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*
Hanson Center for Cancer Research, Adelaide, South Australia;
Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164; and
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B. By using a
mutant HMGI that cannot bind to DNA but can still interact with the
transcription factors, we found that DNA binding by HMGI was not
essential for the promotion of transcription factor binding. However,
the non-DNA binding mutant acts as a dominant negative protein in
transfection assays, suggesting that the formation of functional
HMGI(Y)-containing complexes requires DNA binding as well as
protein:protein interactions. The alteration of HMGI(Y) levels affects
IL-2 promoter activity not only in Jurkat T cells but also in PBL.
Importantly, we also show here that expression of the endogenous IL-2
gene as well as proliferation of PBL are affected by changes in HMGI(Y)
levels. These results demonstrate a major role for HMGI(Y) in IL-2
expression and hence T cell proliferation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel, NF-AT and AP-1 family
proteins (reviewed in Refs. 3 and 4). This
cascade of events results in the coordinate expression of a variety of
genes including the cytokine IL-2, which functions as a major growth
factor for T cells, and expression of IL-2R
, which allows the
formation of a high-affinity receptor for IL-2. The resulting autocrine
loop can act as a strong growth stimulus allowing a specific clonal
expansion of the T cell.
The requirements for activation of IL-2 gene expression in response to
T cell activation have been studied in detail. Initially, two major TCR
responsive regions were identified in the IL-2 promoter (reviewed in
Refs. 3 and 5). These were termed Ag response
elements (ARRE),4
i.e., ARRE-1 (or NF-IL-2A) and ARRE-2 (or NF-IL-2E). The ARRE-2 region
was the first to be identified as a composite binding site for NF-AT
and AP-1 proteins, whereas ARRE-1 was thought to function via the
binding of AP-1 and Oct transcription factors (6, 7, 8, 9, 10).
Recently, the ARRE-1 region has been found to bind a complex of NF-AT
and AP-1 as well as Oct (11). Several other regions of the
promoter have also recently been identified as binding sites for NF-AT
alone or as complexes with AP-1 (11). The mechanism of
costimulation through the CD28 receptor involves enhanced nuclear
expression of NF-B and c-Rel transcription factors
(12, 13, 14), as well as phosphorylation of c-Jun by JNK
kinase, resulting in increased transactivation competence for AP-1
(15, 16). The region of the IL-2 gene that responds to
CD28 (the CD28RR) is composed of a variant NF-B site (referred to as
the CD28RE), which has a high affinity for c-Rel-containing complexes,
as well as an adjacent AP-1 site. These elements cooperate to lead to
increased IL-2 promoter activity (17, 18, 19, 20).
The highly inducible nature of the IL-2 promoter seems to be the result of coordinate binding of many transcription factors to their recognition sequences on the promoter leading to the assembly of a functional unit (3, 4, 11, 14, 21, 22, 23, 24). Such a unit has been termed an enhanceosome on the IFN-ß promoter (25). The family of nuclear proteins known as high-mobility group I(Y) (HMGI(Y)) are known to play a major role in the assembly of the IFN-ß enhanceosome (26, 27, 28). We have previously shown that HMGI(Y) is essential for the selective binding of c-Rel to the IL-2 CD28RE and that modulating HMGI(Y) levels affects not only the activity of the CD28RR but of the entire promoter (14).
HMGI(Y) proteins are small nonhistone nuclear proteins that bind the
narrow minor groove of A:T sequence-rich B form DNA and have numerous
modes of action by which they modify gene transcription (reviewed in
Ref. 29). The HMGI(Y) family of proteins consist of three
members, HMGI and HMGY, which are produced by alternative splicing of
mRNA from the same gene locus, and HMGIC, which is coded for by a
related gene at a separate locus (29). These proteins have
been classed as architectural transcription factors because they do not
act as transactivators in their own right but modify the function of
other proteins (30). HMGI(Y) proteins can interact
directly with several families of transcription factors including
Rel/NF-B, bZip, Ets, homeodomain, and Pou domain proteins and lead
to an alteration in their DNA binding to sites that either overlap or
are adjacent to A:T-rich HMGI(Y) binding sites (29). The
binding of HMGI(Y) also induces structural changes in DNA substrates
(31, 32) that, in turn, often leads to alterations in the
assembly of transcription factors into higher order functional
complexes (29). In addition, HMGI(Y) appears to play a
critical role in chromatin architecture (33) and has been
shown to interact specifically with isolated nucleosome core particles
(34), to alter the rotational setting of DNA on the
surface of nucleosomes (35), and to antagonize H1-mediated
transcriptional repression (36, 37).
