(Received for publication, May 16, 1995; and in revised form, June 21, 1995)
From the
Expression of the major histocompatibility complex (MHC) class
II-associated invariant chain (Ii) is required for efficient and
complete presentation of antigens by MHC class II molecules and a
normal immune response. The Ii gene is generally co-regulated with the
MHC class II molecules at the level of transcription and a shared SXY
promoter element has been described. This report defines the proximal
promoter region of Ii which may regulate Ii transcription distinct from
MHC class II. In vivo genomic footprinting identified an
occupied, imperfect CCAAT box and an adjacent GC box in the proximal
region. These sites are bound in Ii-ositive cell lines and upon
interferon- induction of Ii transcription. In contrast, both sites
are unoccupied in Ii-egative cell lines and in inducible cell lines
prior to interferon-
treatment. Together these two sites synergize
to stimulate transcription. Independently, the transcription factor
NF-Y binds poorly to the imperfect CCAAT box with a rapid off rate,
while Sp1 binds to the GC box. Stabilization of NF-Y binding occurs
upon Sp1 binding to DNA. In addition, the half-life of Sp1 binding also
increased in the presence of NF-Y binding. These findings suggest a
mechanism for the complete functional synergy of the GC and CCAAT
elements observed in Ii transcription. Furthermore, this report defines
a CCAAT box of imperfect sequence which binds NF-Y and activates
transcription only when stabilized by an adjacent factor, Sp1.
One of the central steps in elicitation of an immune response is
the presentation of foreign antigenic peptides by the major
histocompatibility complex (MHC) ()class II molecules on the
surface of cells(1, 2) . Recognition of the
peptide
MHC class II complex by the T-cell receptor on class
II-restricted T lymphoid cells leads to activation of the T cells and
the initiation of the immune response(3, 4) . Binding
of peptides to the class II molecules is regulated in part by the MHC
class II-associated invariant chain (Ii)
protein(5, 6) . The Ii is an integral membrane protein
which associates with class II
/
molecules in the endoplasmic
reticulum(7, 8) . This association has multiple
affects on class II molecules, all of which optimize the binding and
presentation of foreign peptides derived from the extracellular
environment (reviewed in Refs. 9, 10). The Ii also appears to play a
role in targeting the class II molecules to endosomal or endosomal-like
compartments where peptide is
bound(11, 12, 13, 14) . The critical
role of Ii was demonstrated in mutant mice lacking a functional Ii
gene(15, 16) . MHC class II function was impaired
resulting in inefficient antigen presentation and a deficiency in
CD4+ T cells.
The tight functional association of Ii and class
II is reflected in their coordinate transcriptional regulation. Both
molecules are only expressed in a select group of cells including B
cells, activated human T cells, thymic epithelial cells, and
macrophages(17, 18, 19) . Ii and class II are
also coordinately induced by interferon- (IFN-
) in several
cell types including macrophages and brain glial cells (20, 21, 22, 23) . The Ii promoter
and the class II promoters share a common DNA motif, S/X/Y, which
interacts with at least three distinct transcription
factors(4, 24, 25, 26) . Several
studies have clearly demonstrated that this motif is responsible for
both the constitutive and IFN-
-induced coordinate
regulation(27, 28, 29, 30) . In
class II promoters, this motif is located proximal to the TATA box and
is generally sufficient for transcriptional activation. In contrast,
the Ii promoter has over 170 base pairs (bp) between the TATA box and
the S/X/Y motif. There are several cases of differential regulation
between Ii and class II
genes(17, 18, 31, 32) . The
additional Ii promoter sequences between the S/X/Y and TATA domains may
harbor elements mediating the discordant transcription. In the murine
Ii promoter, an NF-
B site has been identified which mediates basal
and TNF
induction of Ii transcription(33) . In addition,
promoter deletion analysis implicated a GC box as important for the
basal activity of the murine gene(30) . Recently, two NF-
B
sites have been characterized at position -172 and -118
base pairs of the human Ii promoter(34) . The function of these
elements is dependent on cell type-specific differences in the binding
of NF-
B subunits. Despite these studies, little is known about the
Ii proximal promoter and the factors bound there which may be required
for transcription as well as potentially mediating the effects of the
distal S/X/Y motif.
