(Received for publication, March 26, 1997, and in revised form, May 1, 1997)
From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
The granzyme B gene is induced in cytotoxic T
lymphocytes in response to antigenic stimulation. Previous studies have
identified several distinct regions in the granzyme B promoter which
may be important in either the induction or the T cell specificity of
the gene. These regions contain the canonical transcription factor
binding sites AP1, cyclic AMP-responsive element (CRE), Ikaros, and
core-binding factor (CBF/PEBP2). Each protein binding site was
disrupted by site-directed mutagenesis to investigate its role in
granzyme B promoter function. Mutations were introduced alone, or in
various combinations, within the context of a 243-base pair promoter
fragment known to confer high levels of reporter gene expression.
Transfection assays revealed that all of the single binding site mutant
promoters were capable of sustaining moderate to high levels of
transcriptional activity in primary activated T lymphocytes, whereas
certain mutants were more impeded in a T cell clone. A quadruple mutant
promoter, with only the CRE binding site intact, showed background
expression levels. This drop in expression was found to be mostly due
to mutations in AP1 and the 3 CBF binding sites. Their close proximity
and requirement in promoter function suggest an important role for protein-protein interaction between these two factors.
In the immune system, the role of cytotoxic T lymphocytes (CTLs)1 is to identify virus-infected or transformed cells and destroy them. T cells recognize foreign antigen on target cells via T cell receptor complexes in the context of major histocompatibility molecules (1). This recognition event induces a variety of molecular signal transduction pathways that activate the cell to proliferate (2). The expanding population of T cells acquires killing potential through the de novo induction of a specific subset of genes that encode the proteins that constitute the killing machinery (3). One form of T cell-mediated killing involves the production of specialized granules that accumulate in the cytoplasm following activation. These granules contain the pore-forming protein perforin (4) and a number of cytotoxic proteinases (granzymes) (5). Upon contact of an activated T cell with a target cell displaying foreign antigen, the granules and their contents are directionally exocytosed into the intercellular junction in a calcium-dependent process. Target cells are believed to take up granule components in a process that is facilitated by perforin. The exact cytotoxic nature of granzymes remains unclear, but granzyme B has been implicated as a mediator of DNA fragmentation once inside the target cell, through the activation of CPP32-like proteinases (6).
Following T cell activation, granzyme B gene expression is induced at
the level of transcription, and its mRNA level peaks after 3 or 4 days. This pattern of expression correlates well with the onset of
potent killing potential under a variety of conditions and is a useful
marker of CTL activation in vivo (7). Previous studies have
indicated that transcriptional regulation may be controlled by
5-flanking promoter sequences (8). A series of promoter deletion
fragments ranging from 108 bp to 5 kilobases were examined for their
ability to confer transcriptional activity on a reporter gene following
transfection into T cells. It was found that two promoter fragments
consistently generated the highest reporter gene activity:
828 and
243. The 243-bp granzyme B promoter contains five regions in which we
have found evidence of transcription factor binding both in
vitro and in vivo. Several distinct regions of
protection were evident in DNase I footprinting experiments with
nuclear extracts derived from T cells. These coincide with consensus
sequence binding sites for two known ubiquitous transcription factors,
activating transcription factor/cyclic AMP-responsive element-binding
proteins (ATF/CREB) and activator protein-1 (AP1), and two
lymphoid-specific factors, core-binding factor (CBF/PEBP2) and Ikaros.
In vivo evidence that these sites are important in promoter
function was demonstrated by in vivo footprinting and DNase
I hypersensitivity analyses (9) in T cell lines and in activated
primary CD8+ T cells. Moreover, several of these
transcription factor binding sites are evolutionarily conserved between
the murine and human granzyme B promoters (10-12).