Here we show that HMGI(Y) can bind to numerous sites across the IL-2 proximal promoter region and modulate the binding of the major transcription factor families that are thought to control IL-2 gene transcription. Functional studies in both Jurkat T cells and primary T cells show that HMGI(Y) plays a major role in the regulation of the IL-2 gene and hence T cell proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The basic medium for Jurkat cell culture was RPMI 1640 medium containing 10% FCS, supplemented with L-glutamine and penicillin-gentamicin antibiotics (RPMIJ). The basic medium for PBL culture was as above but contained 20% FCS, 100 µM 2-ME, and 5% conditioned media (RPMIL). Mononuclear cells were isolated from peripheral blood using lymphoprep (Nycomed Pharma AS, Oslo, Norway) and cultured for 4 days in RPMIL and PHA (5 µg/ml) (Boehringer Mannheim, Mannheim, Germany). Cells were then stimulated with 5 ng/ml of IL-12 (PharMingen, San Diego, CA) for 4 h to promote transition to G1 and panned to remove macrophages. Nonadherent PBLs were pelleted and resuspended in RPMIL at 1 x 107 cells in 400 µl media. PBLs were assayed for CD3, CD4, and CD8 expression using the Cyto-Stat assay kit from Coulter (Palo Alto, CA). Ninety-four percent of the cells were CD3 positive, 74% were CD4 positive, and 20% were CD8 positive. Cells were transfected by electroporation using a Bio-Rad Gene Pulser II (Richmond, CA) at 290 V with a capacitance of 975 µF. Jurkat cells were resuspended at 5 x 106 cells in 400 µl media and electroporated at 270 V, 975 µF capacitance. The efficiency of transfection was determined using the pCMVGFP expression plasmid and flow cytometry (Epics XL-MCL; Coulter) and ranged from 2 to 4% green fluorescence protein (GFP) positive in PBLs and 15 to 25% GFP positive in Jurkat cells. Transfected cells were incubated in RPMIL with 10% conditioned media (PBLs) or RPMIJ media (Jurkats) for 24 h. Cells were then sorted for high fluorescence using a FACStarPlus cell sorter (Becton Dickinson, Mountain View, CA), and the top 1–2% for PBLs and 10–15% for Jurkats were collected and used in IL-2 ELISA and proliferation assays.
Reporter assay, ELISA, and proliferation assay
pRcCMV and pcDNA3.1/Zeo were obtained from Invitrogen (San
Diego, CA), and pRcCMVIGMH, pIL-2luc, and pHIVluc have been previously
described (14). Standard site-specific mutagenesis
procedures, the details of which will be reported elsewhere (Li et al.,
unpublished observations), were used to create a non-DNA binding mutant
form of the HMGI protein designated HMGI(mII,mIII) starting with the
wild-type human HMGI cDNA, clone 7C (38). Briefly,
HMGI(mII,mIII) had four proline to alanine substitutions introduced at
amino acid residues 57, 61, 83, and 87 located in its second and third
DNA-binding domains, the primary regions of the protein that interact
with the minor groove of A:T-rich substrates (39). As a
consequence, the recombinant HMGI(mII,mIII) protein lacks the ability
to specifically bind with high affinity to A:T-rich DNA sequences in
vitro but retains its ability for specific protein-protein interactions
with other transcription factors (Li et al., unpublished observations).
For expression in transfected mammalian cells, the mutant
HMGI(mII,mIII) cDNA was subcloned into the pcDNA3.1/Zeo plasmid vector
(Invitrogen) and used as described above. The HMGY cDNA was also
subcloned into pcDNA3.1 to generate pcDNAHMGY for expression of the
protein in cells. Jurkat cells used in the reporter assay were
cotransfected with 5 µg of pIL-2luc or pHIVluc together with
pRcCMVIGMH, pcDNAHMGI(mII,mIII), or pcDNAHMGY, in combination with the
amount of parent plasmid needed to normalize DNA at 10 µg. At
24 h posttransfection, cells were pelleted and resuspended in
phenol red free RPMIJ media at 1 x 106
cells/ml. Then, 100 µl of cells/well were plated in 96-well culture
plates and stimulated with 50 ng/ml PMA and 1 µM
Ca2+ ionophore (P/I) (A23187; Boehringer
Mannheim) or with the above and a 1/10,000 dilution of ascites fluid
containing activating Ab to the CD28 receptor (-CD28) (Bristol-Myers
Squibb, New York, NY) for 9 h. Cells were then harvested and
assayed for luciferase activity using a 96-well plate Luminometer
(TopCount; Packard, Meriden, CT) as previously described
(14). PBLs were cotransfected with 5 µg of pIL-2luc and
either 10 µg of pRcCMVIGMH, pcDNAHMGI (mII,mIII), or pcDNAHMGY and
the appropriate parent plasmid. At 24 h posttransfection, cells
were stimulated with PHA (5 µg/ml) for 16 h. Cells were then
harvested and cell extracts assayed for luciferase activity as
previously described (40).
ELISA and proliferation assays were performed with PBL or Jurkat cells
transfected with 10 µg of the appropriate expression plasmid together
with 5 µg of pCMVGFP for cell sorting. Then, 1 x
104 sorted cells in 100 µl were plated in
96-well tissue culture plates and stimulated with either PHA for
24 h (PBL) or P/I+-CD28 for 8 h (Jurkat), and supernatants
were assayed for IL-2 using a Quantikine ELISA kit (R&D Systems,
Minneapolis, MN). Proliferation was assayed in similarly treated cells
using the chemiluminescent bromodeoxyuridine incorporation kit from
Boehringer Mannheim.
Production and purification of recombinant proteins
Recombinant hexahistidine-tagged full-length Fos and Jun
proteins, as well as truncated Fos116–211 and
Jun224–334 proteins, were prepared and purified
by affinity chromatography using a Ni-NTA-agarose column (Qiagen,
Chatsworth, CA) as described (41). Fos and Jun proteins
were corenatured in vitro into the heterodimer transcription factor
AP-1 by step-wise dialysis from 6 M urea with the final buffer
containing 25 mM sodium phosphate, pH 7.6, 5% glycerol, and 5 mM DTT
(42). Full-length recombinant human HMGI protein (i.e.,
the unspliced member of the HMGI(Y) protein family; Ref.
