(Received for publication, July 5, 1995)
From the
T cell expression of interleukin 3 (IL-3) is directed by positive and negative cis-acting DNA elements clustered within 300 base pairs of the transcriptional start site. A strong repressor element, termed nuclear inhibitory protein (NIP), was previously mapped to a segment of the IL-3 promoter between nucleotides -271 and -250. Functional characterization of this element demonstrates that it can mediate repression when linked in cis to a heterologous promoter. DNA binding experiments were carried out to characterize the repressor activity. Using varying conditions, three distinct complexes were shown to interact specifically with the NIP region, although only one correlates with repressor activity. Complex 1 results from binding of a ubiquitous polypeptide that recognizes the 3` portion of this sequence and is not required for repression. Complex 2 corresponds to binding of transcription factor (upstream stimulatory factor) to an E-box motif in the 5` portion of the NIP region. DNA binding specificity of complex 3 overlaps with that of upstream stimulatory factor but is clearly distinct. To determine which of the latter two complexes represents NIP activity, we incorporated small alterations into the NIP site of an IL-3 promoter-linked reporter construct and examined their effects on NIP-mediated repression. Functional specificity for repression matches the DNA binding specificity of complex 3; both repressor activity and complex 3 binding require the consensus sequence CTCACNTNC.
Human interleukin 3 (IL-3) ()is a potent growth
factor, which supports early hematopoietic progenitor cells and
potentiates lineage-specific effects of later acting growth
factors(1, 2) . IL-3 expression is restricted to
activated T cells and NK cells (3, 4, 5, 6) and is regulated
primarily at the level of
transcription(7, 8, 9) . cis-acting
promoter elements governing tissue-specific and activation-dependent
expression are found within 300 base pairs (bp) of the transcriptional
start site (8, 9, 10, 11, 12, 13) .
Promoter deletion experiments have identified two activating sites
referred to as ACT-1 (NFIL-3) and
AP-1/Elf-1(8, 9, 10, 12) . In
addition, there is a powerful repressor site, which binds an activity
termed nuclear inhibitory protein (NIP)(9) . Functional
experiments in transfected T cells localized the NIP element between
nucleotides -271 and -250 upstream of the transcriptional
start(9) . These experiments showed that, in the absence of
AP-1 and Elf-1 sites, the NIP element blocks activation mediated by the
downstream ACT-1 and CBF sites, thus silencing IL-3 expression in
activated T cells(9) .
The nature of the NIP repressor has remained elusive subsequent to this initial characterization. Studies with the human interleukin 2 (IL-2) promoter identified a repressor site, NRE-A, which interacts with a zinc finger DNA binding protein(14) . Since the promoters of the IL-2 and IL-3 genes have common characteristics and function exclusively in activated T cells(5) , it was possible that NIP and NRE-A sites might be related. A second possible identity for NIP was suggested by sequence analysis, which revealed that the NIP site contains a consensus E-box sequence (15, 16) that might be recognized by a helix-loop-helix transcription factor.
To better define how NIP interacts with the IL-3 promoter and to characterize the protein mediating repressor activity, we have determined the nucleotide requirements for NIP function. In parallel we have investigated three proteins that bind to the repressor site to determine which correlates with the repressor activity of NIP. Taken together, our results show that NIP and NRE-A are distinct elements and that the repressor protein NIP does not belong to the E-box binding class of transcriptional regulators.
Figure 2:
Sequence of wild-type and NIP
oligonucleotides and other probes used in EMSA. Only the top strand is indicated. Wild-type nucleotides are shown in capitalletters, mutations in boldface.
Nucleotides added for the purpose of labeling duplexes using Klenow
polymerase are shown in lowercase. Numbering of the
nucleotides is relative to the start site of the IL-3
gene(18) . Sequences used for non-NIP oligonucleotides were
obtained from published reports: E2(21) , USF site in the
adenovirus major late promoter(29) . The region corresponding
to the E-box in the various oligonucleotides is shaded.
Figure 1:
The NIP repressor site functions in a
different position in the IL-3 promoter and in a heterologous promoter.