Several distinct signal transduction pathways are activated via the T cell receptor and costimulatory molecules. These signals are integrated in the nucleus in the form of a particular subset of activated transcription factors that converge on a specifically targeted promoter and activate transcription. By studying the specialized requirements for T cell-specific gene expression in terms of transcription factor necessity we can further understand the molecular events that are associated with T cell activation. To determine whether any of the above mentioned binding sites are required for granzyme B transcription, point mutations were systematically introduced into each, either individually or in various combinations, and were assessed for their effect on the 243-bp promoter. We analyzed our promoter mutations by transient transfection assays in two distinct populations of T cells, freshly activated primary lymphocytes and an IL-2-dependent T cell clone (MTL). Primary lymphocytes were activated by a variety of nonchemical stimulation conditions and then transfected. MTL cells constitutively express the granzymes, and transfections were performed without any additional stimulation other than IL-2. Whether this immortalized state of activation reflects the state of primary activation is unknown; however, our transfection studies in these distinct cell types revealed some differences. These experiments enabled us to observe the effects of removing each transcription factor from the native promoter complex and assess its importance in the regulation of the granzyme B gene.
Site-directed mutagenesis was performed using the SculptorTM in vitro mutagenesis system (Amersham Corp.). All mutations were confirmed by sequencing.
PlasmidsGranzyme B promoter fragments were obtained by
restriction enzyme digestion or polymerase chain reaction
amplification. They were inserted upstream of the promoterless
luciferase reporter gene of the basic pGL2 vector (Promega), and
orientation was confirmed by sequencing or directional primer
amplification. SV-gal contains the bacterial
-galactosidase gene
under the control of the SV2 viral promoter (Promega). Plasmids were
grown in DH5
Escherichia coli, and high quality
supercoiled DNA was purified for transfection by CsCl2
density gradient centrifugation. DNA concentration and covalently
closed circular content were determined by ethidium bromide
fluorometric analysis in a Sequoia-Turner model 450 fluorometer.
Primary splenocytes were obtained from 6-12-week old
BALB/c mice. Spleen tissue was ground through a fine wire screen in
RHFM/IL-2 medium, and the cells were pelleted. Red blood cells were
lysed with buffered ammonium chloride lysis buffer. The
IL-2-dependent cytotoxic T cell line MTL 2.8.2 was
generated from CBA/J mice as described (13). MTL 2.8.2 cells and
primary splenocytes were cultured in RHFM (RPMI supplemented with 20 mM HEPES (pH 7.5), 100 mM -mercaptoethanol,
and 10% fetal bovine serum) in the presence of 60 units/ml human
recombinant IL-2. Primary splenocytes were stimulated with 5 µg/ml
concanavalin A (ConA; Sigma) and 1:750 to 1:1,000 dilution (determined
empirically) of hamster anti-mouse
CD3 monoclonal antibody
supernatant.
Transient transfections were performed using
a DEAE-dextran transfection procedure optimized for cytotoxic T cells
(8). Primary splenocytes were cultured at 3.0 × 106
cells/ml in RHFM plus 60 units/ml IL-2, 1:750 dilution CD3, and 5 µg/ml ConA for 20 h prior to transfection. Basically, 1.0 × 107 exponentially growing cells (MTL) or 2.0 × 107 (whole splenocytes) were washed twice in serum-free
medium and resuspended in 1.0 ml of TBS (25 mM Tris-HCl (pH
7.5), 137 mM NaCl, 5 mM KCl, 0.6 mM
Na2HPO4, 0.7 mM CaCl2,
and 0.5 mM MgCl2 (pH 7.0)) with 500 µg/ml
DEAE-dextran (Sigma), 15 µg of covalently closed circular luciferase
reporter plasmid, and 5 µg of
-galactosidase control plasmid. The
DNA was adsorbed for 15 min at room temperature. Cells were washed
twice in serum-free medium and cultured at 2.5 × 105
cells/ml (MTL) or 2.0 × 106 cells/ml (splenocytes) in
RHFM + 60 units/ml IL-2 and incubated at 37 °C in 5%
CO2. Primary splenocytes were stimulated with additional
CD3 and ConA following transfection. The cells were harvested after
48 h, washed twice in phosphate-buffered saline, lysed in Triton
lysis buffer (1% Triton X-100, 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol), and luciferase and
-galactosidase
assays were performed.
Three aliquots of
cell lysates (10-20 µl) were measured for 20 s following the
injection of Luciferase Reagent (Luciferase Assay, Promega) by a LUMAT
LB9501 luminometer (Berthold Systems Inc.). -Galactosidase assays
were performed as described (14). Final activities are given as
luciferase/
-galactosidase values.
