(Received for publication, October 19, 1994; and in revised form, December 14, 1994)
From the
YB-1 is a member of a newly defined family of DNA- and
RNA-binding proteins, the Y box factors. These proteins have been shown
to affect gene expression at both the transcriptional and translational
levels. Recently, we showed that YB-1 represses
interferon--induced transcription of class II human major
histocompatibility (MHC) genes(1) . Studies in this report
characterize the DNA binding properties of purified, recombinant YB-1
on the MHC class II DRA promoter. The generation of YB-1-specific
antibodies further permitted an analysis of the DNA binding properties
of endogenous YB-1. YB-1 specifically binds single-stranded templates
of the DRA promoter with greater affinity than double-stranded
templates. The single-stranded DNA binding sites of YB-1 were mapped to
the X box, whereas the double-stranded binding sites were mapped to the
Y box of the DRA promoter, by methylation interference analysis. Most
significantly, YB-1 can induce or stabilize single-stranded regions in
the X and Y elements of the DRA promoter, as revealed by mung bean
nuclease analysis. In concert with the findings that YB-1 represses DRA
transcription, this study of YB-1 binding properties suggests a model
of repression in which YB-1 binding results in single-stranded regions
within the promoter, thus preventing loading and/or function of other
DRA-specific transactivating factors.
YB-1 is a member of a recently defined family of DNA-binding
proteins, the Y box factors, also known as the cold shock domain
factors(2, 3) . These proteins represent a multigene
family identified in a number of eukaryotic and prokaryotic organisms.
Members of this family include human YB-1, dbpA, dbpB, NSEP-1, and
BP-8; frog FRGY1, FRGY2, YB3, p56, and p54; rat EF1A; murine MUSY1,
MSY1; avian EF1A, RSV-EF-1, chkYB-1; bovine yEF1A#1; and bacterial cspA
and cspB(3, 4, 5, 6, 7) . Y
box proteins are highly conserved, with 97% amino acid homology between
rat EF1A and human YB-1. These factors have been shown to regulate gene
expression at both the transcriptional and translational levels and
several have been suggested to have roles in DNA repair as well as DNA
and RNA condensation. Two closely related Y box proteins, YB-1 and
EF1A, have been shown to regulate transcription. EF1A can activate
transcription through the Rous sarcoma virus long terminal
repeat(8, 9) , while YB-1 has been shown to activate
transcription through the HIV, HTLV1, JCV
promoters(10, 37) . Recently, we showed that YB-1
represses transcription of human major histocompatibility (MHC) ()class II genes(1) . Several other Y box proteins
(FRGY2, FRGY1, MSY1) were shown to repress translation and protein
expression by sequestering mRNA in gametes (11, 12, 13) and somatic cells(14) .
Y box proteins have a broad specificity for nucleic acids, binding
double-stranded DNA, depurinated double-stranded DNA, single-stranded
DNA and
RNA(3, 4, 5, 6, 8, 12, 13, 15, 16, 17, 18) .
Studies to date indicate that the Y box factors are likely to affect
gene expression by various different mechanisms.
YB-1 was originally
cloned by screening a human B cell expression library using a
double-stranded oligonucleotide probe spanning the human DRA X and Y
elements(15) . These authors found that YB-1 binding was strong
to the Y box and weaker to the X box. Binding to the Y box was
dependent on the inverted CCAAT sequence in the Y box. The X and Y
elements are highly conserved in murine and human MHC class II
promoters and are necessary for basal, as well as interferon-
(IFN-
)-induced transcription. We have since shown that YB-1
represses MHC class II gene expression(1) . Transfection of
cells with a YB-1 expression vector repressed endogenous,
IFN-
-induced class II mRNA and protein expression, as well as
IFN-
-induced class II DRA promoter-driven reporter gene
expression.
Sequences within eukaryotic promoters have been
described that are sensitive to reagents that cleave single-stranded
DNA and have been implicated in transcriptional regulation, including
the promoters of the c-myc, -globin, platelet-derived
growth factor A, epidermal growth factor receptor, and decorin
genes(19, 20, 21, 22, 23) .