The Y box of the S/X/Y motif is an inverted CCAAT site which binds the heteromeric transcription factor NF-Y (also known as CBF, CP1, and YEBP)(35, 36, 37, 38) . NF-Y is clearly distinct from the other known CCAAT box-binding proteins, CTF/NF-1 and C/EBP. NF-Y is composed of an A, B, and C subunit of 42, 36, and 40 kDa, respectively(39, 40) . NF-Y is highly conserved through evolution(41) . The yeast transcription factors HAP3, HAP2, and HAP5 are homologues of the A, B, and C subunits, respectively, and are functionally interchangeable in a DNA binding assay(42) . In addition to MHC class II promoters, NF-Y-binding sites are found in many unrelated promoters; e.g. HSV-tk (37) , collagen(36) , and albumin(43) . Typically the binding sites are located in the proximal promoter region between -60 and -80 base pairs upstream of the transcription initiation site(44) . Functional analysis has also shown that NF-Y can be important in transcription reinitiation as well as in activation, suggesting a role in basal transcription(43, 45) .
In the MHC class II promoters, NF-Y also functions to stabilize and/or recruit additional transcription factors to the S/X/Y complex. In vitro, NF-Y and the X box-binding protein, RFX, co-stabilize binding to their DNA sites(46) . Furthermore, in vivo mutation of the NF-Y-binding site abrogates factor binding to the upstream X1 and X2 sites(47) . In addition, NF-Y requires that the DNA stereospecifically align the X and Y boxes for activation(48) . NF-Y has also been studied at the serum albumin promoter and in that context functions synergistically with C/EBP to activate transcription(43) . The interaction between these factors partially destabilizes NF-Y binding but results in a large synergistic increase in the formation of stable preinitiation complexes. These studies of NF-Y suggest that although found in many different promoters, its mode of function may depend upon the local environment of that specific promoter.
In this report we now describe the in vivo characterization of the elements and factors involved at the proximal promoter of the human Ii gene. Through the use of in vivo genomic footprinting, two adjacent binding sites with homology to a GC box and an imperfect CCAAT box are identified. The GC and CCAAT elements act synergistically to promote Ii transcription and are bound by the transcription factors Sp1 and NF-Y, respectively. NF-Y and Sp1 bind cooperatively at the Ii promoter to mediate, at least in part, the observed functional synergy. Thus, maximal Ii transcription requires the action of two NF-Y factors; one in the distal S/X/Y motif and one described here in a cooperative binding complex with Sp1 in the proximal promoter. These findings describe a mechanism of transcriptional activation by the ubiquitous transcription factor NF-Y and define a new promoter region regulating Ii expression.
Figure 1: In vivo protein-/DNA interactions at the Ii gene proximal promoter reveal an occupied GC box and imperfect NF-Y site. A, In vivo footprint analysis of the upper strand of the Ii proximal promoter. Cont., control in vitro methylated deproteinized DNA; In vivo, in vivo methylated DNA. Open arrows, protections; solid arrows, enhancements. Weak interactions are depicted by arrowheads. The functional elements as defined in this report are indicated by brackets on the left. The Raji B cell line and the H9 T cell line constitutively express Ii, and the contact points at the GC box and Y-proximal site are the same. The Ii-negative Jurkat T cell line only displayed a single weak protection at position -50. B, lower strand in vivo footprint analysis. Lane markings are the same as in panel A. Five protections were detected across the GC box and Y-proximal site in the constitutively expressing Raji and H9 cell lines. No contacts were detected in Ii negative Jurkat cell line. C, sequence of the Ii proximal promoter and summary of the in vivo contact points. The functional elements are indicated by shaded boxes and the arrows and arrowheads are as in panels A and B.
Ii gene transcription is
strongly induced by IFN- in a number of non-lymphoid cell types.
