(Received for publication, June 14, 1995)
From the
The PU.1 protein is an ets-related transcription factor
that is expressed in macrophages and B lymphocytes. We present evidence
that PU.1 binds to the promoter of the I-A gene, i.e. a
PU box located next to the Y box. Transfection of PU.1 in B lymphocytes
or in interferon-
-treated macrophages represses I-A
gene
expression. The inhibitory effect of PU.1 was obtained with the DNA
binding domain of the protein, but not with the activation domain.
Using the gel shift retardation assay we found that in vitro transcribed/translated NF-YA and NF-YB bind to the Y box of the
I-A
promoter. When PU.1 was added to the assay, a supershifted DNA
band was found, indicating that PU.1 and NFY proteins bind to the same
DNA molecule. We conclude that I-A
gene expression is repressed by
PU.1 binding to the PU box domain.
MHC ()class II proteins are tissue-specific and play
a key role in the immune system. These proteins are critical for the
generation of the T cell repertoire in the thymus (positive selection)
and for antigen presentation to T helper cells(1, 2) .
The lack of expression of class II proteins in humans (3) or in
animal models (4, 5) leads to a severe combined
immunodeficiency.
The regulation of the expression of these genes involves at least three cis-acting elements located 5` upstream of the transcription initiation site(6, 7, 8) . The elements have been referred to as W, X, and Y, and nuclear factors have been shown to bind to each element. The W sequence is also known as H, S, or Z.
The Y
element contains an inverted CCAAT motif, and the binding factor, NF-Y,
has been characterized as a binding factor whose activity depends on
the CCAAT sequence as well as on the flanking
bases(9, 10) . This NF-Y factor is composed of two
subunits named NF-YA and NF-YB(11) . The two subunits have been
cloned by protein purification and microsequencing of the NF-Y that
binds to the E promoter(12) . The protein sequence reveals
stretches with 70% sequence homology to regions of the yeast
transcription factors HAP2 and HAP3, which control cytochrome gene
transcription in yeast. These cloned factors NF-YA and NF-YB bind to
the Y box of the I-A
gene. (
)
Using an
oligonucleotide containing the Y box of the IA promoter, the
protein PU.1 was identified through a system of expression cloning
(
gt11)(13) . PU.1 is present in B lymphoid cells and
macrophages, and it binds to a purine-rich sequence that contains a
central core with the sequence 5`-GGAA-3`. The DNA-binding domain,
which is located near the carboxyl terminus is homologous to the ets family of DNA-binding proteins and is rich in basic amino
acids(14) . Using antibodies, PU.1 protein has been detected in
the nuclei. (
)
PU.1 has been shown to be a transcriptional
activator(13) . However, little is known about the genes that
are regulated by it. A number of promoters have been analyzed and found
to contain at least one binding site for PU.1 within promoter or
enhancer. Some of the genes believed to be regulated by PU.1 include
the immunoglobulin light chain gene (15, 16) ,
the immunoglobulin
2-4 enhancer gene(17) , the
immunoglobulin heavy chain µ(18) , the immunoglobulin J
chain gene (19) , the mb-1 gene that is expressed in
early B cell differentiation(20, 21) , the Fc
R1b
gene(22) , the M-CSF receptor gene(23) , the CD11b
gene(24, 25) , and the macrophage inflammatory protein
1
(26) . PU.1 can function as an activator by itself or by
interacting with other transcription factors. It has been shown that
PU.1 interacts with the B cell factor NF-EM5 and stimulates
transcription from the immunoglobulin
3` enhancer(15) .
These results point to the complex nature of both DNA-protein
interactions and protein-protein interactions.
Recently, it has been
reported that IFN-, a cytokine that induces the expression of MHC
class II antigens, down-regulates the expression of PU.1(27) .
Due to the proximity of the PU and the Y boxes in the I-A
gene, we
have examined the ability of PU.1 to regulate the expression of the
I-A
gene. Our results indicate that PU.1 represses I-A
expression, probably by binding to the PU.1 box which is located
between the Y box and the transcription start site.