It was previously established by in vitro footprinting
that within the 243-bp granzyme B promoter, there existed five distinct regions that were protected from DNase I digestion (9). These regions
correspond to transcription factor binding sites for ATF/CREB, AP1,
CBF, and Ikaros, and specific DNA-protein interactions were observed as
in vivo footprints in activated CD8+ T cells and
MTLs. This region corresponds to a strong DNase I-hypersensitive site
that was only detectable in activated T cells. To determine whether any
of these binding sites were important for the high levels of reporter
gene expression observed from the 243-bp promoter, we constructed a
series of promoters with three nucleotide substitutions in each binding
site. This strategy conserved the spacing and the helical orientation
of the binding sites. The bp changes were primarily transversions in
the consensus sequence binding sites and were chosen on the basis of
previous methylation interference data, in vivo footprinting
data, and site-directed mutagenesis data collected either on the
granzyme B promoter or on similar binding sites in other gene promoters
(Fig. 1). In addition to the single binding site
mutations, various double mutant promoters were constructed, as well as
a quadruple mutant promoter in which the AP1, Ikaros, and both CBF
sites were abolished, leaving only the CRE intact. Each mutant binding
site, with the exception of the CRE and 5 CBF, was tested by
electrophoretic mobility shift competition assays for the inability to
compete for the wild type binding site with up to a 200 molar excess of
mutant probe (data not shown). Following the mutagenesis reactions each
construct was confirmed by sequencing and subcloned into a promoterless luciferase reporter gene plasmid. Plasmid DNA was purified by cesium
chloride density gradient centrifugation to obtain high quality
supercoiled DNA. The concentration and percentage of covalently closed
circular DNA was assessed by fluorometric analysis prior to every
transfection, and the covalently closed circular DNA content was not
less than 90% for any test plasmid used.
Mutational Analysis of the Murine Granzyme B Promoter in Stimulated Primary Lymphocytes
We have shown that activated CD8+
cells isolated by immunomagnetic separation were able to drive high
levels of reporter gene expression from the granzyme B promoter in
transient transfections. Typically, the T cell portion of a whole
splenocyte population expanded in culture to approximately 75%
following stimulation with CD3 or ConA for 3 days, as assessed by
fluorescence-activated cell sorter
analysis.2 The CD8+ population
proliferated and expanded to approximately 50% of the total culture,
whereas the CD4+ population remained constant at
approximately 25%. Most of the non-T lymphocyte population did not
survive in this type of culture, and we do not believe that they
contributed significantly to reporter gene expression observed from the
granzyme B promoter.
The promoter/luciferase plasmid series was transfected, along with a
-galactosidase control plasmid, into whole primary lymphocyte cultures that were stimulated for 1 day prior to transfection and for 2 days following. The cells were harvested and assayed for luciferase and
-galactosidase activities. The luciferase reporter gene activity was
significantly high in primary cells with the average relative light
unit (RLU) value for the full-length
828 to +68 promoter being over
2,000 and background (promoterless plasmid) being approximately 200 RLU. Also,
-galactosidase activities were very reproducible within
each transfection experiment.
The averaged corrected values for two independent primary cell
transfection experiments are shown in Fig. 2. These
cells were stimulated with CD3, ConA, and IL-2. The values on the
y axis represent the RLU of the luciferase assay divided by
the absorbance of the
-galactosidase assays × 1,000. Because
the
-galactosidase expression and incubation times of the assay
differ between each transfection, the corrected values are only
relative within a single experiment and are not compared between
experiments or cell types. The promoterless pGL2 basic vector served as
the negative control and gave luciferase values that were essentially
background. The next three bars depict the promoter deletion series
108 to +29,
169 to +29, and
243 to +68. As transcription factor
binding sites were added by increasing the length of the promoter to
243 bp, the luciferase activity increased substantially. The next eight
bars represent the mutant promoter series in the context of the 243-bp
promoter (
243 to +68). The largest effect for any single point
mutation was observed for the CRE binding site, which lowered total
promoter activity to 60% of wild type. A surprising finding was that
the mutation of the AP1 binding site appeared to have relatively little
impact on overall promoter activity (73% of wild type). Mutations in
the 3
CBF or Ikaros binding sites had slight negative effects on
transcription, whereas the mutation of the 5
CBF site had no effect on
promoter activity. Overall, none of the single binding site mutations
reduced activity to an extent where it was possible to conclude that
any one factor was necessary for the expression of the 243-bp granzyme
B promoter.