In the glioblastoma cell line U105-MG, the induction is mediated
through a 5-6-fold increase in the rate of Ii gene
transcription(34) . In vivo footprint analysis of the
Ii proximal promoter in U105-MG cells revealed no protein-DNA
interactions prior to IFN-
induction (Fig. 2). Four h after
the addition of IFN-
, protections are clearly visible at the GC
box and Y-proximal site. The contacts are identical to those observed
in the Raji and H9 cells. A similar pattern of interaction was observed
at 18 and 48 h post-induction. This region of the Ii promoter has not
been implicated in the IFN-
response; rather two upstream
elements, the ISRE and S/X/Y domain, appear to mediate the induction (27, 29, 60) . Analysis of those
IFN-
-responsive elements as well as the two NF-
B sites
displayed in vivo occupancy only after IFN-
treatment(34, 49) . Interestingly, protection at these
distal elements did not become maximal until 24 or 48 h post-induction
(see ``Discussion''). This indicates that the entire Ii
promoter is shielded from transcription factor binding in the uninduced
state, and IFN-
treatment relieves this repression.
Figure 2:
Induction of Ii expression by
interferon- induces in vivo protein-DNA interactions at
the Ii proximal promoter. The glioblastoma cell line, U105-MG, was
treated with 500 units/ml recombinant IFN-
and examined by in
vivo footprint analysis at the times indicated above each lane.
Lane markings are as described in Fig. 1. At time 0 h, no
contacts were detected. Interactions at both the GC box and Y-proximal
site were clearly detectable at 4 h and continued for 18 and 48 h. The
interactions were indistinguishable from those detected in the
constitutively expressing B and T cell
lines.
Figure 3: The Ii GC box and Y-proximal site synergize to activate transcription. The wild type (WT) Ii promoter construct contains 790 base pairs of the promoter fused to the CAT reporter gene. Site-specific mutation of the GC box (mtGC), Y-proximal site (mtYprox), or both (mtGC/Yprox) are derived from the wild type parent construct as described under ``Experimental Procedures.'' Constructs were transiently transfected into the Raji B cell line and assayed by CAT activity. The results are normalized to one for expression from the wild type construct. Each bar represents data from seven independent experiments, and the S.E. is indicated by the error bars.
Figure 4: In vitro binding to the Ii proximal promoter detects complexes with the GC box, the Y-proximal site, and both together. A, EMSA analysis with the wild type Ii probe, IiGC/Yprox (-32 to -85 bp). Shaded triangle indicates increasing amounts of Raji nuclear extract (0.2-2 µg) added to the binding reaction and the shaded rectangle represents a constant 1 µg of nuclear extract. Specific complexes are labeled A-D while n.s. indicates the nonspecific band. Oligonucleotide competitors are indicated above lanes 5-10 and are described under ``Experimental Procedures.'' B, EMSA competition analysis demonstrates that the complex binding to the Y-proximal site is related to NF-Y. The wild type Ii probe (IiGC/Yprox) is used in lanes 1-5, and the high affinity NF-Y site DRA Y-box is used for lanes 6-10.
Although the Y-proximal site is divergent at the highly conserved second C residue of the CCAAT box, the remainder of the sequence conforms to an 11-bp NF-Y consensus. The consensus was derived from 17 different published NF-Y-binding sites (Table 1). The MHC class II DRA Y box is a high affinity NF-Y-binding site and was used in EMSA competition to access the relationship between the Y-proximal binding activity and NF-Y (Fig. 4B). Competition of the Ii GC/Yprox oligonucleotide probe with the DRA Y box eliminated the D and B bands (lane 3). This pattern of competition is identical to competition with the Ii Y-proximal site as shown in Fig. 4A, lane 6. The unrelated DRA octa oligonucleotide did not compete for any of the bands. The Y-proximal site also has weak homology to the OTF-1 binding consensus TAATGARAT. However, none of the Ii-specific bands were competed for by a TAATGARAT oligonucleotide (lane 5). An identical pattern of competition was observed when a probe corresponding to the DRA Y box was used (lanes 6-10). Both Ii GC/Yprox and DRA Y box oligonucleotides competed the NF-Y band. Neither the DRA octa or TAATGARAT competed for the band. Together, the competition results suggest that NF-Y interacts at the Y-proximal site. Furthermore, both NF-Y and Sp1 can bind simultaneously to the Ii promoter as evidenced by the D band.