Figure 3:
Repression of I-A promoter expression
by PU.1 protein. A reporter plasmid (15 µg) (top)
containing a fragment of the I-A
promoter (WXY -124 to
-26) or the same fragment with a mutation in the Y box (CTGATTGG
by CTGATTTT) was transfected into A20-2J cells, together with the
-galactosidase expression plasmid and 2 µg of the pECE vector,
the PU.1, the PU.1 binding domain, the PU.1 activation domain, or the
retinoic acid receptor expression vectors as indicated. The amount of
cell extract used for CAT assays was normalized according to the level
of
-galactosidase expression. The CAT enzymatic activity was
quantitated using an imaging system (AMBIS,
Inc.).
The PU.1 protein was cloned using an expression library
(gt 11) and an oligonucleotide covering the Y box binding site
that contains a PU.1 box (GAGGAA). To determine whether the PU.1
protein could bind to the oligonucleotide containing the Y box a 320-bp
fragment containing the I-A
promoter was used for gel retardation
assays. When the in vitro transcribed/translated PU.1 protein
was added to the 320-bp radiolabeled fragment, gel electrophoresis was
performed, and a retarded complex was observed (Fig. 1). This
complex was effectively displaced by a 50-fold molar excess of a cold
oligonucleotide containing the PU.1 box. Oligonucleotides that
contained the Y box and modified PU.1 core sequence did not compete
effectively for binding of the PU.1 protein to labeled probe. These
results suggest that PU.1 protein recognized the PU.1 box in a
sequence-specific manner.
Figure 1: Gel electrophoresis DNA binding assay of in vitro transcribed/translated PU.1. Competition analysis was performed using 2 ng of the 320-bp fragment containing the Y and PU boxes. Cold competitor oligonucleotides were present at 50-fold molar excess. At the bottom of the figure there is the sequence of the 320-bp fragment used as probe and the double-stranded oligonucleotides used as competitor. The dashes indicate those bases that are identical in the different sequences. The boxed bases show the Y box and the underlined bases the core PU box.
To delineate the binding of PU.1 on the
I-A promotor, we performed methylation protection experiments
using the 320-bp fragment (Fig. 2). Binding of the PU.1 to the
I-A
promoter resulted in the protection of two guanine residues on
the upper strand within the core of the PU.1 box. No guanine residue on
the lower strand was protected. These results confirmed the data
obtained with the gel retardation assay, showing that the PU.1 protein
binds to the PU box next to the Y box.
Figure 2: Methylation protection experiments. The 320-bp fragment was labeled on the upper strand and incubated with in vitro produced PU.1, and then the DNA was partially methylated using dimethyl sulfate. Free and bound DNA was separated using a gel retardation analysis. Bound and free bands were isolated, cleaved with piperidine, and analyzed on 8% acrylamide, 8 M urea gels. At the bottom of the figure, the sequence is shown with the protected G.
To examine the possible role
of PU.1 protein in I-A expression, we attempted to determine
whether transcription from the I-A
promoter is affected by the
PU.1 protein. Using the I-A
promoter linked to the CAT gene, we
observed a low CAT activity when this construct was transfected into
the B cell line, A20-2J. As we were interested in the cell
type-specific enhancing activity of the I-A
promoter, we linked a
124-bp fragment of the I-A
promoter containing the W, X, and Y
boxes to the SV40 promoter (Fig. 3), which then gave us a better
signal in the CAT assay. This type of construct has been used by others
to obtain a more efficient expression of other MHC class II genes,
including the I-E
and I-A
genes(35, 36, 37) . Each CAT construct was
cotransfected with the
-galactosidase expression plasmid, pCH110,
into the B cell line A20-2J. All CAT values were then normalized
to the level of
-galactosidase expression to correct any
differences in transfection efficiency. The KS1 vector gave a level of
CAT activity of <1%, while KS1 containing the W, X, and Y boxes gave
a CAT activity of 19.5% (Fig. 3). When a mutation of the Y box
was made, CAT activity was lowered to 13.1%, suggesting that the Y box
plays an important role in the I-A
gene expression. In fact the Y
box contributes 33% of the expression of the KS1-WXY construct. The Y
box mutation was designed to disrupt the binding of the NF-Y to the Y
box as measured by a gel electrophoresis DNA binding
assay(10) .
