constitutive and interferon-induced. The interferon-induced complexes have been well characterized. Two of these complexes are termed interferon-stimulated gene factors (ISGFs), or in alternate nomenclature called M and E (Levy et al., 1988; Imam et al., 1990). ISGF-3 is a complex composed of a 48-kDa DNA binding protein and three nonbinding polypeptides and appears to be the primary mediator of type I interferon-stimulated transcription of class I genes (Levy et al., 1988; Dale et al., 1989; Fu et al., 1990; Levy et al., 1989). It is induced shortly after treatment of cells with type I interferon, even in the absence of protein synthesis. ISGF-2 has been shown to be identical to IRF-1, a protein that binds specifically to the upstream regulatory region of the human IFN- gene. Like ISGF-3, ISGF-2 is also induced in response to IFN-, but induction requires protein synthesis. ISGF-2 also can be induced by IFN- (Imam et al., 1990). ISGF-2 is likely an ancillary factor in the positive regulation of IFN-, although its exact role is unknown (Levy et al., 1988; Harada et al., 1989; Miyamoto et al., 1988; Pine et al., 1990).

There have been several factors identified that bind constitutively to the ISRE of interferon-inducible genes, most of which belong to a family of DNA binding proteins termed the IRF-1 family. One factor identified in the ISG54 gene is ISGF-1 or C (Levy et al., 1988). The function of ISGF-1 is unclear at this time, although recently, a component of ISGF-1 was identified to be IRF-2, which has been shown to be a negative regulator of the interferon gene (Parrington et al., 1993; Harada et al., 1989). Interferon consensus sequence binding protein, another member of the IRF-1 family, binds constitutively to the ISRE of many interferon-inducible genes and also acts as a negative regulator (Weisz et al., 1992; Nelson et al., 1993). A third factor that binds constitutively, although weakly, to the ISRE is the p48 DNA binding subunit of the ISGF-3 complex (Darnell et al., 1994). Finally, two factors have been identified that bind constitutively to the ISRE of the 9-27 gene. These proteins, which are 73 and 84 kDa in size, belong to a family of DNA-binding proteins that has been previously shown to bind specifically to the distal regions of the U1 small nuclear RNA gene promoter and the promoter of the transferrin receptor gene (Wedrychowski et al., 1992). The function of these two proteins in the regulation of the 9-27 gene is unknown at this time.

We have examined the role of the ISRE consensus sequence in the constitutive expression of the HLA-A2 gene. We have confirmed previous studies that have shown that the HLA-A2 locus ISRE consensus sequence by itself is insufficient to confer transcriptional induction by either type I or type II interferons (Hakem et al., 1991; Burns et al., 1993). These data demonstrate that the HLA-A2 promoter does not contain a true ISRE because it does not mediate an interferon response. Nevertheless, because the HLA-A2 ISRE promoter does contain a consensus ISRE sequence and because this sequence element has been referred to as the HLA-A2 ISRE in the literature (Schmidt et al., 1990; Hakem et al., 1991; Koller and Orr, 1985; Zachow and Orr, 1989), that term will be used in this paper. Using polymerase chain reaction-based mutagenesis techniques, we deleted the ISRE from the promoter of the HLA-A2 gene, and stably and transiently transfected the resulting mutated constructs into K562, a human leukemia cell line, and the Jurkat T cell line. Our results indicate that deletion of the ISRE greatly reduces HLA-A2 constitutive expression. Gel mobility shift assays, in vitro DNA-protein footprints, and SDS-polyacrylamide gel molecular mass determinations demonstrate a constitutively produced 105-kDa protein in K562 cell nuclear extracts that binds to the HLA-A2 ISRE core consensus sequence. This binding activity is unaffected by treatment of the cells with IFN-. We discuss these results in the context of locus-specific and cell-type-specific regulation of the HLA class I genes.


MATERIALS AND METHODS

Plasmid Constructions

The pA2-CAT plasmid construct was made as described previously (Burns et al., 1993). This construct contains 525 base pairs of HLA-A2 5`-flanking sequence up to, but not including, the first exon. All site-directed 5` base pair deletions or mutations were constructed using polymerase chain reaction overlap extension techniques as described by Ho (Ho et al., 1989). For the deletion ISRE construct, the deleted base pairs were CAGTTTCTTTTCT, which span -148 to -160. For the pA2/B7ISRE-CAT construct, the HLA-A2 ISRE sequence from -148 to -160 was replaced with the sequence GAGTTTCACTTCT. For the pA2/54ISRE-CAT construct, the HLA-A2 ISRE sequence from -144 to -163 was replaced with the sequence TTCTAGTTTCACTTTCCCTT. The relevant regions of mutant and wild-type constructs were sequenced.

For RNase protection assays, single-stranded probes were generated from the following plasmid constructs. For the Neo gene, a 360-base pair PvuII fragment was subcloned into pGEM I (Promega). For the A2 genomic probe, a 768-base pair PstI fragment from pUC 9 HLA-A2 (Koller et al., 1988) was subcloned into pGEM I. The latter probe protects 160 nucleotides of exon 4 in HLA-A2.

Synthetic Oligonucleotides

The HLA-A2, ISG54, HLA-B7, M1, M2, and M3 oligonucleotides were purchased from National Biosciences Inc. The oligonucleotides were synthesized with an automated synthesizer and purified by high performance liquid chromatography. The HLA-A2 ISRE oligonucleotide spans from -144 to -163 of the HLA-A2 promoter. The ISG54 ISRE oligonucleotide spans from -75 to -114 of the ISG54 promoter. The HLA-B7 ISRE oligonucleotide spans from -109 to -128 of the HLA-B7 promoter. The sequences of the HLA-A2; HLA-B7; and ISG54 M1, M2, and M3 ISRE oligonucleotides are shown (see Fig. 6B). For labeling of oligonucleotides for the UV cross-linking, a small primer was synthesized that is complimentary to the upper strand of the HLA-A2 or ISG54 ISRE oligonucleotides. The HLA-A2 ISRE primer has the sequence 5`-CGCAAGCT-3`, while the ISG54 ISRE primer has the sequence 5`-CGTTACAA-3`. These primers were used to uniformly label the oligonucleotide with [-P]dATP.