A schematicdiagram of the wild-type IL-3 promoter is
shown at the top. Reporter genes indicated on the right were transfected into MLA 144 cells, and expression of these
constructs was monitored by RNase protection using a modified IL-3
first exon probe(9) . The filledarrowhead indicates a 226-nt protected fragment resulting from expression of
the reporter gene(9) . The prominent 151-nt fragment (in the lowerpart of the gels) results from protection of
the probe by gibbon endogenous IL-3 mRNA and serves as internal control
for loading(9) . A, constructsa and b contain a truncated portion of the IL-3
promoter(-173), containing the known regulatory sites
ACT-1(10) , CBF(11) , and DB1 (13) and other
potential sites (CK1, CK2, and CACC)(9, 11) . In constructb, the NIP segment was fused to the
-173 promoter, thus altering the position of the NIP site
relative to the wild-type promoter. B, in constructsc and d, the IL-3 promoter was replaced by a
GM-CSF promoter fragment(-215) known to direct regulated
expression of GM-CSF in activated T cells(28) . Constructd contains the NIP site upstream of the -215 GM-CSF
promoter. Lanea, -173/IL3; laneb, (NIP)-173/IL3
; lanec, -215GM/IL3
; laned,
(NIP)-215GM/IL3
.
Figure 3: The NIP site binds three distinct complexes. Nuclear extracts from unstimulated (panelsA, B, and C) and from stimulated MLA 144 cells (panelD) were tested in EMSA with the probes indicated at the bottom of each panel. Two different unstimulated MLA 144 nuclear extracts were used in panelsA and B. Buffer A was used for panelsA-C, and buffer B was used for panelD. All probes are listed in Fig. 2. Competitors are listed above each lane. Identical results were obtained with nuclear extracts from unstimulated or stimulated Jurkat cells (data not shown). C1, complex 1; C2, complex 2; C3, complex 3.
To analyze formation of complexes 1 and 2 over the NIP region, NIP-3` and NIP-5` probes (Fig. 2) were used in EMSA (Fig. 3, B, C, and D). The NIP-3` probe specifically bound complex 1 (Fig. 3B), while the NIP-5` probe failed to do so (Fig. 3C). Instead, NIP-5` bound complex 2. Complex 2 is specific and often gives rise to two bands, one major band and a slightly faster migrating minor band. The two bands exhibit identical DNA binding specificity in EMSA competition experiments ( Fig. 5and data not shown).
Figure 5: Binding specificity of complex 2 is identical to the E-box consensus. Nuclear extract from unstimulated MLA 144 cells was used in an EMSA with the NIP-5` probe. The binding reaction was performed in buffer A in the absence or presence of various competitors, as indicated above each lane. These conditions preclude detection of complex 3 (see Fig. 3, C and D). Sequences of the competitors used are given in Fig. 2. Identical results were obtained with nuclear extracts derived from Jurkat cells. The deduced binding consensus for complex 2 is shown at the bottom.
To maximize
detection of all proteins binding to the NIP region, we carried out
EMSAs under a variety of different conditions. There was a striking
difference in the pattern of complexes detected when
poly(dA)poly(dT) was used as nonspecific carrier DNA in place of
poly(dI-dC). As shown in Fig. 3D, an additional complex
(complex 3) was detected using the 5` portion of the NIP sequence,
which migrated differently from complex 2 described above. The
assignment of the slower migrating band as complex 2 is based on
competition experiments using a variety of mutated probes ( Fig. 2and 8 and data not shown). The faster migrating band in Fig. 3D is not consistently seen in our different
nuclear extracts (see Fig. 8), and it cannot be fully competed
by excess unlabeled oligonucleotide. Thus, three distinct, specific
complexes (complexes 1, 2, and 3) are reproducibly detected over the
NIP site.
Figure 8: Binding of complex 3 correlates with repression function. Nuclear extracts from various cell lines were tested in EMSA using the probes shown above each lane. Sequences for the various probes are listed in Fig. 2. A, unstimulated Jurkat T cells. B, the source of nuclear extract is indicated for each panel. Only the area of interest is shown. Migration of complex 2/USF and of NIP-C3 is indicated for each panel. stim., stimulated; unstim., unstimulated.
Figure 4:
Complex 1 is not involved in repression. A, nuclear extracts from unstimulated Jurkat cells were used
in an EMSA with probes shown at the bottom of the gels (NIP probe and mutant NIP- probe (see Fig. 2). The
presence or absence (No) and the type of competitor
oligonucleotides are indicated above each lane.