To determine the effect of PU.1 protein on
I-A expression we cotransfected an expression plasmid of PU.1 into
A20-2J cells together with the CAT constructions (Fig. 3).
There was a decrease in CAT activity from 19.5% to 11.2% with PU.1 (42%
reduction). This suggested that PU.1 abolishes the contribution of the
Y box-binding proteins to the transcriptional activity of the I-A
promoter. The reduction in CAT activity seems to be proportional to the
amount of PU.1 transfected. Using 2 µg of PU.1 DNA, the CAT
activity was 11.2% while with 1 µg, CAT activity was 16.4%. We also
tested the effect of plasmids containing either the binding domain or
the activation domain of the PU.1 transcription factor. When the
plasmid containing the PU.1 binding site was cotransfected with the CAT
constructs, the acetylation dropped to 12.2% (37% reduction). However,
the cotransfection of the activation domain did not reduce the CAT
activity (18.5% acetylation). As a control, we used a plasmid vector
that expresses the retinoic acid receptor. Under these conditions, the
levels of CAT activity were not modified (20.0% acetylation). When the
Y box was mutated in cells cotransfected with PU.1, the CAT expression
activity was not modified: 12.5% versus 11.0% (PU.1), 11.3%
(binding domain) and 12.0% (activation domain). Thus, PU.1 was able to
repress CAT activity in those constructs containing the Y box sequence.
This effect seems to be related to the binding activity of PU.1.
To
ascertain whether the suppressive effect of PU.1 was specific to the
I-A promoter and not due to a general down-regulation of
transcription, the effect of the plasmid containing PU.1 was tested on
a vector containing the PU.1 binding site. For these experiments, we
used a fibroblast cell line that did not express PU.1. The vector
pBLCAT2, which contains the CAT gene linked to the thymidine kinase
promoter, transfected into fibroblasts produced 1.2% acetylation (Fig. 4). Cotransfection of an expression vector with PU.1 (pECE
PU.1) did not modify the basic levels of CAT expression. Only if the
pBLCAT vectors contained a PU.1 box in the presence of pECE PU.1 did
the CAT activity increase 4-fold, suggesting that PU.1 is a
transcription factor enhancing this particular promoter, which contains
the binding site of PU.1. Transfection of the KS1-WXY vector produced
an acetylation of 38.3%. Cotransfections of PU.1 expressing vector with
the KS1-WXY vector reduced the CAT activity in this cell type to 24.2%
(37% of the original activity) (Fig. 4).
Figure 4:
Expression of the PU.1 DNA-binding protein
is not toxic. A reported plasmid (15 µg) containing the PU.1
binding site linked to the thymidine kinase promoter was transfected
into L929 fibroblasts cells, which are deficient for PU.1 protein, with
the galactosidase expression plasmid and 2 µg of the pECE
vector or the PU.1 expression vector. The KS1 constructions were also
transfected in the absence or presence of PU.1 expression
vector.
We also tested the
effect of the PU.1 protein on I-A expression induced by IFN-. Bone
marrow derived macrophages were transfected with the KS1-WXY and
incubated with 300 IU/ml murine IFN-
for 24 h. Under these
conditions, IFN-
induced the expression of mRNA for I-A
and
I-A surface expression(29) . In these cells, the KS1 vector
containing the W, X, and Y boxes gave a CAT activity of 1.5%, which
rose to 15.3% in the presence of IFN-
(Fig. 5). The
transfection of PU.1 showed an inhibitory activity on IFN-
induction of CAT activity (43% reduction for the wild type and 39% for
the binding domain). The repressive activity of PU.1 protein on the CAT
expression of the KS1-WXY construction is probably mediated through the
binding to DNA in the PU sequence, because the binding domain, but not
the activation domain, reduced the CAT activity. Thus, the PU.1 protein
was able to repress CAT activity only when it bound to DNA.
Figure 5:
The
PU.1 protein represses the IFN- induction of I-A
expression
in macrophages. Bone marrow-derived macrophages were transfected with
15 µg of the KS1-CAT plasmids described in Fig. 1, together
with 2 µg of the pECE vector or the pECE containing the sequence
for the PU.1 protein, the binding or activation domains of PU.1, or the
retinoic acid receptor.