A direct test for the presence of NF-Y and Sp1 on the Ii
promoter is available through the use of antibodies specific for the
two transcription factors. Specific antibodies or preimmune sera are
added to the EMSA binding reaction and then resolved by gel
electrophoresis. A specific antibody interaction either diminishes the
gel shift band (blocking) or shifts the complex higher in the gel
(supershift). We first tested the antibodies on the wild type Ii probe (Fig. 5, lanes 1-5). The preimmune sera has no
effect on any of the complexes. However, the antisera specific for the
A subunit of NF-Y results in a supershifted band concomitant with a
decrease in the intensity of the D and B bands. This is consistent with
the association of the D and B bands with binding at the Y-proximal
site. The anti-Sp1 antibody also produced a supershifted complex and
diminished the D and C bands. However, the B band was unaffected. This
is also consistent with the D and C bands representing GC box binding.
An unrelated antibody specific for NF-B-p50 did not effect any of
the bands. The antibodies were also assayed on the NF-Y complex derived
from the DRA Y box probe (lanes 6-10). Only the antibody
specific for NF-YA subunit supershifted the NF-Y complex. To simplify
the gel shift-antibody pattern, the same experiment was done with gel
shift probes mutated in one or the other binding sites. As shown in lanes 11-15, when the Y-proximal binding activity is
present alone only the NF-YA and B antibodies recognize this band (lanes 12 and 13). A probe that contains only the GC
box binding activity forms both the A and C bands (lane 16).
The antibody specific for Sp1 supershifts the C band but not the A band (lane 19). This confirms the identity of the C band as Sp1,
while the much less abundant A band is an immunologically unrelated GC
box-binding protein. Combined, the gel shift and antibody reactivity
clearly defines NF-Y binding at the Y-proximal site. Furthermore, this
indicates Sp1 is the predominant GC box-binding protein, although at
this point a role for the A band protein cannot be excluded.
Figure 5: EMSA-antibody supershift analysis identifies NF-Y interacting at the Y-proximal site and Sp1 at the GC box. The antibodies used are indicated above each lane. -, no antibody; PI, preimmune antiserum. The probes used are marked above each gel. Other markings are as described in Fig. 4.
Figure 6:
The DNA binding half-lives of NF-Y and Sp1
are stabilized when both factors are complexed with the DNA together. A, off-rate analysis of NF-Y independently bound to the Ii
Y-proximal site, t= 1.7 min (squares), or bound as a complex with Sp1 at the Ii proximal
promoter, t = 7.5 min (circles). B,
comparison of NF-Y binding stability at the consensus DRA-Y box, t = 6.0 min (squares), and the Ii NF-YSp1 complex, t = 7.5 min (circles). C, off-rate
analysis of Sp1 bound either independently to the Ii GC box, t = 3.2 min (squares) or in a complex with NF-Y, t = 8.9 min (circles). Where the horizontal line at 50 intersects the plot indicates the
calculated half-life for that plot. Values were determined from
PhosphorImage analysis of EMSA competition gels as described under
``Experimental Procedures'' and are plotted as the percent of
complex shifted over the initial complex formed before competition versus the time of competition. Band D was
quantitated for the half-life of Sp1 and NF-Y after competition with
either the GC box or a consensus Y-box, respectively (see lane
1, Fig. 5for example gel prior to competition). Band B of the Ii-Y-proximal probe was quantitated for the half-life of
NF-Y alone (see lane 11, Fig. 5for example of gel
prior to competition). Band C of the Ii GC probe was
quantitated for the half-life of Sp1 alone (see lane 16, Fig. 5for example of gel prior to competition). Band
``NF-Y'' of the DRA-Y box probe was quantitated for the
half-life of NF-Y on the consensus Y box (see lane 6, Fig. 5for example gel prior to competition). Each point is the
average of at least three independent experiments and the S.E. in
indicated by the error bars.