Identical results were obtained with extracts from stimulated Jurkat
cells or extracts from MLA 144 cells before and after T-cell activation
(data not shown). B, the functional consequence of the 4-bp
deletion (NIP-
) engineered in the NIP site was tested in MLA 144
cells as described in Fig. 1. Expression of the IL-3 reporter
gene (9) mediated by a wild-type IL-3 promoter fragment
containing the NIP site (-267/IL3
) and by
the same fragment containing the NIP-
mutation was compared with
that obtained with a promoter fragment lacking a full NIP site (-250/IL3
). The level of expression of this
latter construct is equivalent to that of -173/IL3
(data not shown). The protected fragment corresponding to the
reporter gene is indicated by the arrowhead. As in Fig. 1, the prominent 151-nt fragment at the bottom of the gel
(resulting from protection by endogenous IL-3 mRNA) indicates equal
loading for RNA.
The NIP- mutation was incorporated into the -267/IL3
reporter construct to determine
whether loss of complex 1 binding correlated with a change in repressor
activity at the NIP site. Its activity was compared with -267/IL3
(wild-type), which shows
repression of the reporter gene, and a construct in which the NIP site
has been deleted, -250/IL3
(NIP
minus), which shows no repression. As displayed in Fig. 4B, the 4-bp NIP-
deletion had no effect on
repression function. Thus, complex 1 does not correlate with NIP
activity, and its binding is not required for repression to take place.
Figure 6:
Complex 2 is related to or identical to
USF. A, nuclear extracts from unstimulated Jurkat cells were
tested in EMSA with a NIP-5` probe in buffer A. The nuclear extract was
either directly added to the binding reaction (firstlane), incubated with an antibody raised against the 20
C-terminal amino acids of USF (laneUSF-Ab), or
incubated with the same antibody blocked with excess peptide against
which the antibody was raised (lane
USF-Ab + peptide) prior to the addition of the probe. B, the same
experiment was performed except that a USF binding site derived from
the adenovirus major late promoter (see Fig. 2) was used as a
probe. Experiments in panelsA and B were
run on the same gel. As apparent from the intensity corresponding to
free probe, exposure time for panelA was longer than
for panelB. C, binding of purified USF
(from HeLa cells) to NIP-5` and USF probes. The two reactions were run
on the same gel. The lane with NIP-5` as a probe was exposed
longer.
Figure 7:
The
functional consensus for NIP repression is different from the E-box
consensus. Reporter gene constructs with different mutations in the
-267 IL-3 promoter fragment (see Fig. 2for sequences)
were assayed as described in Fig. 1. Migration of the protected
fragment derived from expression of the reporter gene is indicated by
the arrowhead. Plasmid -173/IL3 (lane labeled NIPminus), which lacks
the NIP site, and plasmid -267/IL3
(lane labeled wild-type) containing an intact NIP element
served as controls. Equal loading for RNA was achieved as indicated by
the prominent 151-nt fragment at the bottom of the gel. Results from the eight mutants shown here and three
additional mutants (data not shown) define the consensus for NIP
repressor function indicated at the bottom.
The IL-2 promoter, which also directs T cell-specific expression, contains a repressor site, NRE-A, which binds a zinc finger protein (14) . Comparison of the complex(es) binding to the NRE-A and NIP sites showed them to have different mobilities in EMSA. Furthermore, NIP oligonucleotides did not compete specific binding to the NRE-A probe; nor did NRE-A oligonucleotides compete NIP-C3 binding to its cognate site (data not shown). This suggests that the IL-2 repressor and NIP-C3 are distinct from each other.
The presence of a negative regulatory element within the IL-3 promoter was originally recognized when a truncated IL-3 promoter lacking the AP-1/Elf-1 sites was tested in T cell lines(8, 9, 12) . These experiments localized this element between nt -244 and -270 of the IL-3 promoter and showed that it also functions as a repressor in primary T lymphocytes(23) , B cells, and HeLa cells(12) . We have shown that the repression function of the NIP region (-267 to -244) is preserved when placed in a different location within the IL-3 promoter or transferred in cis to a heterologous promoter (Fig. 1). Thus, repression through the NIP site is not cell-restricted and may play a more general role than the one involved in IL-3 regulation.