To
investigate whether there is a competitive binding between PU.1 and
NF-Y, the proteins that bind to the Y box of the I-A promoter,
that could account for the transcriptional repression of the I-A
gene, a gel electrophoresis DNA binding assay was performed. Using
nuclear extracts prepared from the B cell line A20-2J and a 320-bp
probe containing the Y box, a gel electrophoresis DNA binding assay was
performed, and a retarded band was found. The addition of a cold
oligonucleotide covering the Y box displaced the binding of nuclear
factor. However, an oligonucleotide with a mutation of two GG within
the Y box (CTGATTGG) was not able to compete for binding, showing that
the binding to the Y box is specific. We had previously shown that the
nuclear factor that bound to the I-A
Y box was composed of two
components, named factor A and B, which were separated by FPLC using a
monoQ column(11) . These factors correspond to the proteins
NF-YA and NF-YB.
The genes for NF-YA and NF-YB were
expressed in vitro using T7 polymerase to generate RNA and
rabbit reticulocyte lysate for the preparation of protein. When added
individually to a labeled fragment of DNA containing the Y box, none of
the proteins bound well to DNA (Fig. 6). When NF-YA was mixed
with NF-YB, however, the complex bound to the DNA containing the Y box
with high affinity. A retarded band was also found when we used in the
assay transcribed/translated PU.1 or the binding domain of PU.1. The
retarded complex DNA-NF-Y proteins was almost at the same position as
the retarded complex with PU.1. This is probably due to the large
fragment (320 bp) used as a probe. Competition experiments showed that
the retarded bands were specific for the Y box or the PU.1 box. The
bands were eliminated when cold oligonucleotides with the sequence of
the Y box or PU.1 box were added but not when oligonucleotides with the
mutated Y or PU.1 boxes were added to the assays. However, no binding
was detected when we added the activation domain of PU.1. The relative
amount of proteins produced by in vitro transcription/translation was calculated taking into account the
amount of radiolabeled methionine incorporated during the in vitro translation. We then tested the ability of PU.1 to compete with
NF-YA or NF-YB for binding to DNA. When both NF-YA and NF-YB were added
with PU.1 the retarded band was more intense than the band obtained
with NF-YA and NF-YB proteins together (Fig. 6). This suggests
that PU.1 binds to either the same or a different molecule of DNA, but
the binding of PU.1 does not inhibit the binding of NFY proteins to the
Y box.
Figure 6:
Gel eletrophoresis DNA binding assay of
nuclear extracts or in vitro transcribed/translated NF-YA,
NF-YB, or PU.1 proteins. Nuclear extracts were prepared from A20-2J
cells. The probe was a 320-bp fragment of the I-A promoter thet
contained the Y and the PU box. Retarded complexes were detected by
autoradiography. The relative concentration of protein for NF-Y or PU.1
was measured taking into account the amount of radiolabeled methionine
incorporated during the in vitro translation and was
quantitated using an imaging system (AMBIS, Inc.) and then diluted to
equal relative concentration.
In order to characterize the interaction between PU.1 and NFY
proteins and the DNA Y box, we used a 33-bp synthetic oligonucleotide
containing this area. When we incubated in vitro transcribed/translated PU.1 or NF-YA + NF-YB proteins with
the Y box oligonucleotide, the proteins produced a retarded band (Fig. 7). When we included NF-YA+NF-YB and PU.1 proteins a
supershift band was observed. The amount of supershifted DNA was
proportional to the amount of PU.1 included in the assay. These data
demonstrated that, at least in some cases, PU.1 bound to the same DNA
molecule as the NF-YANF-YB complex.
Figure 7: Gel electrophoresis DNA binding assay of in vitro transcribed/translated NF-YA, NF-YB, and PU.1 proteins. The probe was a 70-bp fragment from a vector in which a 33-bp synthetic oligonucleotide containing the Y box sequence was cloned. A supershift band was found when NF-YA, NF-YB, and PU.1 proteins were added together with the probe.