Figure 6: A, gel mobility shift competition assay done with the HLA-A2 ISRE probe using K562 nuclear extracts. Five units of heparin were used as a nonspecific competitor. A 12.5-, 25-, and 50-fold molar excess of unlabeled double-stranded oligonucleotide was used for each specific competitor titration. Lane1 contains no extract, while lane2 shows the HLA-A2 ISRE oligonucleotide probe incubated with K562 nuclear extracts with no specific competitor. Lanes3-5 show results with HLA-A2 ISRE oligonucleotide as a specific competitor; lanes6-8 show results with with ISG54 ISRE; lanes9-11 show results with with HLA-B7 ISRE; lanes12-14 show results with with M1 ISRE; lanes15-17 show results with with M2 ISRE; and lanes18-20 show results with with M3 ISRE as a specific competitor. B, oligonucleotide sequence of the HLA-A2 ISRE, the ISG54 ISRE, HLA-B7 ISRE, and the M1, M2, and M3 ISRE oligonucleotides. The nucleotide differences between the HLA-A2 ISRE oligonucleotide and the other ISRE oligonucleotides are underlined and highlighted in italic. C, quantitative assay of specific competition of the HLA-A2, ISG54, HLA-B7, M1, M2, and M3 ISRE oligonucleotides to the complex B1 bound to the HLA-A2 ISRE probe. B1 from the mobility shift in Fig. 6A was scanned using a Molecular Dynamics densitometer, and the percent protein bound on the y axis was determined by dividing the value of B1 in the lanes with competitors by the value of B1 in the lane with no specific competitor.



Tissue Culture

Jurkat, a T leukemia/lymphoma cell line, and K562, a human leukemia cell line, were maintained in suspension cultures as described previously (Chen et al., 1986; Burns et al., 1993).

Transfection

K562 cells were transfected by electroporation at 250 V at a capacitance setting of 800 microfarads with a resistance of R1 using a BTX electro cell manipulator, model number 600. For K562 transfections, 30 µg of the plasmid was transfected into 450 µl of cells at a concentration of 2 10 cells/ml. Jurkat cells were transfected by the DEAE-dextran method modified as described previously (Burns et al., 1993). The cells were harvested 48 h following transfection.

For stable transfections, the electroporation conditions were the same as above with the exception that the neomycin resistance gene (Neo), linked to the Rous sarcoma virus (RSV) promoter, was co-transfected with the HLA-A2 plasmids at a molar ratio of 30:1, A2 plasmid to RSV-Neo. Following transfection, the cells were grown in RPMI 1640 media with 10% fetal calf serum for 48 h, whereupon they were selected in media containing 0.5 mg/ml G418 (Life Technologies, Inc.).

CAT Assays

CAT assays were analyzed by thin-layer chromatography, as described by Gorman et al.(1982), or by liquid scintillation counting according to the Promega CAT enzyme assay system. Each CAT assay was standardized by adding a constant amount of protein from each cellular extract, and the assays were done on the linear portion of the enzyme reaction curve. The CAT assays were also controlled for transfection efficiency by co-transfection with pRSV-Luc, a plasmid containing the luciferase reporter gene driven by the RSV promoter-enhancer, or by pGL2-Control, which is a plasmid containing the luciferase reporter gene driven by the SV40 promoter.

RNA Isolation and RNase Protections

RNA isolation and RNase protections were done as described previously (Chen et al., 1986).

Densitometry

The autoradiographs for the RNase protections and CAT assays were analyzed by densitometry on a GS 300 transmittance-reflectance scanning densitometer (Hoefer Scientific Instruments). The peaks were integrated using the Hoefer software package. The mobility shift assays were analyzed by a Molecular Dynamics Densitometer SI, or by a Molecular Dynamics PhosphorImager SI.

Nuclear Extract Preparation

K562 and Jurkat nuclear extracts used in the gel mobility shift assays, in vitro footprints, and UV cross-linking were prepared by the method of Dignam et al.(1983). For the IFN--induced nuclear extracts, the cells were stimulated with 100 units/ml of IFN- 24 h before cell harvest. The HeLa, Namlawa, and EW nuclear extracts were a gift from Dr. Brian Van Ness.

Gel Mobility Shift Assays

The target oligonucleotide was end-labeled with [-P]ATP by using T4 polynucleotide kinase; 0.1 ng of end-labeled DNA probe was incubated with 10-20 µg of nuclear extract protein/binding reaction in the presence of 1-3 µg of pUC19 vector, and 5 units of heparin, 5 µg of salmon sperm, or 5 µg of poly(dI-dC) at room temperature for 30 min. The DNA-protein complexes were separated in 6% polyacrylamide gels. Competition experiments with unlabeled oligomers were performed by the addition of up to 125-fold excess of the double-stranded oligomer to the reaction mix.

In Vitro Footprints

The methylation interference footprints were as described previously (Baldwin and Sharp, 1987). Piperidine cleavage and sequencing gel analysis were performed according to published Maxam and Gilbert protocols (Maxam and Gilbert, 1980). The copper orthophenanthroline footprinting was done exactly according to the method of Dale et al.(1989).

UV Cross-linking

The UV cross-linking was carried out following the protocol of Treisman(1987). The only modification was that reactions were irradiated for 5 min in a Stratagene Stratalinker UV cross-linker 2400 to a total energy of 1.2 10 microjoules/cm.

Southwestern Blotting Assay

The Southwestern blotting assay was carried out following the protocol of Miskimins (Miskimins et al., 1985).

RESULTS

The ISRE Sequence Does Not Act as an Interferon Response Element But Does Play a Positive Role in Constitutive HLA-A2 Gene Expression in K562 and Jurkat Cells

Previous evidence has shown that in certain cell types, the ISRE is insufficient to confer type I or type II IFN induction of HLA-A2 and HLA-A3 genes (Hakem et al., 1991; Burns et al., 1993). However, the role of the ISRE in constitutive regulation of MHC class I genes has not been studied. To investigate the potential role of the ISRE in the IFN- response of the HLA-A2 gene, a construct consisting of the HLA-A2 promoter and 525 base pairs of 5`-flanking sequence linked to the CAT gene was transfected into both untreated and IFN--treated K562 and Jurkat cell lines. As shown in Fig. 1, IFN- treatment had no effect on the expression of the HLA-A2 CAT construct in either cell line. We have previously shown that endogenous HLA class I genes in both of these cell lines respond with a 3-10-fold increase in transcription under identical treatment conditions (Burns et al., 1993; Radford et al., 1991).