Control of Ii gene transcription plays a critical role in the
presentation of antigens by MHC class II molecules and the maintenance
of a normal immune response. This report now defines a new region of
the Ii promoter required for expression of this gene. Examination of
the in vivo occupancy of the Ii promoter in constitutively
expressing cell lines identified a bound GC box and imperfect CCAAT box
(Y-proximal) at positions -74 and -53, respectively,
relative to the transcription initiation site. Only 11 base pairs or
one helical turn of the DNA separate these two recognition sites. In
addition, binding at the TATA box at position -24 was detected
when an alternative in vivo footprinting protocol was utilized
to reveal contacts at adenine residues (data not shown). Combined with
our previous studies(34, 49) , these observations now
complete the in vivo footprint analysis of the entire 300-base
pair Ii promoter. There are eight elements clearly bound in
vivo; TATA box (-24 bp), Y-proximal (-53 bp), GC box
(-74 bp), ISRE (-96 bp), B-1 (-118 bp),
B-2
(kB-2 bp), Y (-207 bp), and X (-236 bp). Additionally, the
S element (-260 bp) has been defined functionally but does not
display interaction contacts in vivo. This is consistent with
the related S element in the class II DRA promoter which also did not
show in vivo contacts (54, 62) .
The GC
box and Y-proximal sites were occupied in both B and T cell lines which
constitutively express Ii. However, neither site was bound in the
Ii-negative Jurkat T cell line. The lack of in vivo binding
occurs despite the presence of the transacting factors Sp1 and NF-Y in
the nuclei of these cells, as detected by in vitro binding
assays(34, 54) . ()Furthermore, none of the
elements within the 300-bp Ii promoter are occupied in vivo in
the Ii-negative cell line. However, when the Ii promoter is transiently
introduced into these cells, it displays a significant level of
transcriptional activity. This finding suggests a higher order
inhibition of DNA binding at the endogenous Ii promoter, potentially at
the level of chromatin reorganization. This is consistent with the
nuclease hypersensitivity studies done on many genes, which
demonstrated that inactive genes are often found in condensed,
nuclease-resistant regions of the
chromatin(63, 64, 65) . Interestingly, the
functionally related class II DRA gene promoter is also unoccupied and
not expressed in the Jurkat cell line. Although the class II genes are
located on a separate chromosome from Ii, a similar mechanism may be
responsible for the inaccessibility of these promoters in
non-expressing cells.
Interferon- is a potent inducer of Ii and
class II transcription (23, 66, 67, 68) . In the
IFN-
-inducible cell line U105-MG, the Ii proximal promoter
elements GC and Y-proximal sites are unoccupied prior to the induction.
IFN-
treatment up-regulates binding at both proximal elements by 4
h, and binding continues at 18 and 48 h after addition IFN-
.
Loading of the proximal elements appears to occur prior to loading at
the distal elements. The distal X and Y boxes display only the very
weak beginnings of interaction at 4 h but by 24 h show maximal
binding(49) . Loading at the negative Ii element kB-1 appears
to be delayed even further, until 48 h. This finding suggests that
interactions at the Y-proxmal site and/or the GC box occur first and
may be required to open the promoter for additional factor binding.
Thus, the Ii promoter minimizes basal level expression by preventing
binding of the transacting factors in the uninduced state. This is in
direct contrast to the class II DRA gene in which all elements are
bound prior to IFN-
induction in this cell line(54) .
Treatment with IFN-
then induces an up-regulation of the weak
binding activity at the X box of the DRA S/X/Y domain to a strong
interaction. The Ii promoter also utilizes an S/X/Y domain for the
IFN-
response (27, 29) and to a lesser degree an
ISRE element(60) . Thus, the induction pathway for Ii and class
II are likely to be highly related. However, the mechanism by which the
Ii promoter remains unoccupied until IFN-
treatment is unclear. In vivo analysis of several other promoters has revealed
situations analogous to both the all or none effect observed for Ii and
the limited effect detected for class II DRA (53, 69, 70, 71) . It is somewhat
surprising to find two coordinately regulated genes invoking different
mechanisms. Interestingly, an in vivo study of the class II
DRA promoter demonstrated a central requirement for NF-Y in the loading
of the promoter(47) . Stable introduction of a DRA promoter
construct with a mutated Y/CCAAT box blocked in vivo binding
at all promoter elements in the IFN-
-induced U105-MG cells. In
contrast mutation of the other promoter elements S, X1, and X2 only has
a local effect on binding in vivo. The findings from that
study implicated the transcription factor NF-Y binding to the Y/CCAAT
box as critical for recruitment of the other promoter factors and/or
opening the chromatin structure across the promoter region. This has
potentially important implications for the Ii promoter since two
NF-Y-binding CCAAT boxes are present in this promoter, the distal Y box
of the S/X/Y domain and the Y-proximal element. It will be interesting
to determine if one or both of these elements modulate accessibility to
the Ii promoter.