To further characterize the interaction between NFY and PU.1 proteins, we quantified the binding activities of the factors, both separately and together (Fig. 8). NF-YA alone bound only small amounts of DNA, while NF-YB alone bound little or no DNA. When increasing amounts of NF-YA were added to a constant amount of NF-YB (4 µl), a linear relationship between the amount of NF-YA and protein-DNA complex was observed. Under these conditions NF-YB was apparently in excess, since even when high amounts of NF-YA were added to the reaction there was no evidence that the percentage of probe bound reached a plateau (Fig. 8). These results indicate that NF-YA alone binds to DNA, but that the affinity between DNA and NF-YA is much greater when NF-YA is associated with NF-YB. There was a significant binding when either PU.1 or the PU.1 binding site were added to DNA (Fig. 8). In the presence of a constant amount of NF-YB there was a linear increase proportional to the amount of NF-YA and PU.1 present in the reaction (Fig. 8). At each concentration in the presence of PU.1 or the PU.1 binding domain, the binding of the NF-YA+NF-YB complex was higher than in the absence of PU.1. In contrast, in the presence of the PU.1 activation domain the binding of NF-YA + NF-YB remained unchanged.
Figure 8: Titration of DNA binding of recombinant proteins. Gel electrophoresis DNA binding assay was done with increasing amounts of in vitro transcribed/translated NF-YA, NF-YB, and PU.1 proteins. There was binding complementation between NF-YA and NF-YB and cooperative binding with PU.1 or the binding domain of PU.1. The retarded complexes were quantitated using the AMBIS radiographic imaging system. Percentage binding is referred to the total amount of DNA added to the assay. There was binding complementation between NF-YA and NF-YB and cooperative binding with PU.1 or the binding domain of PU.1. In the assays marked as NF-YA + NF-YB, NF-YB was at a constant concentration of 4 µl while the amounts of NF-YA were those indicated in the figure.
Transcription is regulated by gene-specific transcription factors that bind to regulatory elements in gene promoters and enhancers, stimulating the intrinsic basal rate of transcription initiation. In addition, transcriptional repression comes from a set of molecules that inhibit transcription in a gene- specific manner(38, 39) . Repressors are of two different types, passive and active. Passive repressors inhibit the effect of positively acting transcription factors by, for example, competing for their DNA binding sites or making inactive complexes. Active repressors posses intrinsic repressing activity and inhibit transcription directly.
Depending of the genes and the tissues, different
mechanisms have been implicated in MHC class II gene regulation.
Numerous studies have reported factors that exert a negative effect on
the transcription of a wide range of genes(40, 41) .
In some cases, a silencing factor might be operative in suppressing the
transcription of class II genes in nonexpressing cells. This is
suggested by fusion studies that have demonstrated extinction of class
II expression in hybrids of class II-negative and -positive
cells(42) . The fusion of L929 fibrosarcoma cells with splenic
B lymphocytes retained the E gene at the genomic level but none of
the clones had any detectable basal or inducible class II message or
gene product. A more recent study found reactivation of low levels of
class II transcription in plasmacytoma cells and human T cells on
transient fusion with B lymphoblastoid cells and, to a much lesser
extent, with splenocytes(43) . These two studies demonstrated
the presence of different mechanisms controlling the tissue specificity
of MHC class II gene expression.
In cells that do not express MHC
class II genes, a cis-acting regulatory element silences
expression of I-A gene(44) . Using competition
electrophoretic mobility shift assays, the core protein binding site
was localized within a region of an 8-10-bp response element
designated A
NRE at -543 to -534 bp. A nuclear extract
from B cells does not bind to this element and mutation of this site
abrogates the transcriptional silencing activity of this region in
epithelial cells. The A
NRE is not found in the A
gene, but is
present 480 bp upstream of the transcription initiation site of
A
's analogous human gene DQ
. These data suggest that
the A
NRE repressor acts in a gene- and tissue-specific manner.