Figure 1: CAT assay of extracts from K562 and Jurkat cells transfected with pA2-CAT. Cells were either maintained in IFN-free media (control) or media to which interferon was added at concentration of 100 units/ml (+ IFN GAMMA). Cells were stimulated with interferon for 48 h before harvesting for CAT activity. Controls were as described under ``Materials and Methods,'' and the autoradiograms shown are representative of at least six independent repeats of each assay.



To determine the role of the ISRE in constitutive transcriptional control of the HLA-A2 gene, deletions were made in the promoter of the A2 gene (Fig. 2). Using the polymerase chain reaction overlap extension technique (Ho et al., 1989), the ISRE was deleted from the A2 5` promoter region. The wild-type promoter segment includes 525 base pairs of the A2 5` promoter and extends to, but does not include, the first exon of the gene. For the deletion ISRE construct, the deleted base pairs were from -148 to -160, which includes the core binding region for the known ISRE binding proteins (see ``Materials and Methods'').


Figure 2: A, schematic illustration of the HLA-A2 promoter from -148 to -196 base pairs. The barsabove the gene map illustrate the class I regulatory element (enhancer A) and the ISRE. The barsbelow the graph show the deletions made for the clone HLA-A2 del ISRE. B, sequence differences between the HLA-A2 ISRE motif, and the ISRE binding region in the promoters of other interferon-responsive genes. The sequences shown are the consensus ISRE, and the ISRE sequences from HLA-A2, ISG54, ISG15, H-2D, H-2K, H-2L (Levy et al., 1988), 6-16 (Reis et al., 1992), IFN- (Harada et al., 1989), and HLA-B7 genes (Hakem et al., 1991).



The promoter lacking the ISRE, as well as a wild-type promoter, were then linked to a CAT reporter gene, and these constructs were transiently transfected into K562 and Jurkat cells. Transfectants were cultured for 48 h and then harvested for CAT assays. Deletion of the ISRE from the HLA-A2 promoter significantly reduced the overall level of CAT expression in both K562 and Jurkat cells (Fig. 3). The CAT assay results from at least six independent transfections for each construct, in each cell line was quantitated with a densitometer, and mean values were calculated. When the wild-type CAT levels were standardized to a value of 1, deletion of the ISRE reduced relative CAT expression to a level of 0.32 ± 0.07 in Jurkat and 0.2 ± 0.05 in K562 cells (Fig. 3).


Figure 3: Expression levels of the HLA-A2 promoter upon deletion of the ISRE sequence in K562 and Jurkat cells. In each experiment, wild-type levels were set to a value of 1; the expression levels of HLA-A2 del ISRE are relative to the wild-type expression level. The errorbars represent the calculated standard deviation for each set of mutants and cell lines. The results are expressed as the average of at least six independent transfections for each construct and each cell line.



These same constructs were also stably transfected into K562 cells, and the amount of HLA-A2 CAT fusion RNA was measured by RNase protection assay. The results confirmed the CAT assay data, in that deletion of the HLA-A2 ISRE from the HLA-A2 gene significantly reduced the level of correctly initiated HLA-A2 CAT RNA produced (data not shown). In stable transfections, expression levels were corrected for copy number of the transfected gene.

At least one report has shown a constitutive positive transcriptional effect mediated by sequences downstream from the transcription start site of an MHC class I gene (Ganguly et al., 1989). In order to test for the function of the ISRE core sequence in the context of an intact HLA-A2 gene, the ISRE was also deleted from a genomic clone of HLA-A2, which contains 525 base pairs of the 5` untranslated region, all 8 exons, and approximately 1,100 base pairs of 3`-untranslated region (Koller et al., 1988). This construct was then stably co-transfected into K562 cells with an RSV-Neo antibiotic resistance selection plasmid. Following selection of the cells in G418 media, cytoplasmic RNA was harvested, and the level of HLA-A2 RNA produced was assayed by RNase protection assays. The results were standardized for RNA loading and stability relative to expression of the co-transfected internal control, RSV-Neo. As shown in Fig. 4, deletion of the ISRE caused an approximate 6-fold reduction in the amount of HLA-A2 RNA levels. The HLA-A2 probe used in the RNase protection assay protects 160 nucleotides of exon 4. The bands at 130-140 nucleotides, which migrate below the protected band of 160 nucleotides, are derived from K562 endogenous HLA class I mRNA to which the HLA-A2 RNA probe cross-hybridizes. RNase protections using a 5` probe have shown correct initiation at the HLA-A2 start site (Burns et al., 1993). A DNA slot blot assay, performed using a plasmid vector (pGEMI) probe, showed that all four of the stably transfected K562 cell lines illustrated had the same number of copies of the transfected plasmids (data not shown). Thus, the ISRE consensus sequence appears to be required for high level constitutive expression of the HLA-A2 gene even in the context of extensive 5` and 3` flanking, coding, and intervening sequences.


Figure 4: RNase protection assay of RNA from four separate pools of K562 cells stably transfected with either a plasmid containing a wild-type HLA-A2 genomic clone or the HLA-A2 del ISRE clone. The 160-nucleotide band is uniquely derived from the HLA-A2 RNA transcript, while the lower bands are derived from endogenous K562 class I RNA that cross-hybridizes with the HLA-A2 probe as demonstrated in protection assays with untransfected K562 cells (not shown).