29) was produced using the expression vector pET7C
carrying the wild-type human HMGI cDNA (38) as described
(43, 44). NF-ATp and c-Rel were prepared as described
(45, 46). The purity of each recombinant preparation was
assessed by SDS-PAGE (47) Protein concentrations were
determined spectrophotometrically employing either a Bio-Rad protein
assay kit or using the extinction coefficient
220 = 74,000 L/mol · cm for wild-type HMGI
protein (48).
Preparation of nuclear extracts
Nuclear extracts were prepared from Jurkat T cells stimulated with PMA (20 ng/ml), Ca2+ ionophore (1 µm), and CD28 Ab (1:10,000) for 1 or 6 h. Nuclear extract preparation was as previously described (14).
In vitro DNase I footprinting
A cloned 410 bp XhoI/HindIII restriction
fragment encompassing nucleotides (nt) -360 to +50 of the human IL-2
gene proximal promoter (49) was the starting fragment for
use in in vitro protein footprinting. Promoter subfragments were
isolated by either selective restriction enzyme digestion or PCR
amplification techniques (47). A fragment from -180 to
-60 was used for some of the footprinting experiments. Standard gel
electrophoretic procedures (47) were used to isolate all
DNA fragments followed by purification on a Qiagen column as described
by the manufacturer. Restriction enzyme fragments were 5'-end
radiolabeled with T4 polynucleotide kinase and
[
-32P]ATP. The ends of PCR fragments were
selectively radiolabeled by incorporating, during the final few cycles
of the amplification reaction, either one or the other of the two PCR
primers that had been 5' radiolabeled with T4 polynucleotide kinase and
[-32P]ATP.
Footprinting of both the HMGI and AP-1 recombinant proteins on promoter DNA fragments radiolabeled on one 5' end, employing the nuclease DNase I, followed published protocols (35, 44). For each protein and DNA substrate, optimal conditions for footprinting were empirically determined. Single-stranded DNA cleavage products were then separated by electrophoresis on a 6% sequencing gel with Maxam-Gilbert "G-lane" chemical cleavage products of control DNA fragments serving as reference standards (47). Band intensities were quantitatively analyzed using a PhosphorImager machine and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
EMSA
Oligonucleotides for gel shift analysis had the following sequences: IL-2 CD28RR, 5'-TGGGGGTTTAAAGAAATTCCAGAGAGTCATCAG-3'; CD28APm, 5'-TGGGGGTTTAAAGAAATTCCAGAGAGagATCAG-3'; CD28REm, 5'-TGGGGGTTTAAAGccATTCCAGAGAGTCATCAG-3'; IL-2 NF-IL-2A, 5'-AAGTCTTTGAAAATATGTGTAATATGTAAAACATTTTGA-3'; GM170, 5'-GATCCTGTAGGAAACAGGGGCTTGAGTCACTCCAG-3'; TRE, 5'-GTGGAATTCGGGGAAGTGACTCAGCGCTCGGACG-3'. The lower case letters represent the mutations introduced into the CD28RR to generate the AP mutant or the RE mutant. Double-stranded oligonucleotides were end labeled using T4 polynucleotide kinase (40). Affinity-purified recombinant NF-ATp and truncated Fos and Jun proteins were used in binding reactions with the NF-IL-2A or GM170 radiolabeled oligonucleotides (0.1 ng) in a buffer containing 20 mM HEPES, 5 mM KCl, 20 mM NaCl, 2 mm MgCl2, 2 mM DTT, and 5 µg purified BSA (New England Biolabs, Beverly, MA) (50). Protein:DNA complexes was resolved on a nondenaturing 5% polyacrylamide gel containing 0.5x TBE buffer (0.5 mM Tris, 42 mM boric acid, 1 mM EDTA, pH 8.3). Binding of recombinant proteins to the CD28RR was conducted in 10 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM EDTA, pH 7.5, 10 mM DTT, 0.2% Nonidet P-40, 1% glycerol, 0.4% sucrose, 0.5 mg/ml BSA, and 100 ng poly(dG:dC) in 20 µl reactions. A total of 0.2 ng of radiolabeled probe was generally used with the amounts of recombinant proteins indicated for individual experiments. Reactions were separated on 5% 0.5x TBE polyacrylamide gels. EMSAs for nuclear extract were conducted as previously described except that 5% glycerol was used instead of Ficoll and 0.5 mM PMSF was included. Competitor double-stranded oligonucleotides or Abs was added to the binding reactions at the concentrations indicated in individual experiments and incubated for 10 min before the addition of the radiolabeled probe. All gels were dried and exposed to x-ray film. Digital images were produced on a Fujifilm LAS1000 Plus CCD camera (Tokyo, Japan) and Image Gauge software (Fujifilm).