The NIP region was used as a probe to identify
proteins that might mediate its repression function. Using various
assay conditions for DNA binding in vitro, we were able to
detect three specific complexes (Fig. 3). The most prominent
complex is complex 1. Partial characterization and purification of
complex 1 indicate that it is a ubiquitous protein of with a molecular
mass of 63 kDa. ()It forms on the 3` half of the NIP site
and is dispensable for repression function (Fig. 4). Its role in
IL-3 regulation remains unknown. A complex with similar properties has
been described, which binds a site in the stromelysin gene promoter and
appears to mediate phorbol ester activation in cooperation with a
nearby AP-1 site(24) .
The 5` half of the NIP site interacts with two distinct protein complexes to form complex 2 and complex 3. Both complexes are expressed ubiquitously ( Fig. 8and data not shown) and appear to be conserved across different species. Since a consensus sequence for an E-box binding protein is present in the 5` half of the NIP site, we sought to determine whether either of the two complexes might be a member of the basic helix-loop-helix family of transcription factors. Eleven mutant oligonucleotides of the NIP-5` probe were tested as competitors in EMSAs revealing that the binding specificity of complex 2 (CACNTG) matched the general consensus sequence of an E-box site (CANNTG). The identity of complex 2 was explored using antibodies directed against ubiquitous E-box proteins. Complex 2 was disrupted by an antiserum recognizing the general transcriptional activator USF. Additional experiments established that complex 2 is identical or closely related to USF.
Complex 2 cannot be the NIP repressor, however, since its DNA-binding specificity differs from the functional repressor consensus sequence CTCACNTNC. Other E-box binding proteins are also unlikely candidates, since a mutation altering the invariant G in the NIP E-box core had no effect on repressor function. Since neither complex 1 nor complex 2 binding correlated with repressor activity, we focused on complex 3, the only remaining candidate for this function. The use of different informative mutant oligonucleotides as DNA probes in EMSA showed that, among the three complexes that form on the NIP region, only complex 3, designated NIP-C3, displayed the appropriate specificity for being the repressor.
NIP-C3 is expressed in a wide array of tissues and species ( Fig. 8and data not shown). Its binding in vitro is
exquisitely sensitive to the type of nonspecific DNA present in the
binding reaction. We were unable to detect NIP-C3 when poly(dI-dC) was
used as carrier. This behavior is reminiscent of the zinc finger
transcription factor EF1, which was originally identified as a
transcriptional activator of the
-crystallin enhancer and was
subsequently found to repress E2-box-mediated gene activation through
competition of binding. Although NIP-C3 and
EF1 share similar
binding specificities, they differ in recognition of at least one
important position, where the presence of a specific nucleotide
inactivates NIP repressor function while it favors
EF1
function(25) . Although the two proteins may share similar
mechanisms of repression, the above difference strongly argues that the
two factors are different.
There is accumulating evidence that a growing number of proteins function by displacing helix-loop-helix transcription factors from their cognate E-box sequences. Indeed, another zinc finger protein, ZEB, was recently shown to be able to silence the IgH enhancer by displacing the E2A complex from its cognate site(26) . Since binding sites for complex 2/USF and the repressor significantly overlap, a dynamic equilibrium of occupancy by complex2/USF and NIP-C3 over the 5` portion of the NIP site may dictate appropriate IL-3 expression in an analogous manner(27) .
Why should the IL-3 promoter contain a strong repressor element? IL-3 is a potent growth factor, which plays multiple, complementary roles in normal hematopoiesis(1) . Meticulous control of its production may be necessary to prevent unbridled proliferation of progenitor cells and to meet the body's fluctuating needs for differentiated cells. IL-3 may be an example of a protein that is so deleterious when overproduced that blocking its expression may be of greater importance than activating its expression. It has previously been shown that the proximal portion of the IL-3 promoter activates a basal level of transcription in a variety of cell types(12) , yet IL-3 is produced only by activated T cells(5) . The NIP repressor may serve as a clamp that normally prevents IL-3 transcription in all cells. Its effect, however, can be specifically abrogated when the upstream AP-1 and Elf-1 sequences engage proteins expressed only in activated T cells. Future studies must be aimed at understanding the mechanism by which factors acting at the AP-1 and Elf-1 sites relieve NIP repression.