Gel Mobility Shift Assays Indicate Specific Binding to the HLA-A2 ISRE

The sequence of the HLA-A2 ISRE compared with that of HLA-B7, ISG54, ISG15, H-2L, H-2D, IFN-, 6-16, and the consensus ISRE is shown in Fig. 1B. As can be seen, there is considerable sequence divergence among the consensus ISREs of these genes. In order to determine what nuclear proteins, if any, bind to the ISRE of the HLA-A2 gene, a labeled double-stranded oligonucleotide containing the HLA-A2 ISRE sequence was tested for binding in mobility shift assays using K562 nuclear extracts. This sequence extends from -144 to -164 relative to the transcription start site and includes all of the HLA-A2 ISRE sequence, but it does not include the enhancer A sequence. The oligonucleotide bound two complexes, designated B1 and B2 (Fig. 5A). Lane1 shows the ISRE probe bound with uninduced K562 nuclear extract, using poly(dI-dC) as a nonspecific competitor. Other investigators have shown that different polyanion competitors can increase the binding affinity of certain proteins to their sequence-specific DNA binding sites (Schwartz and Lee, 1992). Therefore, we examined the effect of adding heparin as a nonspecific competitor. Lane3 illustrates the results of incubation of the ISRE oligonucleotide with K562 nuclear extracts in the presence of 5 units of heparin and 1 µg of pUC19 as nonspecific competitors. While the same pattern of binding complexes was seen with heparin and poly(dI-dC), heparin greatly increased binding of the lower band, B1. Heparin does not increase binding of the upper band, B2. The same complexes bound to the ISRE were also seen when salmon sperm DNA was used as a nonspecific competitor (data not shown). These complexes were specifically competed with a 100-fold molar excess of unlabeled ISRE oligonucleotide, as shown in lanes2 and 4. A 100-fold molar excess of an unlabeled oligonucletide that corresponds to the enhancer A of the HLA-B7 gene was unable to compete either B1 or B2 (data not shown).


Figure 5: A, gel mobility shift assay done with an oligonucleotide containing the HLA-A2 ISRE sequence incubated with K562 nuclear extracts. In lanes1 and 2, 5 µg of poly(dI-dC) were used as a nonspecific competitor, while in lanes3 and 4, 5 units of heparin were used as a nonspecific competitor. In addition, all binding assays included 1 µg of pUC-9 plasmid DNA. Lanes1 and 2 were from an autoradiogram exposed overnight, while lanes3 and 4 were from an autoradiogram exposed for 1 h. Lanes2 and 4 show the results obtained from assays containing a 100-fold molar excess of unlabeled ISRE oligonucleotide as a specific competitor. B, gel mobility shift assay done using the HLA-A2 ISRE motif incubated with K562 nuclear extracts from control or IFN-treated cells. The nuclear extracts were from cells that were either uninduced (lanes1 and 2) or induced for 24 h with IFN-, at a concentration of 100 units/ml. A 100-fold molar excess of the double-stranded ISRE oligonucleotide was used as a specific competitor in lanes2 and 4.



Although 525 base pairs of 5`-upstream flanking sequence, including the ISRE consensus sequence, was shown to be insufficient to confer IFN- responsiveness (Fig. 1), mobility shift assays were performed to determine if the binding pattern of the HLA-A2 ISRE changed upon IFN- induction. K562 nuclear extracts from untreated cells or cells treated for 24 h with IFN- were used in the assay. Fig. 5B shows that an equivalent amount of binding to the ISRE oligonucleotide was observed for the B1 complex in the presence of either uninduced extracts or extracts from K562 cells treated with IFN-. The level of binding complex B2 showed a moderate but reproducible increase in IFN--induced extracts. Fig. 5B also shows a third complex binding to the HLA-A2 ISRE consensus sequence labeled B3. This complex appeared inconsistently in the mobility shift assays, depending on the particular extract used, and its significance is unknown.

In order to determine if the complex that binds to the HLA-A2 ISRE corresponds to any of the known ISRE binding proteins, an ISG54 ISRE oligonucleotide was used as a specific competitor. This sequence was selected because many of the studies of ISRE binding proteins have been carried out using the ISG54 gene promoter (Levy et al., 1988). Fig. 6A, lanes3-5 show, respectively, a 12.5-, 25-, and 50-fold molar excess of unlabeled HLA-A2 ISRE, which specifically competes the labeled HLA-A2 ISRE binding complexes, while lanes6-8 show that a 12.5-, 25-, and 50-fold excess of unlabeled ISG54 ISRE is unable to compete either the B1 or B2 complex. Lanes9-11 show a 12.5-, 25-, and 50-fold molar competition with the HLA-B7 ISRE oligonucleotide. While the HLA-B7 oligonucleotide is able to compete for the B2 and B3 complexes, it does not compete the B1 complex as efficiently as the HLA-A2 ISRE (Fig. 6A, lanes3-5versuslanes9-11 and Fig. 6C). The fact that the B1 complex does not bind to the ISG54 ISRE sequence suggests that it is not ISGF-1, or any of the other ISRE binding complexes identified in the ISG54 gene. Also, because the B1 complex has a greater affinity for the HLA-A2 ISRE than for the ISG54 or HLA-B7 ISRE regions, it appears to have preferential affinity for the HLA-A locus.

The sequences of the ISG54 ISRE and the HLA-B7 ISRE oligonucleotides are shown in Fig. 6B. In order to determine which base pairs were necessary for binding of the B1 complex to the HLA-A2 ISRE, mutated oligos designated M1, M2, and M3 were used as competitors against the HLA-A2 ISRE in mobility shift assays (Fig. 6B). Fig. 6A, lanes12-20 and Fig. 6C show that while the mutant ISRE oligos do not compete for the B1 complex as well as the HLA-A2 ISRE, none of the individual mutations completely abrogate the ability of the mutant ISRE oligos to bind B1. Thus, it would appear that all of the base changes between the HLA-A2 ISRE and the ISG54 ISRE are necessary to completely disrupt binding of the B1 complex to the ISRE-like element.

Since the ISG54 ISRE oligonucleotide does not compete the complexes that bind to the HLA-A2 ISRE, the reverse experiment was done to determine if the HLA-A2 ISRE could compete complexes that bind to the ISG54 ISRE. The labeled ISG54 ISRE oligonucleotide was incubated with K562 nuclear extracts, and a single complex was formed, which was competed with either unlabeled ISG54 ISRE or HLA-A2 ISRE. The mobility shift gel was scanned on a PhosphorImager, and the results are graphed in Fig. 7. As can be seen in Fig. 7 , a 12.5-fold molar excess of unlabeled ISG54 ISRE reduces the amount of complex bound to the ISG54 oligonucleotide to 20.8%, and 50-fold reduces the amount of complex bound to 6.1% compared with no specific competitor, while the equivalent amounts of HLA-A2 ISRE oligonucleotide reduces the amount of complex bound to the ISG54 oligonucleotide to 66.8 and 26.3%, respectively. Thus, the complex bound to the ISG54 ISRE oligonucleotide has a 3-4-fold higher affinity for the ISG54 ISRE than for the HLA-A2 ISRE.