Western blot analysis
For Western blot, Jurkat cells were transfected, sorted, and stimulated as above, except 1 x 106 cells were recovered from the GFP-positive population. A similar analysis using PBLs was not feasible due to the low transfection efficiency of these cells and the large numbers of cells required for the assay. Nuclei were isolated from cells and HMG I(Y) proteins were extracted with 5% perchloric acid and precipitated with TCA (44). Proteins were separated on 18% denaturing polyacrylamide gels, and HMGI(Y) was detected by Western blot using a 1:250 dilution of a rabbit polyclonal anti-HMGI(Y) Ab (51). The enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL) was used, and the resultant bands were detected by x-ray film and scanned by densitometry (Bio-Rad).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that transcription from a transfected
IL-2 promoter reporter construct was inhibited by coexpression of
antisense RNA for HMGI(Y) proteins (14). A comparison of
the sensitivity of the promoters for a number of T cell-expressed genes
(IL-2, IL-3, GM-CSF, and the HIV long terminal repeat (LTR)) to HMGI(Y)
depletion found that IL-2 was the most significantly affected by
changes in HMGI(Y) levels (data not shown). The IL-2 proximal promoter
contains 
65% A:T base pairs and has many potential binding sites
for HMGI(Y) with A:T sequences present within or adjacent to several
known transcription factor binding sites. To identify the binding sites
for HMGI(Y) on the IL-2 promoter, DNase I footprinting was performed on
a fragment of the IL-2 promoter from nt -360 to +50 (Fig. 1
). DNase I footprinting was conducted on
both DNA strands by labeling each end of the DNA fragment
independently. Fig. 1A represents footprints obtained with
the DNA labeled at -360, and in Fig. 1B the DNA was labeled
at +50. As shown in Fig. 1, A and B, footprints
for HMGI(Y) were observed at a number of locations across the proximal
promoter (indicated by vertical bars and filled arrowheads). Fig. 1C shows the location of these HMGI(Y) binding sites in
relation to the sequence and the known transcription factor binding
sites on the promoter. Because different binding sites within the
promoter have different affinities for HMGI(Y) (data not shown), only
those sites that are consistently seen in independent replicate
experiments are indicated. The HMGI(Y) footprints are within or
adjacent to the -45 NF-AT/TATA-2 site (11), the NF-IL-2A
region, the NF-IL-2B region, the CD28 response region (CD28RR), and the
-285 NF-AT site in ARRE-2. All of these elements have been shown to be
important in IL-2 promoter activity (reviewed in Refs. 3
and 5). Interestingly, at the concentrations used in this
assay, HMGI(Y) was also observed to specifically footprint at the -30
TATA-1 sequence (Fig. 1), a region of the IL-2 promoter that is known
to be protected in vivo by proteins in both unstimulated and stimulated
T cells (21). As a control for these experiments, a
Fos/Jun (AP-1) heterodimer was also footprinted to the promoter DNA,
both alone and in combination with the HMGI(Y) protein (Fig. 1, A and B). AP-1 was used at two different
concentrations, which were empirically determined, in Fig. 1, A and B. As expected, at the high concentration
of input protein used in Fig. 1A, the Fos/Jun complex
altered the footprinting pattern across the entire promoter but
specific footprints were seen across known AP-1 sites in the promoter
region (indicated by vertical dashed lines), including the low-affinity
NF-IL-2A (ARRE-1) site located between nt -80 and -90. These specific
footprints were confirmed in Fig. 1B at lower concentrations
of AP-1. However, when both HMGI(Y) and Fos/Jun were combined together
in the reaction mixture, binding of Fos/Jun to the NF-IL-2A site and
the CD28RR was considerably reduced, and this is most clearly seen at
the higher concentrations of protein used in Fig. 1A (see
below).
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HMGI(Y) has previously been shown to promote or inhibit transcription factor binding to their regulatory elements overlapping or adjacent to the HMGI(Y) binding sites (reviewed in Ref. 29). We have previously shown that HMGI(Y) can modulate the binding of c-Rel to the CD28RR on the IL-2 promoter. We wished to determine whether HMGI(Y) affected the binding if other transcription factors to the IL-2 promoter. Several of the regions on the IL-2 promoter to which HMGI(Y) binds have been described as NF-AT and AP-1 binding sites (reviewed in Refs. 3 and 4). We chose to examine the effect of HMGI(Y) on NF-AT and AP-1 binding to two of these sites because of their distinct properties. The NF-IL-2A (ARRE-1) region has previously been reported to cooperatively bind NF-AT and AP-1 to form a higher order NF-AT complex on the NF-IL-2A region (11). The second site we examined, the CD28RR, has also been reported to bind NF-ATp (11, 46) and AP-1, but the binding sites are in a distinct configuration and cooperative binding is not observed (46).
At low protein concentrations, recombinant truncated Fos/Jun alone does
not stably bind to the low-affinity AP-1 site in the NF-IL-2A site in
EMSA (Fig. 2A, lane
1). However, the addition of recombinant HMGI(Y) resulted in the
appearance of an AP-1 band (Fig. 2A, lanes 2–5).
The intensity of the AP-1 band initially increased (up to 2 ng HMGI(Y))
and then decreased with the addition of increasing amounts of HMGI(Y)
(Fig. 2A). These results demonstrating that high
concentrations of HMGI(Y) inhibit Fos/Jun binding are consistent with
the previously described footprinting results obtained with varying
concentrations of these proteins (Fig. 1
A). Recombinant
NF-ATp protein was added to determine the effect of HMGI(Y) proteins on
formation of the NF-AT/AP-1 complex. Under the binding conditions used
here, NF-ATp did not bind alone to the probe and the formation of the
higher-order NF-AT complex did not occur (Fig. 2A,
lane 6). Addition of HMGI(Y) protein resulted in a
dose-dependent increase in formation of the NF-AT complex as well as
the previously observed increase in AP-1 binding (Fig. 2A,
lanes 7–10). At higher levels of HMGI(Y), the intensity of
the NF-AT complex did not significantly decline as was seen for AP-1
alone (Fig. 2A, lanes 7–10). The amount of AP-1
and NF-ATp used in these experiments was sufficient for strong binding
to the consensus sites contained in an oligonucleotide from the GM-CSF
enhancer (GM170) that can bind NF-ATp or AP-1 alone and also form a
higher-order complex (Fig. 2A, lanes
11–15). This oligonucleotide showed no binding of
HMGI(Y), and neither the binding of AP-1 or NF-ATp alone nor the
formation of the NF-AT higher-order complex were affected by addition
of HMGI(Y) at any concentration (Fig. 2A, lanes
11–15).