In addition, eukaryotic single-stranded DNA-binding proteins have
recently been described such as FSB, STR, MF3, p70, ERDP-1 (20, 21, 24, 25) and are implicated
in transcriptional regulation. FSB specifically binds single-stranded
FUSE, the far upstream element of the c-myc promoter, and
transactivates FUSE-CAT reporter constructs(24) . In addition,
activation of the c-myc gene correlates with the induction of
single-stranded sequence in FUSE in vivo.
In this study, the binding properties of YB-1 on the MHC DRA promoter are characterized. We demonstrate that YB-1 specifically binds single-stranded templates of the DRA promoter with much greater affinity than the double-stranded template. Through methylation interference analysis the contact points of YB-1 in the DRA promoter are identified. The possible mechanisms by which YB-1 represses DRA transcription are explored. Mung bean nuclease analysis reveals that YB-1 can stabilize single-stranded regions within the X and Y elements of the DRA promoter. We propose a model of transcriptional repression in which YB-1 binding results in single-stranded regions in the DRA promoter and as a consequence prevents the binding and/or function of the X and Y box transactivating factors.
Whole cell extracts were
prepared by lysing cells in 10 mM Hepes (pH 8.0), 1 mM EDTA, 7 mM -mercaptoethanol, 50 mM KCl,
0.4% Nonidet P-40, 1 µM pepstatin, 1 µM leupeptin, 1 µM E-64, and 100 µM phenylmethylsulfonyl fluoride (Boehringer Mannheim). The lysates
were centrifuged for 30 min at 13,000
g at 4 °C,
and the supernatants were used in EMS reactions. Protein concentrations
for whole cell and nuclear extracts were determined by the Bio-Rad
protein assay (Bio-Rad).
Figure 1: Summary of probes used. The nucleotide sequence of the DRA promoter is shown with the Servanius (W), X (X1X2), and Y elements shaded. The position of the X, Y, W, X + Y, and WXY probes relative to the promoter is shown.
Mung bean nuclease was used to detect
single-stranded regions in double-stranded probes. For mung bean
nuclease, an 187-base pair probe (Fig. 1, WXY) spanning
the DRA promoter was generated by restriction enzyme digest of the
plasmid, 5` 152 DRA-CAT described previously(31) . The
plasmid was digested with XbaI, end-labeled with T4
polynucleotide kinase, and digested with BstYI to generate a
187-base pair, single end-labeled probe. This fragment was purified by
polyacrylamide electrophoresis and electroelution. The probe (4
10
cpm) was incubated with rYB-1 (1 µg) in the presence
of 1 µg of poly(dI-dC) in the EMS buffer in a 10-µl reaction at
37 °C for 20 min. The volume of the reaction was expanded five
times and 0.10 volume of both a 10
mung bean nuclease buffer
(New England Biolabs) and 10 mM ZnSO
were added.
Mung bean nuclease reactions were carried out at saturating
concentrations of mung bean nuclease activity, as determined by
previous titrations of mung bean nuclease on the WXY probe. One unit of
mung bean nuclease (New England Biolabs) was added to the reaction and
incubated at 37 °C. Ten-µl aliquots of the reaction were
stopped at different time points by incubation for 20 min at 37 °C
in 240 ml of stop buffer (100 mM Tris (pH 8.0)), 100 mM NaCl, 20 mM EDTA, 0.1% SDS, 100 µg/ml proteinase K,
200 µg/ml glycogen). The samples were then precipitated and
separated on a 6% polyacrylamide urea, wedge gel. Sequencing reactions (32) of the WXY probe were run simultaneously with the mung
bean nuclease reactions.
YB-1 was originally cloned by its binding to a probe containing the DRA X (comprised of X1 and X2) and Y proximal promoter elements, of which all are required for MHC class II transcription(15) . We have since shown that YB-1 can repress both endogenous DR protein and mRNA levels, as well as DRA promoter-driven, reporter gene transcription (1) . In order to examine the mechanism of YB-1-mediated repression, we produced recombinant YB-1 protein (designated rYB-1) and characterized its binding activity on the DRA promoter. We also used the rYB-1 to generate YB-1-specific antibodies to identify endogenous YB-1.
