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(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
MHC ( 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 Using an
oligonucleotide containing the Y box of the IA 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 Recently, it has been
reported that IFN-
Figure 3:
Repression of I-A
The PU.1 protein was cloned using an expression library
(
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
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 To determine the effect of PU.1 protein on
I-A To
ascertain whether the suppressive effect of PU.1 was specific to the
I-A
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
We also tested the
effect of the PU.1 protein on I-A expression induced by IFN-
Figure 5:
The
PU.1 protein represses the IFN-
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
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
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-YA
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 In cells that do not express MHC
class II genes, a cis-acting regulatory element silences
expression of I-A In our experiments, we show that PU.1 is able to repress the
expression of the I-A The PU box is present only in one of the MHC class II gene
promoters, the I-A 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 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- 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.
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24385-24391
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gene Expression by the Transcription Factor PU.1 (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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.
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. (
) 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. (
)
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., 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.
Cells
In these studies, the cell lines A20-2J
(mouse B lymphocyte) and L929 (mouse fibroblast) were used. Mouse bone
marrow-derived macrophages were produced in vitro using L
cell-conditioned medium as previously described(28) . After 6
days in culture, bone marrow-derived macrophages were washed and
incubated in the absence of L cell-conditioned medium for 4 days. For
IFN- stimulation studies, 300 IU/ml of murine IFN-
(a gift
from Genentech, Inc., South San Francisco, CA) were added to the medium
for 24 h. Under these conditions IFN-
induces the expression of
mRNA for I-A
and I-A surface expression(29) .Nuclear Extract Preparation
Nuclear extracts were
prepared as described previously(10) . In brief, cells were
lysed in a homogenizer (Dounce). The crude nuclei were extracted at 4
°C with a buffer containing 0.4 M NaCl for 30 min with
continuous stirring followed by centrifugation at 10
g for 1 h. The supernatant was dialyzed, and
extracts were cleared by centrifugation at 10
g for 10 min. The supernatant (protein concentration of 1-5
mg/ml) was frozen in aliquots and stored at -70 °C.Transfection Assays
A20-2J (mouse B
lymphocyte) cells were transfected in suspension in 1 ml using the
DEAE-dextran method as described previously(30) . 15 µg of
the experimental DNA and 3 µg of the plasmid, pCH110, a
-galactosidase expression vector used to measure transfection
efficiency and 30 µg of DEAE-dextran were added to each plate. In
the cotransfection experiments with PU.1 or the retinoic acid receptor,
2 µg of the indicated plasmid were used(30) .
Chloramphenicol acetyltransferase (CAT) assays were performed using a
standard protocol(31) . Briefly, cells were isolated 48 h after
the DNA was added and subjected to three freeze-thaw cycles in dry
ice/ethanol at 37 °C. The extracts (10-60 µl standardized
by -galactosidase activity) were incubated with
[
C]chloramphenicol and acetyl coenzyme A for 60
min at 37 °C, incubated for 7 min at 60 °C, followed by
extraction with ethyl acetate. The samples were dried and resuspended
in 20 µl of ethyl acetate for thin layer chromatography.