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B, lanes 1–3). An increase in AP-1 binding was
observed with the addition of HMGI(Y) (Fig. 2B, compare
lanes 2, 5, and 8 or lanes
3, 6, and 9), but relatively high levels of
HMGI(Y) were required to observe this effect (20 ng compared with 2 ng
for the promotion of AP-1 binding to the ARRE-1 region). We have
previously shown that HMGI(Y) promotes the binding of c-Rel to an
adjacent site (CD28RE) in the CD28RR (14). NF-AT proteins
can also bind to the CD28RR, and we have previously shown that HMGI(Y)
can promote truncated recombinant NF-ATp binding to the CD28RR
(46). Therefore, we examined whether HMGI(Y) could promote
the formation of an NF-ATp/AP-1 complex on this site. At the
concentrations used to promote AP-1 binding here (20 ng), the binding
of NF-ATp was in fact inhibited (Fig. 2B, lanes
10–13). Similar results were observed whether full-length AP-1
(Fig. 2C) or truncated AP-1 (data not shown) was used in the
binding reactions. These results show that HMGI(Y) can modulate the binding of transcription factors that are required for IL-2 promoter function to at least two major control regions of the IL-2 proximal promoter. However, it appears that the ratio of HMGI(Y) to AP-1 or NF-ATp may be important in determining the functional outcome of the interaction.
HMGI(Y) modulates nuclear AP-1 binding to the CD28RR
The CD28RR consists of an AP-1 binding site as well as a c-Rel
binding site (CD28RE). We have previously shown that the
c-Rel-containing complexes from activated T cell nuclear extracts
require HMGI(Y) to bind to the CD28RE (14). To determine
whether nuclear AP-1 was also dependent on HMGI(Y), we examined binding
of AP-1 from nuclear extracts of P/I/CD28-activated Jurkat T cells.
Because the Rel and AP-1 complexes migrate at the same position on EMSA
gels using the intact CD28RR (17), it was necessary to
generate EMSA probes with mutations in either the CD28RE (RE mutant) or
the AP-1 site (AP mutant). Binding of nuclear extracts to these probes
showed that an inducible complex could bind to the RE mutant in
extracts made both 1 and 6 h following stimulation with P/I/CD28
(Fig. 3A, lanes
4–6). These complexes migrated at the same position as the
complexes binding to the wild-type probe or the Rel-containing
complexes binding to the AP mutant (Fig. 3A, lanes
1–3 and lanes 7–9). The identity of the complexes
binding to the RE mutant was confirmed by competition experiments where
the RE mutant, the wild-type CD28RR, and a consensus AP-1 site (TRE)
were able to compete for complex formation but the AP mutant was not
(Fig. 3B). Thus, these complexes contain AP-1-like proteins.
To determine whether the formation of the AP-1-like complex was
dependent on HMGI(Y), an anti-HMGI(Y) Ab was added to the binding
reactions. The addition of the HMGI(Y) Ab to the binding reactions
reduced AP-1 binding to the RE mutant (Fig. 3C, lanes
1–3), as it did the c-Rel complexes binding to the AP mutant
(Fig. 3C, lanes 5–7). A control Ab (C) did not
affect complex formation (Fig. 3C, lanes 4,
8, and 11). We have previously shown that HMGI(Y)
Ab removes the inducible complexes containing c-Rel from the CD28RR but
does not affect NF-B complexes binding to a distinct NF-B site
(14). AP-1 binding to a TRE was not significantly reduced
by the addition of the HMGI(Y) Ab (Fig. 3C, lanes
9 and 10).
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D, lanes 7 and
8). In contrast, a dose-dependent increase in AP-1 binding
was observed with increasing levels of HMGI starting at 1 ng on the RE
mutant (Fig. 3D, lanes 9–16). We also observed
that HMGI(Y) no longer bound to the RE mutant (Fig. 3D),
probably because the RE mutant affects the A:T stretch within the
CD28RE to which HMGI(Y) most likely binds. These results 1) show that
AP-1 binding in nuclear extracts is influenced by HMGI(Y) and 2) imply
that the ability of HMGI(Y) to promote AP-1 binding is not dependent on
DNA binding by HMGI(Y). HMGI(Y) binding to DNA is not required for promotion of transcription factor binding to the CD28RR
The results above imply that the promotion of transcription factor
binding may not always require DNA:HMGI(Y) interactions. To test this
further, site-specific proline to alanine mutations were introduced
into the second and third DNA binding domains of HMGI (two
substitutions in each binding domain) to create a form of the protein
(designated HMGI(mII,mIII)) that could not specifically bind to A:T-DNA
under the assay conditions employed. We tested the ability of
HMGI(mII,mIII) to bind to the IL-2 promoter both in footprinting and in
gel shift assays. At concentrations of protein where recombinant HMGI
or HMGY bound specifically to the CD28RR, HMGI(mII,mIII) protein did
not bind (Fig. 4A). The same
result was obtained in footprinting assays across the IL-2 promoter
from -180 to -60 (Fig. 4B). Four footprints for HMGI were
observed across this region of the promoter (Fig. 4B,
lanes 2–5). In contrast, HMGI(mII,mIII) did not form
specific footprints across any of these regions (Fig. 4B,
lanes 6–9).