Figure 2:
Purification of recombinant YB-1. Samples
of lysates from bacteria induced with
isopropyl--D-thiogalactopyranoside to express GST (lane 2), the GST/YB-1 fusion protein (lane 3),
GST/YB-1 purified on glutathione-Sepharose beads (lanes 4 and 6), and YB-1 cleaved from GST/YB-1 beads with thrombin (lane 5) and molecular weight markers (M) were
separated by SDS-polyacrylamide gel electrophoresis and stained with
Coomassie Brilliant Blue.
Figure 3:
A, anti-YB-1 antisera reacts specifically
with rYB-1. Anti-YB-1 (-YB-1) or preimmune sera (Pre-IM) were incubated with immunoblots containing 1 µg
each of GST, rYB-1, NF-YB, or hXBP. rYB-1 is marked by an asterisk. B, identification of endogenous YB-1 in
nuclear extracts. Immunoblots from gels electrophoresed with 45 µg
of either Raji or HeLa nuclear extracts were incubated with anti-YB-1
or preimmune IgG and visualized by ECL. The solid arrowhead marks endogenous YB-1, and the open arrowhead marks the
70-kDa specific band.
Figure 4:
YB-1 forms a stronger complex on
single-stranded DNA with a greater affinity than double-stranded DNA.
YB-1 binding properties were analyzed on single-stranded and
double-stranded X + Y probes by EMS. Increasing concentrations of
rYB-1 (500-5 ng) were incubated with X + Y probes and
separated on nondenaturing gels. Probes were: X + Y single strand,
strand 1 (X + Y), X + Y
single strand, strand 2 (X + Y
), X
+ Y double strand, annealed oligonucleotides (X + Y
), and X + Y double strand probe
isolated from plasmid (X + Y
).
Putative YB-1 multimers are marked by the solid arrow and
monomers by the asterisk.
In contrast to strand 1, rYB-1 bound to X
+ Y, and to X + Y
with much lower
affinities (Fig. 4, lanes 8-19). To rule out the
possibility that YB-1 binding on the double-stranded probe was due to
contaminating single-stranded species, the double-stranded probe was
isolated directly from a plasmid (Fig. 4, lanes
20-24). Binding to this probe was extremely weak, indicating
that YB-1 binds poorly to double-stranded DNA containing the X and Y
elements. Preferential YB-1 binding to single-stranded DNA is
consistent with the ability of other Y box proteins (dbpB, NSEP-1,
BP-8, and cspB) to bind single-stranded
DNA(5, 16, 17, 34) .
To determine
the specificity of rYB-1 binding, reactions were carried out with
100-fold molar excess cold homologous or heterologous single-stranded
oligonucleotides (Fig. 5A, lanes 1-3). Unlabeled
homologous X + Y DNA competed the complex formation,
whereas an oligonucleotide spanning strand 1 of an unrelated DNA, the W
element of the DRA promoter, did not. Similarly, other single-stranded,
heterologous oligonucleotides, including strand 1 of the DRA octamer
element, strand 2 of the W element, and strand 1 of the myb binding
site of the c-myc promoter did not compete for rYB-1 binding
(data not shown).
Figure 5:
A, endogenous YB-1 binding to
single-stranded DNA is specific. rYB-1 (lanes 1-3) and
whole cell U937 extract (lanes 4-6) were incubated in
the presence or absence of 100-fold molar excess homologous (X
+ Y) or heterologous (W) competitor DNA in an EMS
assay. The asterisk marks putative monomeric rYB-1 and
endogenous YB-1. The solid arrowhead marks the slower
migrating complex in the U937 extract. B, anti-YB-1
supershifts endogenous YB-1. rYB-1 (lanes 2-10) or U937
whole cell extract (lanes 11-17) were preincubated with
5, 2, 1, or 0.5 µl of anti-YB-1 (I) or negative control (C) antibodies for 30 min at 4 °C. The reactions were
incubated with the X + Y probe in an EMS assay.
Monomeric YB-1 is marked by an asterisk, and the specific,
supershifted antibody-YB-1-probe complex is noted with an open
arrowhead.
EMS analysis using whole cell extracts from the
U937 cell line incubated with the X + Y probe
resulted in a complex that co-migrated with rYB-1 (Fig. 5A,
lanes 1 versus 4). In addition to this band, a slower migrating
complex was observed. Both complexes were specifically competed by
homologous but not heterologous probe, as was rYB-1 (Fig. 5A, lanes 5 and 6).