We then looked at the mutant binding sites in combination. Promoters
lacking either or both the 5 and 3
CBF binding sites consistently
showed normal expression in transient assays. The 5
CBF/Ikaros double
mutant contained the same binding site compliment as the
169 promoter
and was approximately as active. When the AP1, Ikaros, and both CBF
elements were all ablated the result was a very dramatic decrease in
overall activity, comparable to the expression observed from the
108
promoter. Therefore, no other elements distal to the CRE were involved
in promoter transcription.
Our primary
cell cultures were initially stimulated with a combination of three
potent activators of T cells (CD3, ConA, and IL-2) in an effort to
maximize granzyme B-luciferase reporter gene expression. This
treatment, however, may mask contributions to overall promoter output
by the individual transcription factors. The
CD3 antibody utilized
in these experiments binds to the
chain of the CD3/TCR complex
(15). This antibody, in combination with IL-2, is a sufficient stimulus
to generate potent cytolytic activity in primary lymphocyte cultures.
ConA is a widely employed mitogen that interacts nonspecifically with
surface glycoproteins and is capable of activating T cells through the
T cell receptor and likely through other glycosylated surface
receptors. At high doses, IL-2 activates primary T lymphocytes to
express perforin and granzyme B mRNAs, and these cells possess
cytotoxic potential (16, 17). Instead of using all three stimuli
simultaneously,
CD3, ConA, and IL-2 were added in culture
individually, or in combination, and the mutant promoter fragments were
transfected as above and assessed for reporter gene expression.
ConA alone or with added IL-2 appeared to be the best stimulus for this
type of culture as the cells proliferated well and expressed high
levels of luciferase activity from the granzyme B promoters. Fig.
3 depicts the average expression profiles of the wild
type and mutant granzyme B promoters in ConA- and ConA + IL-2-stimulated whole primary lymphocytes. In cells treated with ConA
alone (panel A), mutations in the CRE and AP1 binding sites
lowered overall promoter expression to approximately 60% of wild type.
The Ikaros mutation and either CBF binding site mutations were somewhat
inhibitory to promoter activity. Only the 4X mutant promoter, with
mutations in the AP1, Ikaros, and both CBF sites, significantly
decreased promoter activity to near background levels, similar to the
108-bp promoter fragment (data not shown). No major effect of any
individual binding site mutations was evident in these transfection
experiments with ConA stimulation alone.
Continuous exposure to 60 units/ml exogenous IL-2 in addition to ConA
did appear to enhance the effects of certain mutations slightly. Under
these conditions, mutations in the CRE and AP1 binding sites lowered
overall promoter activity to 54 and 40% of wild type, respectively,
the greatest extent observed in any of the primary cell transfections
(Fig. 3B). The Ikaros and 5 CBF were dispensable to
promoter function in these transfections, but the 3
CBF site may have
been somewhat important as the expression of this mutant was only
slightly higher than that of the CRE mutant.
Transfection experiments with cells stimulated with CD3 alone
resulted in a very good transfection efficiency, as determined by the
high levels of
-galactosidase expression from the control plasmid,
but very little granzyme B promoter activity was observed from any of
the mutant or wild type promoters (data not shown). The combination of
CD3 and IL-2 did stimulate the cells enough to detect significant
luciferase levels when approximately twice the normal number of cells
were transfected. The expression pattern was very similar to that
obtained with ConA stimulation. The CRE and AP1 sites were most
important, but neither mutation lowered promoter activity to a
significant extent, and the Ikaros and both CBF binding sites were
dispensable (data not shown).
Finally, primary lymphocytes were treated with 5,000 units/ml human
recombinant IL-2 and transfected with the longer 828-bp promoter after
20 h of initial stimulation. After further IL-2 treatment, no
luciferase activity and only trace amounts of -galactosidase activity were detectable after 1, 2, or 3 days post-transfection, and
the cells failed to proliferate at any significant rate. It is likely
that these cells were resistant to transfection and were unable to
express the reporter gene.