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We then investigated whether HMGI(mII,mIII) could alter transcription
factor binding when added to binding assays using the CD28RR together
with AP-1 and NF-ATp. The addition of HMGI(mII,mIII) lead to a
dose-dependent increase in AP-1 binding but did not inhibit NF-ATp
binding as did wild-type HMGI (Fig. 4C, lanes
10–15). We observed that HMGI(mII,mIII) generated a more
consistent dose dependence compared with the wild-type HMGI (Fig. 4C, compare lanes 4–9 and 10–15).
c-Rel binding to the CD28RR was also increased by HMGI(mII,mIII) (Fig. 4D, lanes 1 and 6–9). In addition,
inhibition of transcription factor binding sometimes seen at high
concentrations of HMGI was never observed with HMGI(mII,mIII). In
summary, these results show that HMGI binding to DNA is involved in the
inhibition of binding but is not required for the promotion of
transcription factor binding.
Alteration of HMGI(Y) expression results in the modulation of IL-2 promoter activity in both Jurkat and primary T cells
To determine whether HMGI(Y) had a functional role in IL-2 gene
transcription, HMGI(Y) levels were altered in the cell by antisense or
overexpression studies. The pRcCMVIGMH plasmid expressing antisense
HMGI was cotransfected into Jurkat T cells with pIL2luc or pHIVluc. The
HIV LTR responds to P/I and CD28 stimulation through NF-B sites that
closely match consensus sites and contain no potential binding sites
for HMGI(Y) (our unpublished observations).
As previously reported (14), the expression of antisense
HMGI RNA resulted in a 72% decrease in reporter activity in
P/I-treated Jurkat cells and a 75% inhibition in P/I+-CD28-treated
cells at the maximum dose of antisense expression plasmid (10 µg;
Fig. 5A). The pHIVluc reporter
transfections showed only a small reduction in promoter activity at the
maximum dose of antisense plasmid (Fig. 5A). To confirm that
the antisense HMGI RNA was reducing the level of HMGI(Y) protein in the
cells, a Western blot, using an anti-HMGI polyclonal Ab, was
performed on nuclear extracts from transfected cells either
unstimulated or P/I-stimulated following transfection. Densitometry
scanning of the blot showed that there was a significant reduction in
the levels of HMGI(Y) protein in the antisense-expressing cells (Fig. 5B). This experiment also showed that the amount of HMGI(Y)
in the nuclei of Jurkat T cells was increased almost 3-fold by P/I
activation (Fig. 5B).
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C). Transfection with pRcCMVIGMH
resulted in an 84% inhibition of the reporter activity in unstimulated
PBLs and an 81% inhibition in PHA-stimulated cells (Fig. 5C). These results show that the activity of a transfected
IL-2 promoter is dependent on HMGI(Y) in either a T cell line or in
primary T cells.
We also tested whether increased levels of HMGI(Y) protein affected
IL-2 promoter activity. Transfection of the pcDNAHMGY expression
plasmid into Jurkat T cells together with the pIL-2luc or the pHIVluc
increased luciferase activity 2-fold for IL-2 but had no effect on
HIV LTR activity (Fig. 6A).
Because the mutant HMGI(mII,mIII) protein also promoted transcription
factor binding, its effect in transfection assays was also tested.
Cotransfection of an expression plasmid for the HMGI(mII,mIII) protein
resulted in significant inhibition of the IL-2 promoter (60% in P/I
and 73% in P/I+-CD28-treated cells; Fig. 6B) at the
highest amounts of transfected plasmid. The pHIVluc reporter plasmid
showed no loss of reporter activity with expression of the
HMGI(mII,mIII) protein (Fig. 6B). Thus, this non-DNA binding
mutant appears to act as a dominant negative protein for IL-2 gene
transcription.
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IL-2 production and primary T cell proliferation are dependent on HMGI(Y)
It is possible that the transfected IL-2 promoter may be more
highly dependent on HMGI(Y) than its endogenous counterpart because of
the likely differences in chromatin configuration on chromosomal and
plasmid DNA. To monitor the effect of HMGI(Y) on expression from the
endogenous IL-2 gene, the amount of IL-2 protein secreted into the
supernatant of cells transfected with pRcCMVIGMH and
pcDNAHMGI(mII,mIII) was assayed by ELISA. To enrich for transfected
cells, 5 µg of pCMVGFP was cotransfected with the HMGI plasmids
above, and the cells were subsequently sorted for high level GFP
expression by FACS. The sorted cells were then stimulated with either
PHA for PBLs or P/I+-CD28 for Jurkat cells to induce IL-2
expression. In PBLs and Jurkats, significant inhibition of IL-2
production was detected in cells transfected with RcCMVIMGH (78 and
88%, respectively) or pcDNAHMGI(mII,mIII) (47 and 55%, respectively)
(Table I). These results imply that the
role of HMGI(Y) may be extended to endogenous IL-2 gene transcription
in both Jurkat T cells and primary T cells.