Figure 7: A, quantitative assay of specific competition for the HLA-A2 and ISG54 ISRE oligonucleotides to the complex bound to the ISG54 ISRE. The values were computed the same as in Fig. 6C. B, gel mobility shift assay done with HLA-A2 and ISG54 ISRE probes using K562 or HeLa nuclear extracts. Five units of heparin were used as a nonspecific competitor. Lanes3-6 show results of assays containing K562 nuclear extracts, while lanes7-10 illustrate experiments in which HeLa cell nuclear extracts were used. Lane1, HLA-A2 ISRE probe only; lane2, ISG54 ISRE probe only; lane3, HLA-A2 ISRE oligonucleotide probe with K562 nuclear extracts; lane4, a 125-fold molar excess of cold HLA-A2 ISRE oligonucleotide; lane5, ISG54 ISRE oligonucleotide probe with K562 extracts; lane6, a 125-fold molar excess of cold ISG54 ISRE oligonucleotide, lane7, HLA-A2 ISRE oligonucleotide probe with HeLa cell nuclear extracts; lane8, with a 125-fold molar excess of cold HLA-A2 ISRE oligonucleotide; lane9, ISG54 ISRE probe with HeLa cell nuclear extracts; lane10, with a 125-fold molar excess of cold ISG54 ISRE oligonucleotide.



The presence of the HLA-A2 ISRE-bound complex was assayed in nuclear extracts from other cell lines as well. Fig. 7B shows that in HeLa extracts, a protein complex binds to the HLA-A2 ISRE and migrates the same as the complex from K562 extracts, designated here as complex B1 (lanes3 and 7). The ISG54 ISRE binds a complex, designated C1, which migrates slower than the HLA-A2 ISRE B1 complex in both K562 and HeLa cell extracts (lanes5 and 9). The same complex, which binds to the HLA-A2 ISRE in K562 and HeLa cells was also seen in Jurkat cell extracts and two B cell lines, Namlawa and EW (data not shown).

Functional Assays Using the ISG54 and HLA-B7 ISREs Confirm Their Inability to Substitute for the HLA-A2 ISRE

Because both the ISG54 and HLA-B7 ISRE oligonucleotides bound the B1 complex with less affinity than the HLA-A2 ISRE, transfection experiments were performed to determine if the ISG54 and HLA-B7 ISRE sequence motifs would be similarly unable to substitute completely for the HLA-A2 ISRE motif. Using polymerase chain reaction site-directed mutagenesis techniques, the HLA-A2 ISRE was mutated to match either the HLA-B7 ISRE or the ISG54 ISRE (see ``Materials and Methods''). These constructs, called pA2/B7ISRE-CAT and pA2/54ISRE-CAT, along with the wild-type pA2-CAT construct, were transfected transiently into K562 cells, and the resulting CAT activity was assayed by liquid scintillation counting. The pGL2-Luc control plasmid was co-transfected as an internal control for transfection efficiency (see ``Materials and Methods''). The results show that both pA2/B7ISRE-CAT and pA2/54ISRE-CAT express at a lower level than the wild-type plasmid, approximately at the same level as the pA2-CAT del ISRE construct (Fig. 8). With the wild-type expression standardized to 1 for each experiment, the pA2/B7ISRE-CAT construct expresses at a level of 0.17, while the pA2/54ISRE-CAT construct expresses at a level of 0.29 compared with wild-type. Thus, the results show that a nuclear factor or complex binds constitutively to the HLA-A2 ISRE and augments transcription of the HLA-A2 gene.


Figure 8: CAT assay of plasmids pA2-CAT, pA2-CAT del ISRE, pA2/B7ISRE-CAT, and pA2/54ISRE-CAT transiently transfected into K562 cells. In each experiment, wild type levels were set to a value of 1; the expression levels of pA2-CAT del ISRE, pA2/B7ISRE-CAT, and pA2/54ISRE-CAT are relative to the wild-type expression level. The errorbars represent the calculated standard deviation for each set of mutants. The results are expressed as the average of at least six independent transfections for each construct.



In Vitro DNA Footprinting Shows Binding of B1 to an Eight-base Pair Region within the Core ISRE Consensus Sequence

DNA footprinting patterns have been reported for some previously characterized ISRE binding proteins. These include the binding patterns of ISGF-1, -2, and -3 to the ISRE of the ISG15 and ISG54 genes (Levy et al., 1988; Pine et al., 1990), IRF-1 to the ISRE of the interferon gene (Harada et al., 1989) and E and C to the 6-16 gene (Dale et al., 1989). To determine which base pairs are contacted within the ISRE by nuclear complex B1, methylation interference footprinting was performed. Fig. 9 shows that on the antisense strand, the adenine residues at -155 through -157 and -150 through -153 are crucial for B1 binding. Partial interference could also be seen for the guanine nucleotides at positions -149 and -154.


Figure 9: Methylation interference determination of ISRE/CBP protein-DNA contact points. Preparative binding reactions were carried out using partially methylated ISRE DNA probes, 5` end-labeled on the antisense strand, and nuclear extracts from untreated K562 cells. Unbound probe (UB) and protein-bound probe (B) were resolved by nondenaturing polyacrylamide gel electrophoresis, the DNA was recovered, and following piperidine treatment the cleavage products were separated on a 15% polyacrylamide gel. The HLA-A2 ISRE sequence is shown to the right of the autoradiogram. Filledcircles represent sites of full methylation interference; opencircles represent partial methlyation interference.



To investigate the pyrimidine-rich coding strand, which could not be assayed by the dimethyl sulfate methylation interference method, a copper orthophenanthroline footprint was performed. The copper orthophenanthroline footprint on the sense strand confirmed the methylation interference footprint on the antisense strand, showing binding of B1 between nucleotides -149 and -157 (data not shown).