In our experiments, we show that PU.1 is able to repress the
expression of the I-A gene. Binding to the PU DNA box, which is
next to the Y box in the I-A
gene, was mediated by PU.1 protein as
demonstrated using electrophoretic mobility shift assays and dimethyl
sulfate protection assays. One possible mechanism of repression is
based on the affinity of different proteins to bind to the same or
different DNA sequences(40, 41) . In this regard, we
can mention that the NF-Y factor and the F2 bind in a mutually
exclusive manner to a critical promoter region of the gene for the
IE110k protein of herpes simplex virus(45) . Moreover, NF-Y
factor can act as a negative regulator competing with factor 3 in the
promoter of ApoA-I gene(46) . Recently it has been reported
that YB-1, a protein identified by using a radiolabeled Y box sequence
to screen a
gt 11 expression cDNA library, represses the IFN-
activation of MHC class II genes(47) . This could explain the
inverse relationship between the levels of YB-1 and MHC class II
induction by IFN-
. The data presented show that PU.1 protein is
able to repress the expression of the class II I-A
gene. In our
experiments we show that PU.1 and NF-Y proteins bind to the same DNA
molecule.
The PU box is present only in one of the MHC class II gene
promoters, the I-A, and therefore the PU.1 protein, may act as a
specific gene regulator. Another PU.1 box has been described in the
promoter of the TAP gene(48) . However, the involvement of this
PU box and the PU.1 protein in the regulation of TAP gene expression is
unknown. The regulatory role of PU.1 in I-A
gene expression in
vivo is more difficult to demonstrate. Like Y-B1, PU.1 is
down-regulated by IFN-
, and both proteins may regulate class II
expression by different mechanisms(27, 47) . Recently,
in murine tissue macrophages, it has been shown that IFN-
induces
binding of PU.1 to the I-A
gene(49) . The binding
increases gradually, plating at 6-9 h and decaying to basal
levels 24 h after stimulation. In IFN-
-stimulated macrophages, the
levels of I-A
mRNA can be detected after 8 h of incubation with
IFN-
, increasing gradually up to 24 h(28) . The data from
these two reports suggest a correlation between PU.1 release from the
I-A
gene promoter and the expression of mRNA.
Some
transcriptional activators are down-regulated by inhibitory proteins
with which they form protein complexes with altered or reduced DNA
binding activity. One example of such a mechanism in the repression of
MHC class II genes is provided by the glucocorticoid receptor. This
protein is able to form heterocomplexes with the X box-binding protein
and represses MHC class II I-A gene expression(30) . It
has been reported recently that PU.1 can specifically repress the
glucocorticoid-induced activation of promoters carrying a
glucocorticoid response element and other nuclear receptors such as the
thyroid hormone or retinoic acid(50) . The glucocorticoid
receptor represses PU.1-mediated transcriptional activation, showing
that, in some cases, PU.1 protein can form heterodimers that inhibit
gene expression.
The binding of the NF-Y proteins to DNA increases
in the presence of the PU.1 protein. This could be due to the binding
to different DNA molecules or to the simultaneous binding of NFY and
PU.1 proteins to the same DNA molecule. In the latter case, the
interaction between NF-YA/PU.1 may explain the repression of IA-
gene expression when cells were transfected with a vector coding for
the PU.1 protein. The activation domain of PU.1 is known to bind to the
transcription factor TFIID in vitro(51) . Due to the
proximity of the PU box to the transcription initiation site, the
interaction between PU.1 and TFIID could explain the repressive effect
of PU.1. However, the binding domain of PU.1 without the activation
domain is able to repress I-A
gene expression, suggesting that the
hypothetical interaction with TFIID is not an important factor. It is
well documented that synergism between different transcriptional
activator has a major role in transcriptional activation(52) .
Regulation of MHC class II gene expression requires three separate
elements, the boxes W, X, and Y with stereospecific and distance
constraints(53) , and it also requires cooperation between the
different factors that bind to these boxes (54, 55, 56) . In this context, the complex
of NF-YA and PU.1 proteins could have an inhibitory effect by
disruption of the interactions between the proteins that bind to the W,
X, and Y boxes of the MHC class II genes, which would involve
interference with the preinitiation complex assembly. It is also
possible that active repressors could promote local chromatin changes
resulting in repression of transcription.
The results reported here are one more example of how transcription factors can interact with DNA resulting in reduced expression of genes that are activated by other transcription factors. These interactions provide an important opportunity for cross-talk between different signal transduction pathways and allow for modification of the responses of particular genes to specific extracellular stimuli.