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, A
and B). To test whether increased HMGI(Y) levels also
altered proliferation, PBLs were transfected with the pcDNAHMGY plasmid
and pCMVGFP to allow for sorting of transfected cells. Overexpression
of HMGY in PBLs increased proliferation of these cells by 2-fold
(Fig. 7C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain genes is an
essential component of the formation of the autocrine loop that drives
T cell proliferation and clonal expansion following an immune stimulus.
We have shown here that HMGI(Y), a protein involved in regulating
promoter architecture, is required for IL-2 promoter activity and T
cell proliferation. This requirement for proliferation appears to stem
from the involvement of HMGI(Y) in controlling transcription from both
the IL-2 and IL-2R-chain genes in response to mitogenic stimuli. It
has previously been shown that HMGI(Y) participates in the inducible
expression of the human IL-2R gene by facilitating the assembly of
multiprotein, enhanceosome-like complexes on both an upstream enhancer
element that has binding sites for HMGI(Y), Elf-1 (an Ets family
protein), Stat5, and a GATA family protein (52) and on the
proximal promoter element that contains binding sites for HMGI(Y),
Elf-1, and NF-B proteins (53). Likewise, the present
results obtained from both in vitro footprinting, EMSA, and
transfection experiments strongly suggest that HMGI(Y) facilitates the
formation of a functional multiprotein complex consisting of a number
of different transcription factors on the proximal promoter of the IL-2
gene during transcriptional activation in vivo. Here, and elsewhere (14), we have demonstrated that HMGI(Y) modulates the binding to the IL-2 promoter of NF-AT, AP-1, and c-Rel; three transcription factor families that play important roles in IL-2 promoter activity. This has been shown both for recombinant proteins and proteins present in nuclear extracts. In each case, the transcription factor binding site constitutes a nonconsensus element that differs markedly in sequence from the high-affinity consensus sites. This is generally, but not always, because of the presence of the A:T sequences required for HMGI(Y) binding; consequently, these sites are weakly recognized by their cognate transcription factors. Therefore, HMGI(Y) serves to lower the threshold at which transcription factors can bind to and activate these weak sites. The presence of these nonconsensus sites in the IL-2 promoter appears to be important for the T cell-restricted expression of the promoter. It has been shown that mutation of some of these sites to consensus high-affinity sites weakens the induction dependence or T cell specificity of the promoter (54, 55, 56). There is no evidence that there are T cell-restricted members of the transcription factor families that may have selectivity for the IL-2 promoter sites. Instead, it is likely that the weak interactions together with the need for proteins such as HMGI(Y) and cooperative binding of many of the protein complexes is a requirement to generate these characteristics. Thus, HMGI(Y) may play an indirect role in the T cell specificity and induction dependence of the IL-2 promoter.
The role of HMGI(Y) in the assembly of a functional enhanceosome has
been best studied for the IFN-ß promoter (57). It has
been shown that HMGI(Y) promotes the coordinate binding of members of
the NF-B, activating transcription factor, and IFN- regulatory
factor families of transcription factors to the IFN-ß promoter
leading to the assembly of a functional complex known as an
enhanceosome (57). This enhanceosome is then thought to
recruit coactivator complexes such as CREB binding protein to lead to
chromatin reorganization and transcription activation
(58). It has recently been shown that the recruitment of
transcription factors to the IFN-ß promoter requires the binding of
HMGI(Y) to DNA and that HMGI(Y):transcription factor interaction is not
crucial for this recruitment (59). The IFN-ß promoter
DNA contains an intrinsic bend that is straightened by HMGI(Y) binding,
and this is important to allow transcription factor binding
(27). However, HMGI(Y):transcription factor interactions
are required for the completion of the enhanceosome assembly process on
the IFN-ß gene (59). We have found here that binding of
either c-Rel or c-Fos/c-Jun to the CD28RR of the IL-2 promoter does not
require HMGI(Y) binding to DNA. We have shown this by using either a
mutant of the CD28RR that can no longer bind HMGI(Y) or a mutant HMGI
protein that does not bind DNA specifically. However, the non-DNA
binding mutant of HMGI can still interact with the NF-ATp and AP-1
transcription factors and may promote transcription factor binding by
protein:protein interactions. The intrinsic structure of the IL-2
promoter region is not known but it is possible that the promoter DNA
does not require structural alteration to allow transcription factor
binding. However, the HMGI(mII,mIII) non-DNA binding mutant acted as a
dominant negative protein in transfection assays. This result implies
that correct DNA binding as well as protein recruitment is important
for the assembly of a functional complex. When expressed at
sufficiently high levels, the dominant negative mutant may compete with
wild-type protein in the cells to form nonfunctional complexes. Thus,
it is likely that for both the IL-2 and IFN-ß promoters, HMGI(Y)
needs to both bind to DNA and interact with transcription factors to
generate a functional complex, although the order of events may
differ.