Molecular Mass Determination of the B1 Binding Protein

The molecular mass of the B1 binding protein was estimated by photoactivated protein-DNA cross-linking analysis. As shown in Fig. 10A, a single intense band was seen at approximately 105 kDa molecular mass. This band was specifically competed with a 100-fold molar excess of unlabeled HLA-A2 ISRE oligonucleotide. A UV cross-linking assay was also carried out using the ISG54 ISRE as a probe. Fig. 10B shows two major bands with the ISG54 ISRE probe, which are an intensely labeled broad band between 45 and 66 kDa and a faint band migrating at approximately 110-130 kDa. The broad 45-66 kDa band is in the same molecular mass range as that reported for the previously characterized binding components of ISGF-1, -2, and -3 and was competed well by specific competitor; in contrast, the higher molecular mass band is much fainter and not competed as well with specific competitor. While the faint high molecular mass band cross-linked to the ISG54 oligonucleotide migrates in a similar molecular mass range as the single intense band seen with the A2 ISRE oligonucleotide, it is only a minor component compared with the broad lower molecular mass complex.


Figure 10: Mass determination of the nuclear protein binding to the HLA-A2 (A) or ISG54 (B) ISRE by UV protein-DNA cross-linking. Binding reactions and UV irradiation were done as described under ``Materials and Methods.'' Lane1 contained no extract; lane2 contained 20 mg of K562 nuclear extract incubated with the HLA-A2 ISRE probe (A) or the ISG54 ISRE probe (B) oligonucleotide; lane3 contained 20 µg of K562 nuclear extract with the addition of a 100-fold molar excess of unlabeled HLA-A2 ISRE (A) or ISG54 ISRE (B) oligonucleotide.



To confirm the molecular mass estimate for the HLA-A2 ISRE binding factor and to exclude the possibility of multiple protein components in the UV cross-linked protein-DNA complex seen in Fig. 10A, a Southwestern blot was performed using a uniformly labeled HLA-A2 ISRE oligonucleotide probe and K562 nuclear extracts from cells either untreated or treated with IFN-. A single band of molecular mass 105 kDa was observed in the Southwestern blot assay (Fig. 11), confirming the results of the UV cross-linking assay. In addition, no change could be seen in the size or the intensity of the ISRE binding protein present in untreated versus IFN-treated extracts, suggesting that this, indeed, represents the protein binding factor in complex B1 in Fig. 5. Thus, the B1 complex binding protein is clearly different than any of the previously characterized ISRE binding proteins; the binding component of ISGF-3 is 48 kDa (Kimura et al., 1986), ISGF-2/IRF1 is 56 kDa (Pine et al., 1990), IRF2 is estimated at 50 kDa (Porter, 1988), and two factors previously identified that bind to the ISRE of the 9-27 gene are 73 and 84 kDa (Wedrychowski et al., 1992). In addition, while this protein may bind with a lower affinity to the ISG54 ISRE sequence, it is the only protein that shows detectable binding to the HLA-A2 ISRE in either the UV cross-linking or Southwestern blot assays.


Figure 11: Southwestern blot analysis of K562 nuclear protein binding to the HLA-A2 ISRE oligonucleotide. 50, 100, or 150 µg of untreated K562 nuclear extracts and 150 µg of IFN--treated K562 nuclear extracts were electrophoresed in an SDS-polyacrylamide gel followed by electroblot to nitrocellulose membrane, incubation with uniformly P-labeled HLA-A2 ISRE probe, and autoradiography. Interferon-treated cells were incubated with 100 units/ml of IFN- for 16 h prior to preparation of extracts.



DISCUSSION

In summary, the major findings of this report are as follows. We have confirmed that the ISRE present in the HLA-A2 gene does not mediate interferon induction and, hence, is not a true ISRE. However, deletion of this ISRE-like element in the HLA-A2 gene does cause a significant reduction in constitutive HLA-A2 expression in both K562 and Jurkat cells, as measured by CAT assays and RNase protections. Mobility shift assays using the HLA-A2 ISRE and uninduced K562 nuclear extracts show the presence of a constitutive factor that binds to the ISRE and that binding is not altered upon IFN- induction. We have designated this factor the ISRE constitutive binding protein (CBP). ISRE/CBP does not bind efficiently to the ISG54 or HLA-B7 ISRE. Replacement of the HLA-A2 ISRE with the ISG54 or HLA-B7 ISRE results in a decrease in the level of constitutive expression of HLA-A2. Thus, the HLA-A2 ISRE and the ISRE/CBP are to our knowledge the first example of constitutive control elements that could account for differential expression of HLA-A versus HLA-B genes. Methylation interference and copper orthophenanthroline in vitro footprints show ISRE/CBP binding specifically to the core of the HLA-A2 ISRE motif with a similar pattern as that shown for ISGF-1 and 2 (Levy et al., 1988; Pine et al., 1990). However, the mass of the ISRE/CBP as determined by UV cross-linking and Southwestern blot assays is 105 kDa. This, coupled with its lack of binding to the ISG54 ISRE, confirms that it is a novel factor.

There is some precedence for a constitutive transcriptional role for an ISRE binding protein in the case of the interferon gene. Using a human fibroblast line, GM-637, it was shown that cells stably transfected with an IRF-1 (ISGF-2) containing expression vector in the sense orientation showed higher levels of IFN- mRNA than control cells, while cells transfected with a vector containing the IRF-1 gene in the antisense orientation produced little or no IFN- mRNA (Reis et al., 1992). However, our functional transfection assays, gel mobility shift competition assays, and molecular mass sizing assays indicate that IRF-1 is not involved in the constitutive expression of HLA-A2.

Previous work in the murine MHC class I system has suggested that the class I regulatory element / enhancer A element is the major sequence motif responsible for constitutive cis-acting regulation of the H-2 genes and the HLA-B7 gene (Henseling et al., 1990; Kimura et al., 1986; Miyazaki et al., 1986; Ganguly et al., 1989; Chamberlain et al., 1991). Deletion of the core region of the class I regulatory element of the enhancer A site in the promoter of the HLA-A2 gene did not significantly affect constitutive expression in K562 and Jurkat cell transfection assays. This result suggests either cell-type specificity or promoter specificity between HLA-A2 and other MHC class I genes in which enhancer A has been shown to have a constitutive enhancer function. There are other examples of MHC class I genes in which an enhancer A is not required for constitutive expression. HLA-Aw24 does not have a consensus H2TF1 binding site in the 5` enhancer A region, while the ubiquitously expressed nonclassical HLA class I gene, HLA-E, lacks the entire enhancer A region (Koller et al., 1988; N'Guyen et al., 1985). In addition, none of the known porcine or rabbit MHC class I sequences contain the precise enhancer A sequence (Singer and Maguire, 1990).