MTL 2.8.2 is an immortalized, IL-2 dependent, cytotoxic T cell clone that
constitutively expresses the endogenous granzymes. These cells were
transfected with the wild type and mutant promoter constructs and
assayed for expression (Fig. 4). The absolute luciferase values were higher in MTL than in primary cells with the RLU reading for the full-length 828-bp promoter being greater than 10,000. The pGL2
promoterless vector served as the negative control and was essentially
background. The activities of the minimal promoter deletion series are
depicted in the next three bars. Luciferase activity increased
proportionately as the length of the promoter was extended to 243 bp.
Of the five individual promoter mutants (next five bars), the CRE
binding site appeared to be the most important controlling factor (36%
of wild type activity). The AP1 site was relatively important as well
in that the promoter was transcribed at 64% of wild type levels in the
absence of AP1 binding. Mutation of the 3 CBF site had no effect on
the promoter, and the 5
CBF site had, if anything, a stimulatory
effect in both the single mutant and the double CBF mutant. Neither the 5
CBF or Ikaros mutation alone was inhibitory, but together they reduced expression to below that of the
169 promoter, to near background levels. This result was not observed in primary lymphocytes. As expected, the 4X mutant promoter was expressed at near background levels and was almost as low as its functional equivalent, the 108-bp
promoter. In MTL cells, the CRE was the most important individual
controlling element, and both the deletion analysis and the
site-specific mutagenesis data indicate an important role for Ikaros
and CBF in granzyme B expression.
AP1 In Conjunction with 3
It was observed
that when the AP1, Ikaros, and both CBF binding sites were abolished,
expression dropped to near background levels. However, neither of the
CBF sites appeared to be important for the observed high activity of
the 243-bp fragment. To determine whether the AP1 and Ikaros binding
sites were responsible for this dramatic loss of activity, a double
mutant promoter was constructed in which both the AP1 and Ikaros sites
were abolished. Additionally, when the AP1 and 3 CBF sites were added
to the
108 promoter (the
169 fragment), activity increased
substantially, indicating that these two factors may be very important
for promoter activity. A second double mutant promoter was constructed
in which both the AP1 and 3
CBF sites were abolished.
The reporter gene activity of these new mutant constructs was compared
with the wild type promoter, each single mutant, and the 4X mutant
promoter. The averaged values for four independent transfection
experiments in activated primary cells and two independent transfections in MTL cells are shown in Fig. 5. In
primary cells, the AP1 mutation appeared to reduce promoter activity to
61% of wild type, and Ikaros had no inhibitory effect (panel
A). The AP1 mutant was much less active in MTL cells (37% of wild
type), and some inhibition was observed for the Ikaros mutant
(panel B). When both the AP1 and Ikaros binding sites are
abolished, promoter expression was reduced to an extent in primary
cells (47% of wild type) and was approximately the same as the AP1
mutant in MTL (40%). When the AP1 and 3 CBF binding sites are
abolished, the promoter is still active in MTL cells (73%), despite
the absence of a functional AP1 binding site. However, expression is
reduced to background levels (20%) in primary cells, indicating that
together these sites are very important in conferring high
transcriptional activity. From this series of transfections we conclude
that the presence of functional binding sites for AP1 and the 3
CBF is primarily responsible for the high levels of expression observed from
the granzyme B minimal promoter in primary lymphocytes. MTL cells,
however, were primarily sensitive to mutations in the AP1 binding site
alone but not in combination with the 3
CBF.
The granzyme B gene is transcriptionally induced upon antigenic stimulation of cytotoxic T cells. We have previously identified several transcription factors that are involved in the induction or maintenance of an active granzyme B promoter and have shown that they bind to their cognate sequences in the endogenous promoter. In our present studies, primary murine lymphocytes stimulated via the T cell and IL-2 receptors were utilized as the principal experimental system. Results from transfection studies in this physiologically relevant system were compared with data obtained from transfections of the same reporters in a T cell clone. Primary lymphocytes express granzyme B mRNA in a stimulation-dependent manner. They require at least two independent signals for proliferation and CTL-specific gene expression. First, they are stimulated through the T cell receptor, by presented antigen, and second by IL-2 through the IL-2 receptor. MTL cells are different in that they are an antigen-independent, IL-2 dependent, cytotoxic T cell line (13). MTL 2.8.2 cells were selected for antigen independence by culturing in the presence of high levels of IL-2 and the phorbol ester PMA (18), and they constitutively express all of the granzyme genes.