The anti-YB-1 antisera was used to identify the endogenous YB-1 in U937 extracts. Addition of YB-1-specific antibodies to the rYB-1 EMS reaction resulted in a slower migrating, supershifted complex (Fig. 5B, lanes 3, 5, 7, and 9). No such complex was observed either with the negative control antibodies (lanes 4, 6, 8, and 10) or with antibodies in the absence of rYB-1 (lane 1). The supershifted band in the U937 extract is of similar migration as rYB-1 and is antibody-specific (compare lanes 11, 13, and 15 versus 12, 14, and 16). Its formation was also dependent on the concentration of antibody used.
When
single-stranded probes spanning either the X box or the Y box were
used, it was found that rYB-1 bound preferentially to X and to Y
(Fig. 6). Titration of rYB-1 on
these probes shows that complex formation was lost at 40 ng of rYB-1 on
Y
, whereas a strong complex formation was observed at
this concentration on X
. This may reflect a stronger
affinity of YB-1 for X
than Y
. Both
X
and Y
complexes were also competed by an
homologous cold competitor, but not by an unrelated probe, indicating
specificity of binding (data not shown). rYB-1 also formed a weak
complex with X
and Y
(Fig. 6, lanes 21-24).
Figure 6:
YB-1 preferentially binds X and Y
. rYB-1 was incubated in concentrations
ranging from 750 to 40 ng with the following single-stranded probes:
X
(lanes 1-5), X
(lanes
6-10), Y
(lanes 11-15), or
Y
(lanes 16-20) and double-stranded
probes: X
(lane 21) and Y
(lane
24) in EMS reactions. The asterisk marks putative
monomeric rYB-1.
Figure 7:
A, methylation interference analysis of
YB-1 binding. Methylation interference was carried out as described
under ``Materials and Methods'' using Y,
Y
, X
, and X
probes. Free and
bound fractions are marked as F and B, respectively.
Strong interference and hypersensitive sites are marked by large
closed and open arrowheads, respectively, whereas weaker
patterns are marked by smaller arrows. The positions of the
bands in the probe are denoted by the nucleotide base number (see Fig. 7C). B, YB-1 promotes single-stranded
regions in the X and Y elements of the DRA promoter. 1 µg of YB-1
was incubated with the WXY
probe in EMS conditions,
followed by incubation with the single-strand-specific mung bean
nuclease for increasing periods of time at 37 °C. Hypersensitive
sites are marked by open circles. C, summary of YB-1
interaction on the DRA promoter. YB-1 contact points on single-stranded
DNA (closed arrows) and YB-1-induced hypersensitive sites (open arrows) on the DRA promoter, as determined by
methylation interference analysis, are illustrated. YB-1-induced
single-stranded sites on a double-stranded probe, as determined by mung
bean nuclease analysis, are indicated by open circles in the lower panel. The W, X, Y, and octamer elements are noted by shaded boxes.
Methylation interference analysis identified
single-stranded YB-1 binding sites on both strands of the X box in the
X2 element, with the strongest protection on X (Fig. 7A, lanes 1-6, summarized in C). This strand bias is consistent with the greatest binding
affinity of YB-1 for X
observed by EMS (Fig. 6).
Four times more rYB-1 was required to generate a methylation
interference pattern on X
. In contrast to the results
with the X probes, no interference pattern was observed on the
Y
nor Y
probes, despite rYB-1 interaction
with Y
seen by EMS (Fig. 6). One obvious
explanation is that methylation of G residues did not interfere with
protein binding.
The EMS results in Fig. 6demonstrate that
rYB-1 binds Y, although with a lower affinity than
Y
(lanes 23 and 24). Previously,
double-stranded Y box binding sites have been reported for the Y box
protein EF1(8, 9) . In those reports, methylation
interference analyses were carried out using chick embryo nuclear
extracts (or fractions of) that most likely contained other Y-binding
proteins, such as NF-YA and NF-YB. Purified, recombinant YB-1 is used
here to address this concern and to define YB-1 double-stranded Y box
binding. Incubation of rYB-1 with Y
resulted in a stretch
of hypersensitive G and A residues on both strands of the probe (Fig. 7A, lanes 7-12). This observation suggests
that rYB-1 preferentially binds methylated G and A residues in the
context of the CCAAT sequence.