Acetylation was quantified using a radioanalytic imaging system (AMBIS,
Inc., San Diego, CA). Each experiment was performed at least three
times, and the figures represent the mean and 1 S.D.Plasmid Constructions
The CAT reporter
constructions were made using the KS-SV2CAT vector (32) in which the SV40 enhancer region (SphI to PstI) was removed. The different constructions, KS1-WXY
(-124 to -26) and KS1 mutant (mutation in the Y box as
indicated in Fig. 3) containing the I-A
promoter, were
generated using specific oligonucleotides or by polymerase chain
reaction using the plasmid 12.33.2, which contains the I-A
promoter(10) . The DNA fragments were ligated directionally
into XbaI-SmaI-digested KS1 vector. DNA fragments
used for the gel electrophoresis DNA binding assay were generated by
cloning. Double-stranded oligonucleotides were prepared using a DNA
synthesizer (380 A; Applied Biosystems Inc., Foster City, CA) and
cloned in the BamHI site of the vector, pGEM1. The plasmid
containing the gene for the retinoic acid receptor (pECE RAR) was
obtained from Dr. M. Pfahl (La Jolla Cancer Research
Foundation)(33) . The plasmids containing NF-YA and NF-YB were
obtained by polymerase chain reaction, using oligonucleotides prepared
from the sequence of the plasmid vectors containing NF-YA and NF-YB as
described previously. Finally, PU.1 and the PU.1 binding
site (170-260) with the CAT construction were as described
previously(13) . The plasmid containing the activation domain
(75-93) was also as described previously(15) . The pECE
vector contains the SV40 promoter and enhancer(34) . In
vitro transcription/translation of the NF-YA, NF-YB, and PU.1 was
performed as described by the manufacturer (Promega, Madison, WI). RNA
was transcribed using T3 RNA polymerase. The RNA template (2 µg)
was added to a rabbit reticulocyte lysate (Promega) to generate
[S]methionine-labeled protein in vitro.
Protein production was confirmed by SDS-polyacrylamide gel
electrophoresis and autoradiography. Gel electrophoresis DNA binding
assays were performed directly with the in vitro translated
product. 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, quantified using
an imaging system (AMBIS, Inc., San Diego, CA), and then diluted to
equal relative concentration. In some cases the gels were dried and
placed into contact with two sheets of Kodak XAR film to distinguish
the
S label from the
P DNA label.
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.).
Gel Electrophoresis DNA-Binding Assay and Dimethyl
Sulfate Protection Assay
The DNA fragment containing the Y box
sequence (320 bp) was used previously to characterize the Y box
DNA-binding protein(10) . In some cases, a plasmid containing a
synthetic 33-bp oligonucleotide
(5`-gaTCCAATGCTGATTGGTTCCTCACTTGGGACg-3`) containing the Y box was
digested with HindIII, labeled by filling the ends with
-P-labeled dNTPs and Klenow polymerase and digested
with EcoRI. A 70-bp fragment containing the desired protein
binding site was thus isolated from a 6% polyacrylamide gel. For gel
electrophoresis DNA binding assays, DNA (10,000 cpm of
P
label, approximately 0.1 ng) and nuclear extract or proteins prepared
by in vitro transcription/translation using a rabbit
reticulocyte lysate system (Promega) were mixed in a total volume of 20
µl with a buffer containing 12 mM HEPES (pH 7.9), 60
mM KCl, 5 mM MgCl
, 0.12 mM EDTA,
0.3 mM phenylmethylsulfonyl fluoride, 0.3 mM
dithiothreitol, and 12% glycerol. Two µg of poly(dI-dC) (Pharmacia
Biotech Inc.) was also added to each reaction mix. Samples were
incubated at 20 °C for 30 min and then loaded in a 6%
polyacrylamide gel (acrylamide:bisacrylamide, 30:1), 0.25 TBE
(1 TBE = 89 mM Tris, 89 mM boric acid,
2 mM EDTA) containing 5% glycerol, and electrophoresis was
carried out at 4 °C and 250 V for 3 h. After electrophoresis the
gels were dried and placed into contact with x-ray film (Kodak XAR-5).
Radioactivity was quantified using radioanalytic imaging system (AMBIS,
Inc.). For dimethyl sulfate protection assays a 320-bp fragment of the
I-A promoter was used according to the protocol previously
described(10) .
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.
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.
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) . 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. 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).
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.
. 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.
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.
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.
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.
NF-YB complex.
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. 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 ANRE 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 ANRE 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 ANRE repressor acts in a gene- and tissue-specific manner. 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., 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. 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.
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.
), interferon-
; bp, base pair(s).
))
We thank John Knight for the synthesis of the
oligonucleotides. We also thank Dr. Magnus Pfahl for the plasmid
containing the retinoic acid receptor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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