It was apparent from our previous transfection studies that reporter
gene expression increased substantially as the length of the minimal
promoter increased in length from 108 bp, to 169 bp, and to 243 bp. The
shortest promoter included only the CRE binding site at 90. The next
largest fragment included one CBF binding site at
125 and an AP1 at
150. The 243-bp promoter included another CBF binding site at
180
and Ikaros at
200. Promoter expression increased substantially in
both primary lymphocytes and MTLs as more binding sites were appended;
however, promoter activity decreased as the fragment was extended to
402 bp. Therefore, our studies focused on the sites contained within
the 243-bp fragment.
The granzyme B CRE is a target for a ubiquitous family of basic leucine
zipper-containing transcription factors (19). These proteins may become
potent transcriptional activators following phosphorylation through the
cAMP-stimulated protein kinase A signal transduction pathway.
Stimulation through the T cell receptor results in increased levels of
intracellular cAMP and the subsequent activation of transcription
factors that are required for cAMP-responsive gene regulation. In the
context of the short 108-bp promoter, the granzyme B CRE did not have
significant transcriptional activation activity. However, it was quite
important when included in the 243 promoter as its mutation reduced
transcription to between 36 and 60% of wild type in T cells. Thus,
CREBs only exerted a substantial positive effect when linked in
cis with the other transcription factors.
The ubiquitous transcription factor AP1 is composed of heterodimers of
the fos and jun family of DNA-binding proteins. Upon T cell activation,
fos and jun are induced by de novo synthesis, and their
transcriptional activation potentials are stimulated by phosphorylation
(20). The DNA binding activity of AP1 is low or absent in resting T
cells (21). Upon stimulation through the T cell receptor, these
proteins were observed to form a strong specific complex with an
oligonucleotide probe containing the granzyme B AP1 binding site and a
distinct in vivo footprint (9). Overall promoter expression
in primary lymphocytes was lowered 73% to 61% of wild type when this
site was altered in the 243 promoter. MTL cells were quite sensitive
to mutations in the AP1 binding site (64% to 39% of wild type
expression). The results with MTL are similar to studies involving the
human granzyme B promoter where the AP1 binding site was found to be
essential for reporter gene expression in
12-O-tetradecanoylphorbol-13-acetate/dibutyryl cAMP-stimulated PEER cells (11). Analogously, the proximal AP1 binding
site in the IL-2 gene promoter was necessary for high levels of
reporter gene expression in both PMA-stimulated human peripheral blood
cells and PMA-stimulated Jurkat T cells (22). The MTL line was
developed from T cells treated with high levels of PMA and IL-2. The
difference between our findings in primary cells and previous data in T
cell clones may be explained by an inflated protein kinase C response
due to stimulation by phorbol esters.
Core-binding factor, also known as polyoma enhancer-binding protein
(PEBP2), is a heterodimer of an evolutionarily conserved family of
three subunits and a single
subunit (23-27). Following stimulation of T cells, the
-CBF subunit is believed to translocate from the cytoplasm into the nucleus where it augments the DNA binding
activity of a lymphoid-specific
-CBF subunit (28). Weak CBF DNA
binding activity was observed in resting T cells, but a distinct
complex was observed to bind to the granzyme B 3
CBF site in activated
T cells. Although both CBF sites in the granzyme B promoter displayed
prominent in vivo footprints in activated T cells, the
mutation of either or both CBF sites had little or no negative effect
on transcription in murine CTLs.