The fact that ISRE/CBP binds with greater avidity to the HLA-A2 ISRE than to the HLA-B7 or ISG54 ISRE motifs suggests that ISRE/CBP is selective for the HLA-A locus versus the HLA-B locus. ISRE/CBP may therefore be an important factor in the differential expression of MHC class I genes. For example, this kind of selectivity could account for the finding that melanoma cell lines, which consistently express high levels of HLA-A antigens display a low but variable level of expression of HLA-B (Marincola et al., 1994). Likewise, different roles of the ISRE and the enhancer A could account for the observation that certain cells express little or no HLA-A antigens but express higher levels of HLA-B antigens (Soong et al., 1991). Further purification of the ISRE/CBP and cloning of the gene that encodes it will allow full characterization of this factor and its exact role in the regulation of HLA class I gene transcription.


FOOTNOTES

*
This work was supported by National Institutes of Health (NIH) Grant R01-CA45634, the Masonic Cancer Center Fund, Inc., and the University of Iowa Genetics NIH training grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medicine and Inst. of Human Genetics, Box 286 UMHC, University of Minnesota, Minneapolis, MN 55455. Tel.: 612-624-7915; Fax: 612-625-8966.

The abbreviations used are: MHC, major histocompatibility complex; HLA, human leukocyte antigen; ISRE, interferon-stimulated response element; IFN, interferon; ISG, interferon-stimulated gene; ISGF, ISG factor; RSV, Rous sarcoma virus; CAT, chloramphenicol acetyl transferase; IRF, interferon response factor; CBP, constitutive binding protein.


ACKNOWLEDGEMENTS

We thank Judy Goetzke for assistance in preparation of this manuscript and Dr. Brian Van Ness for a helpful critique.