High concentrations of HMGI appear to inhibit the binding of certain transcription factors at specific sites, e.g., AP-1 to the NF-IL-2A site and NF-ATp to the CD28RR. This inhibition was dependent on DNA binding by HMGI and would appear to be a direct competitive effect. It has previously been shown that HMGI(Y) can inhibit NF-AT binding to the IL-4 promoter (60, 61). In the case of IL-4, this inhibition of DNA binding translates into the ability of increased HMGI(Y) to inhibit IL-4 promoter function (61). In contrast, our results strongly suggest that HMGI(Y) is an activator of the IL-2 promoter. The data that support this conclusion are 1) antisense HMGI RNA expression strongly inhibits IL-2 promoter activity in both Jurkat T cells and PBLs, 2) increased expression of HMGI, either in response to P/I activation or by cotransfection of an expression plasmid, correlates with increased IL-2 promoter activity, and 3) production of IL-2 from the endogenous IL-2 gene is inhibited by antisense HMGI RNA in both Jurkat T cells and PBLs. These experiments not only show that a promoter in the context of a reporter plasmid, but also the endogenous gene, in both a T cell line and normal T cells is dependent on HMGI(Y) for activity. The ability of HMGI(Y) to promote transcription factor binding may be an important aspect of the positive function of HMGI(Y) on the IL-2 promoter. Whether inhibition of transcription factor binding is an artifact of high concentrations of HMGI or plays a role in the removal of inhibitors or down-regulation of the promoter following activation remains to be determined. One possible explanation for the results observed here is that at high concentrations of HMGI(Y) following T cell activation NF-ATp is displaced by HMGI(Y), which then promotes the binding of c-Rel. The appearance of NF-ATp in the nucleus (4) at early times following activation is likely to proceed the increased levels of c-Rel that requires new protein synthesis (12). A time-dependent change in the proteins that bind to the CD28RR may play a role in correct activation.
We have previously shown that the GM-CSF promoter (14) and the IL-3 promoter (our unpublished observations) are dependent on HMGI(Y) for activity. All of these cytokines are expressed following a primary T cell activation, before the development of effector T cell function (62, 63). In contrast, IL-4 requires at least three cell divisions before expression is detected (64, 65) and is the hallmark of the Th2 effector phenotype (62, 63). Signaling from the IL-4 receptor has also been shown to lead to phosphorylation of HMGI(Y) and a consequent reduction in DNA binding (66). Because IL-4 activation of T cells leads to increased IL-4 synthesis, it is intriguing to speculate that the phosphorylation of HMGI(Y) and its removal from the DNA may be one mechanism by which this increased IL-4 production is achieved (61). The same signaling pathway may consequently decrease IL-2 production. It may be of interest to examine the role of HMGI(Y) in expression of other Th1- or Th2-specific cytokines and to test the consequences of different levels of HMGI(Y) on the expression of these cytokines and the development of the T cell subtypes.
Alteration of the level of HMGI(Y) in PBLs leads to an effect on
proliferation of these cells. It would appear that the expression of
antisense HMGI RNA does not have a general effect on the cell cycle
machinery because antisense HMGI RNA expression had little effect on
the proliferation of Jurkat T cells. This change in proliferation in
PBLs is likely to be the result of changes in IL-2 expression in these
cells, although we have not definitively proven this link. Attempts to
rescue the proliferative defect in HMGI antisense RNA-expressing cells
lead to only a small reversal of this inhibition. This may be due to
the fact that the expression of the IL-2R-chain gene is also
controlled by HMGI(Y) as discussed above. The IL-2R-chain is
required for high-affinity IL-2 receptor complex formation (reviewed in
Ref. 67). Indeed, the PBLs expressing antisense HMGI RNA
had a reduced level of IL-2R-chain on the cell surface as measured
by FACS analysis (data not shown).
We have found here that HMGI(Y) levels increase in Jurkat T cells in response to P/I treatment. PMA treatment has previously been shown to increase HMGI(Y) levels in other cell types (68, 69), as have other signals such as exposure to growth factors such as serum (70, 71) and epidermal growth factor (72) or proinflammatory agent such as endotoxin and IL-1ß (73). The increase observed in HMGI(Y) levels may directly translate into increased promoter activity mediated by HMGI(Y) interactions with the other inducible transcription factors such as c-Rel or AP-1.
The results presented here show that the architectural transcription factor HMGI(Y) is critical for the correct regulation of IL-2. The finding that HMGI(Y) not only affects the function of a transfected IL-2 promoter but also the endogenous IL-2 gene and T cell proliferation implies that the level of HMGI(Y) in cells is a critical determinant of IL-2 gene activation and that modulating such levels either by physiological or pharmacological means may provide a means of modulating the immune response.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology, University of Queensland, Brisbane, Australia.
3 Address correspondence and reprint requests to Dr. M. Frances Shannon, Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra ACT 2601, Australia. E-mail address:
4 Abbreviations used in this paper: ARRE, Ag response element; HMGI(Y), high-mobility group protein I or Y; CD28RR, CD28 response region; CD28RE, CD28 response element; P/I, PMA and calcium ionophore; LTR, long terminal repeat; GFP, green fluorescence protein.
Received for publication September 22, 1999. Accepted for publication January 4, 2000.
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) or P/I+
) before luciferase assays were performed.
Luciferase activity is expressed as counts per second (CPS) readings
from the Packard TopCount luminometer. B, Western blot
showing the decrease in HMGI(Y) levels in cells expressing antisense
HMGI. Cells were transfected with either pRcCMV (lanes 1
and 3) or pRcCMVIGMH antisense expression plasmid
(lanes 2 and 4). Protein was extracted
from nuclei of either unstimulated (lanes 1 and
2) or P/I-stimulated (lanes 3 and
4) cells, resolved on SDS polyacrylamide gels, and
HMGI(Y) was detected by Western blot. Recombinant HMGI protein
(lane 5) was also loaded as a reference. Numbers below
represent the densitometry quantitation of HMGI(Y) protein bands.
C, PBLs, pretreated to generate blasts, were transfected
with the pRcCMV or pRcCMVIGMH plasmids, and the pIL-2luc reporter
plasmid and luciferase activity was measured either before (
