From the
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.
The MHC
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-
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
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-
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
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-
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.
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.).
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.
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.
There is some
precedence for a constitutive transcriptional role for an ISRE binding
protein in the case of the
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.
We thank Judy Goetzke for assistance in preparation of
this manuscript and Dr. Brian Van Ness for a helpful critique.
(
)
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).
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.
(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).
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).
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 discuss these results in the context of
locus-specific and cell-type-specific regulation of the HLA class I
genes.
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.
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.
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.
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.