Ikaros is a zinc finger DNA binding protein that is expressed
throughout hemopoietic development and is essential for the differentiation of the lymphoid lineage of cells (29, 30). This protein
has been observed to bind in vitro to a nonconsensus DNA
binding site in the granzyme B promoter in both resting and activated
CTLs. The murine granzyme B Ikaros element is very similar to those
found in the human granzyme B promoter and the CD3 promoter. Mutations introduced into the Ikaros binding sites of both of these
promoters significantly abrogated their expression in T cells (12, 29),
whereas no negative effect was observed for mutations in the murine
granzyme B Ikaros element.
In theory, a promoter fragment in which the 5 CBF and Ikaros binding
sites are abolished should only be as active as the 169-bp fragment, if
they contained the same compliment of functional binding sites.
Similarly, a promoter in which the AP1, Ikaros, and both CBF binding
sites are abolished should reflect only the activity observed from the
108-bp promoter. In our primary cell transfection experiments the
expression of the 5
CBF/Ikaros double mutant was identical to that of
the
169 promoter. The expression of the 4X mutant promoter was
identical to the
108 promoter in both primary cells and in MTLs.
Therefore, we conclude that no transcription factors, other than the
ones we have identified, are playing a significant role in the
transcription of the minimal granzyme B promoter.
Primary lymphocytes must remain sensitive to a variety of signals so that inducible genes can be precisely controlled by subtly different stimuli. In contrast, cell lines are likely locked into invariably active signal transduction pathways. Recently, striking differences regarding the regulation of the IL-2 promoter were observed in transfection experiments performed in Jurkat T cells and primary lymphocytes, both activated with PMA + phytohemagglutinin. Several mutations were far more inhibitory to transcription in Jurkats than they were in primary cells (22). The most compelling differences between the results of our transfection studies performed in primary cells and those conducted in cell clones are that several transcription factors, such as AP1, CRE, and CBF, were not as important for transcription in primary cells as they have been reported to be under different conditions in cell lines (11, 12, 31). This may reflect the removal from normal growth and transcriptional control mechanisms which is typical of immortalized cell lines. MTLs were more sensitive to mutations in the CRE and AP1 binding sites than primary cells likely because they possess highly active forms of CREBs and AP1 subunits. Hence, the removal of these factors from the promoter complex would significantly affect expression.
These studies strongly suggest a functional interaction between the AP1
and CBF complexes. No other examples of interaction between these
factors have been identified, but functional cooperativity between
either AP1 or CBF and other transcription factors has been observed in
many other promoters (32-38). In most instances, the binding sites for
AP1 or CBF and another factor are separated by not more that 10 bp, and
both intact sites are required for optimal DNA binding by either
factor. This spacing appears to be critical as insertions of 5 or 10 bp
between binding sites destroy cooperative binding and function as a
unit. The granzyme B AP1 and 3 CBF sites are separated by 20 bp, and
both factors bind their respective elements independently of one
another. The removal of either AP1 or CBF from the promoter complex has
a limited negative effect, but the removal of both abrogates
expression. This could denote a form of protein-protein interaction
whereby the proteins associate in the absence of DNA binding, and only one binding site is required to tether both factors to the promoter. A
similar situation is exemplified by the endothelin-1 promoter where the
transcription factor GATA-2 potentiated AP1 activity despite the
absence of a functional GATA binding site and vice versa
(37). Similarly, the AP1 and GATA proteins were found to associate in
the absence of DNA. Alternatively, AP1 and CBF could serve as a docking
site for an adapter protein that requires at least one factor bound to
the promoter for association.
We have successfully identified all of the cis-acting
sequences that are responsible for the high levels of promoter activity observed from the 243-bp minimal promoter. These are the CRE, AP1, 3
CBF, 5
CBF, and Ikaros elements. T cell clones transcriptionally regulate granzyme B promoter expression in a manner that deviates from
primary lymphocytes. Clones are differentially sensitive to certain
mutations and thus may not contain the same compliment of transcription
factors as primary cells. Although no single factor was necessary for
expression, the combination of AP1 and CBF was essential for the
granzyme B promoter. Further studies are required to delineate the
nature of interplay between AP1 and CBF, but it is clear from our
studies that murine granzyme B expression is regulated by transcription
factors acting in concert.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M22526.
We thank Brenda Duggan for help and technical support and Irene Shostak for maintaining cultured cell lines.