Synergistic activation by two distinct
transcription factors is becoming a common phenomenon and several
different mechanisms have been reported (see (72) and
references within). This report defines a synergistic activation of Ii
transcription by Sp1 bound at the GC box and NF-Y bound at the
Y-proximal site. Neither site was capable of activating Ii
transcription in the absence of the other site. However, together they
formed a strong transcriptional activator. At least one basis for their
synergy is that NF-Y binds to the Y-proximal element with a very short
half-life. Sp1 also binds to the Ii GC box with a short half-life.
Stabilization of both NF-Y and Sp1 binding occurs when Sp1 and NF-Y
bind the Ii promoter together. This suggests that in vivo Sp1
and NF-Y may only bind cooperatively to the Ii promoter and thus are
completely dependent on each other to activate transcription. In
vivo footprint analysis of mutant Ii promoters indicates both
sites must be present for binding to occur at either site. ()Interestingly, the human multidrug resistance gene MDR has
a similar arrangement of GC box and CCAAT box in its proximal
promoter(73) . These elements were shown to functionally
synergize (
)although the mechanism has not been
investigated. The MDR CCAAT box matches the consensus NF-Y sequence (Table 1), however, stabilization of DNA binding may also play a
role in MDR activation.
A number of studies have described
cooperative interactions of Sp1 with other factors, but not NF-Y.
Examples include OTF-1(74) , C/EBP(75) ,
NF-B(76) , Ets(77) , E2,
YY1(78, 79) , and Sp1(80, 81) . In at
least one case a direct physical interaction between Sp1 and the E2
factor was clearly demonstrated even in the absence of an E2
DNA-binding site (82) . In addition, Sp1 can multimerize and
thereby synergistically activate transcription(80) . However,
in most cases the mechanisms are less well defined. NF-Y has been
described in two distinct cooperative interactions. The MHC class II
DRA promoter has an NF-Y-binding site which must be stereospecifically
aligned with the X box to function (48) . Reith et al.(46) recently demonstrated that stable binding of NF-Y and
the X box factor RFX required a cooperative interaction between the two
proteins. NF-Y also binds in the serum albumin promoter adjacent to a
C/EBP site. The binding of both proteins is required to promote stable
preinitiation complex formation(43) . Interestingly, in this
case NF-Y DNA binding is weaker in the complex with C/EBP than when
alone. Clearly these studies reinforce the hypothesis that synergy can
occur through many different mechanisms. In particular, NF-Y appears to
have multiple activating functions which are dependent on the local
promoter environment.
In conclusion, maximal transcription from the
Ii promoter requires a GC box and NF-Y site proximal to the TATA box.
These sites function in a synergistic mannar, and it is shown that
cooperativity in DNA binding is at least one mechanism underlying the
synergy. An additional outcome of this study is the identification of a
functional, imperfect NF-Y-binding site that would be undetected in
homology searches for the consensus core sequence CCAAT or CAAT. This
raises the potential for an even greater usage of NF-Y in promoters
that do not have a classical CCAAT box. Finally, the Ii gene exhibits
all or none in vivo binding across the promoter in response to
IFN- induction. With the previous observations that NF-Y can
control access to the DRA promoter in vivo(47) , it
will be important to determine if NF-Y plays a similar role for Ii.
This could have broad implications for the regulation of multiple genes
with an NF-Y/CCAAT box.