Previous experiments have shown that YB-1 can suppress DRA
promoter function in IFN--inducible cell lines. This manuscript
explores the possible mechanism by which this occurs. The most
important finding is that YB-1 induces or stabilizes a single-stranded
region in the Y box. This region coincides with the binding site of
YB-1 on a double-stranded probe, as determined by previous DNA binding
assays(15) . Based on these findings we propose that YB-1 binds
to double-stranded sequences flanking the Y box which may be prone to
single-strandedness and induces or stabilizes the single strand
configuration.
The CCAAT box per se in the Y element binds the NF-Y/CBF family of proteins. Previously, we have shown that the in vivo occupancy of NF-Y is the most critical step in the assembly of the proteins binding the proximal elements(35) . Mutations in the Y/CCAAT sequence prevented in vivo occupancy of adjacent X1X2 elements as determined by genomic footprinting. Taken together we propose that YB-1-induced single-strandedness around the CCAAT element may interfere with the binding of NF-Y/CBF to the CCAAT box and disrupt the assembly of this proximal promoter.
We have also identified YB-1 binding sites by methylation interference analysis within the X1X2 element once this sequence has been made single-stranded. The mung bean nuclease data also demonstrate that YB-1 can induce or stabilize single-stranded sites within the X element on a double-stranded probe. The mung bean nuclease-sensitive sites directly flank the methylation interference contact sites. This suggests that YB-1 may also bind to double-stranded sequences in or around the X element and induce or stabilize a single-stranded region. This structural change and/or occupancy of the X2 element may additionally prevent binding of X box transactivating proteins, e.g. hXBP, X2BP, and RFX(17, 29, 36, 37) .
Single-stranded DNA binding has been described for several Y box
proteins(4, 5, 17, 18) . The binding
of two other Y box proteins, NSEP-1 and BP-8 to single-stranded DNA
that is pyrimidine-rich has been interpreted to indicate binding to
triple helix DNA or H-DNA in the human c-myc and -globin
promoters, respectively. The reagents used here, as well as in these
other studies, cannot distinguish between H-DNA and single-stranded
DNA. There are several CT-rich stretches in the DRA promoter flanking
the X box and the Y box; however, these are most likely too short to
form H-DNA. In addition, the binding sites determined by methylation
interference are not CT-rich. Alternatively, it is possible that the
nuclease-sensitive sites in the c-myc promoter to which NSEP-1
binds are in fact single-stranded DNA, as opposed to H-DNA.
In addition to our study, two studies have shown that another Y box protein, FRGY2, represses gene expression. The mechanism has been elegantly determined and appears to be mediated by binding to and preventing translation of mRNA(11) . In these studies, mRNA levels were either maintained at a steady state or increased. YB-1, however, appears to be acting at the level of the DR promoter, as opposed to sequestering mRNA, in that DRB mRNA levels are reduced, and the inhibition is specific to the MHC class II promoter-CAT constructs, whereas the heat shock 70- and thymidine kinase-CAT constructs are not affected. Although we have shown here that YB-1 binds single-stranded DNA, we cannot rule out that YB-1 may also interact with RNA repressing translation.
An additional contribution of this study is the generation of recombinant YB-1- and YB-1-specific antibody. This antibody recognizes not only rYB-1, but has allowed us to identify endogenous YB-1 in whole cell extracts as well as in nuclear extracts. This will allow us to further characterize YB-1 under varying conditions.
We have shown that YB-1 is a transcriptional repressor and propose that this repression is accomplished by promoting single-stranded regions in the Y and X elements. Significant multimer formation was observed with rYB-1, as has been reported for other Y box proteins(13) . It is possible that one dimer or multimer of YB-1 spans the X and Y elements, with one unit binding strand 1 in the X box while the other unit binds strand 2 in the Y box (Fig. 8). In this situation YB-1 may not only preventing binding of the necessary transactivating factors to X and Y, but may also introduce a contortional constraint in this region of the promoter.
Figure 8: Illustration of a proposed model by which YB-1 represses DRA transcription. This model proposes that in the absence of YB-1, the DRA promoter is in a closed double-stranded form. YB-1 facilitates the opening and/or traps the DNA in the open form by its binding to the single-stranded templates. This prevents binding and/or function of other DRA-specific DNA-binding proteins that activate transcription.