REFERENCES
  1. Ackrill, A., and Blair, G.(1988) Oncogene 3, 483-487 [Medline] [Order article via Infotrieve]
  2. Baldwin, A. S., Jr., and Sharp, P. A.(1987) Mol. Cell. Biol. 7, 305 [Medline] [Order article via Infotrieve]
  3. Baldwin, A. S., Jr., LeClair, K., Singh, H., and Sharp, P.(1990) Mol. Cell. Biol. 10, 1406-1414 [Medline] [Order article via Infotrieve]
  4. Ball, E., Guyre, P., Glynn, J., Rigby, W., and Fanger, M.(1984) J. Immunol. 132, 2424-2428 [Abstract/Free Full Text]
  5. Basham, T., Bourgeade, M., Creasey, A., and Merigan, T.(1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3265-3269 [Abstract]
  6. Burke, P. A., Hirschfeld, S., Shirayoshi, Y., Kasik, J. W., Hamada, K., Appella, E., and Ozato, K.(1989) J. Exp. Med. 169, 1309-1321 [Abstract]
  7. Burns, L. J., Waring, J. F., Reuter, J. J., Stinski, M. F., and Ginder, G. D.(1993) Blood 81, 1558-1566 [Abstract]
  8. Chamberlain, J. W., Vasavada, H. A., Ganguly, S., and Weissman, S. M. (1991) Mol. Cell. Biol. 11, 3564-3572 [Medline] [Order article via Infotrieve]
  9. Chen, E., Karr, R. W., Frost, J. P., Gonwa, T. A., and Ginder, G. D. (1986) Mol. Cell. Biol. 6, 1698-1705 [Medline] [Order article via Infotrieve]
  10. Clark, L., Pollock, R. M., and Hay, R. T.(1988) Genes & Dev. 2, 991-1002
  11. Dale, T. C., Rosen, J. M., Guille, M. J., Lewin, A. R., Porter, A. G. C., Kerr, I. M., and Stark, G. R.(1989) EMBO J. 8, 831-839 [Abstract]
  12. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R.(1994) Science 264, 1415-1421 [Medline] [Order article via Infotrieve]
  13. Dey, A., Thornton, A. M., Lonergan, M., Weissman, S. M., Chamberlain, J. W., and Ozato, K.(1992) Mol. Cell. Biol. 12, 3590-3599 [Abstract]
  14. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G.(1983) Nucleic Acids Res. 11, 1475-1489 [Abstract]
  15. Friedman, R. L., and Stark, G. R.(1985) Nature 314, 637-639 [Medline] [Order article via Infotrieve]
  16. Friedman, R. L., Manly, S. P., McMahon, M., Kerr, I., and Stark, G. (1984) Cell 38, 745-755 [Medline] [Order article via Infotrieve]
  17. Fu, X.-Y., Kessler, D. S., Veals, S. A., Levy, D. E., and Darnell, J. E., Jr.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8555-8559 [Abstract]
  18. Ganguly, S., Vasavada, H. A., and Weissman, S. M.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5247-5251 [Abstract]
  19. Gorman, C. M., Moffat, L. F., and Howard, B. G.(1982) Mol. Cell. Biol. 2, 1044-1051 [Medline] [Order article via Infotrieve]
  20. Hakem, R., Le Bouteiller, P., Jezo-Bremond, A., Harper, K., Campese, D., and Lemonnier, F. A.(1991) J. of Immunol. 147, 2384-2390 [Abstract/Free Full Text]
  21. Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., Furia, A., Miyata, T., and Taniguchi, T.(1989) Cell 58, 729-739 [Medline] [Order article via Infotrieve]
  22. Henseling, U., Schmidt, W., Scholer, H. R., Gruss, P., and Hatzopoulos, A.(1990) Mol. Cell. Biol. 10, 4100-4109 [Medline] [Order article via Infotrieve]
  23. Ho, N. S., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene(Amst.) 77, 51-59 [CrossRef][Medline] [Order article via Infotrieve]
  24. Hood, L., Steinmetz, M., and Malissen, B.(1983) Annu. Rev. Immunol. 1, 529-568 [Medline] [Order article via Infotrieve]
  25. Imam, A. M. A., Ackrill, A. M., Dale, T. C., Kerr, I. M., and Stark, G. R.(1990) Nucleic Acids Res. 18, 6573-6580 [Abstract]
  26. Kimura, A., Israel, A., Le Bail, O., and Kourilsky, P.(1986) Cell 44, 261-272 [CrossRef][Medline] [Order article via Infotrieve]
  27. Koller, B. H., and Orr, H. T.(1985) J. Immunol. 134, 2727-2733 [Abstract/Free Full Text]
  28. Koller, B. H., Geraghty, D. E., Shimizu, Y., DeMars, R., and Orr, H. T. (1988) J. Immunol. 141, 897-904 [Abstract/Free Full Text]
  29. Lenardo, M. J., and Baltimore, D.(1989) Cell 58, 227-229 [Medline] [Order article via Infotrieve]
  30. Levy, D. E., Kessler, D. S., Pine, R., Reich, N., and Darnell, J. E., Jr.(1988) Genes & Dev. 2, 383-393
  31. Levy, D. E., Kessler, D. S., Pine, R., and Darnell, J. E., Jr.(1989) Genes & Dev. 3, 1362-1371
  32. Marincola, F. M., Shamamian, P., Alexander, R. B., Gnarra, J. R., Turetskaya, R. L., Nedospasov, S. A., Simonis, T. B., Taubenberger, J. K., Yannelli, J., Mixon, A., Restifo, N. P., Herlyn, M., and Rosenberg, S. A.(1994) J. Immunol. 153, 1225-1237 [Abstract/Free Full Text]
  33. Maschek, U., Pulm, W., Segal, S., and Hammerling, G.(1989) Mol. Cell. Biol. 9, 3136-3142 [Medline] [Order article via Infotrieve]
  34. Maxam, A., and Gilbert, W.(1980) Methods Enzymol. 65, 499 [Medline] [Order article via Infotrieve]
  35. Miskimins, W. K., Roberts, M., McClelland, A., and Ruddle, F.(1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6741-6744 [Abstract]
  36. Miyamoto, M., Fujita, T., Kimura, Y., Maruyama, M., Harada, H., Sudo, Y., Miyata, T., and Taniguchi, T.(1988) Cell 54, 903-913 [Medline] [Order article via Infotrieve]
  37. Miyazaki, J.-I., Appella, E., and Ozato, K.(1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9537-9541 [Abstract]
  38. Moller, P., Hermann, G., Moldenhauer, G., and Momburg, F.(1987) Intl. J. Cancer. 40, 32-39 [Medline] [Order article via Infotrieve]
  39. Nelson, N., Marks, M. S., Driggers, P. H., and Ozato, K.(1993) Mol. Cell. Biol. 13, 588-599 [Abstract]
  40. N'Guyen, C., Sodoyer, R., Trucy, J., Strachan, T., and Jordan, B. R. (1985) Immunogenetics 21, 479-489 [Medline] [Order article via Infotrieve]
  41. Parrington, J., Rogers, N. C., Gewert, D. R., Pine, R., Veals, S. A., Levy, D. E., Stark, G. R., and Kerr, I. M.(1993) Eur. J. Biochem. 214, 617-626 [Abstract]
  42. Pine, R., Decker, T., Kessler, D. S., Levy, D. E., and Darnell, J. E., Jr.(1990) Mol. Cell. Biol. 10, 2448-2457 [Medline] [Order article via Infotrieve]
  43. Porter, A. C., Chernajovsky, Y., Dale, T. C., Gilbert, C. S., Stark, G. R., and Kerr, I. M.(1988) EMBO J. 7, 85-92 [Abstract]
  44. Radford, J. E., Jr., Chen, E., Hromas, R., and Ginder, G. D.(1991) Blood 77, 2008-2015 [Abstract]
  45. Reis, L. F. L., Harada, H., Wolchok, J. D., Taniguchi, T., and Vilcek, J.(1992) EMBO J. 11, 185-193 [Abstract]
  46. Sanderson, A., and Beverly, P.(1983) Immunol. Today 4, 211-213
  47. Schmidt, H., Gekeler, V., Haas, H., Engler-Blum, G., Steiert, I., Probst, H., and Muller, C. A.(1990) Immunogenetics 31, 245-252 [Medline] [Order article via Infotrieve]
  48. Schwartz, R. J., and Lee, T. C.(1992) Nucleic Acids Res. 20, 140 [Medline] [Order article via Infotrieve]
  49. Sen, R., and Baltimore, D.(1986) Cell 46, 705-716 [Medline] [Order article via Infotrieve]
  50. Singer, D. S., and Maguire, J. E.(1990) Crit. Rev. Immunol. 10, 235-257 [Medline] [Order article via Infotrieve]
  51. Soong, T. W., Oei, A. A., and Hui, K. M.(1991) Semin. Cancer Biol. 2, 23-33 [Medline] [Order article via Infotrieve]
  52. Tanaka, K., Issselbacher, K. J., Khoury, G., and Jay, G.(1985) Science 228, 26-30 [Medline] [Order article via Infotrieve]
  53. Travers, P. J., Arklie, J. L., Trowsdale, J., Patillo, R. A., and Bodmer, W. F.(1982) Monogr. Natl. Cancer Inst. 60, 175-180
  54. Treisman, R.(1987) EMBO J. 6, 2711-2717 [Abstract]
  55. Wallich, R., Bulbuc, N., Hammerling, G., Katzav, S., Segal, S., and Feldman, M.(1985) Nature 315, 301-305 [Medline] [Order article via Infotrieve]
  56. Watanabe, N., Sakakibara, J., Hovanessian, A. g., Taniguchi, T., and Fujita, T.(1991) Nucleic Acids Res. 19, 4421-4428 [Abstract]
  57. Wedrychowski, A., Henzel, W., Huston, L., Paslidis, N., Ellerson, D., McRae, M., Seong, D., Howard, O. M. Z., and Deisseroth, A.(1992) J. Biol. Chem. 267, 4533-4540 [Abstract/Free Full Text]
  58. Weisz, A., Marx, P., Sharf, R., Appella, E., Driggers, P. H., Ozato, K., and Levi, B.-Z.(1992) J. Biol. Chem. 267, 25589-25596 [Abstract/Free Full Text]
  59. Yano, O., Kanellopoulos, J., Kieran, M., Le Bail, O., Israel, A., and Kourilsky, P.(1987) EMBO J. 6, 3317-3324 [Abstract]
  60. Zachow, K. R., and Orr, H. T.(1989) J. Immunol. 143, 3385-3389 [Abstract/Free Full Text]
  61. Zijlstra, M., Bix, M., Simister, N., Loring, J., Raulet, D., and Jaenisch, R.(1990) Nature 344, 742-746 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.