From the
Analysis of the core promoter sequence of the mammalian
ribosomal RNA genes revealed an E-box-like sequence (CACGCTG) to which
the upstream stimulatory factor (USF) binds. Because the core promoter
binding factor (CPBF) binds specifically to the core promoter sequence
of the ribosomal RNA gene (Liu, Z., and Jacob, S. T. (1994) J.
Biol. Chem. 269, 16618-16626) and resembles USF in some
respects, we explored the potential relationship between USF and CPBF.
Competition electrophoretic mobility shift assay using labeled core
promoter probe and several unlabeled competitor oligonucleotides showed
that USF can indeed bind to the core promoter and that only those
oligonucleotides with an E-box sequence could compete in the
promoter-protein complex formation characteristic of CPBF. This complex
formation was thermostable, a unique property of USF. Furthermore,
antibodies raised against USF cross-reacted with the 44-kDa component
of rat CPBF. To prove further the relationship between CPBF and USF, we
examined the effects of the unlabeled USF and ribosomal gene core
promoter oligonucleotides on polymerase (pol) I and pol II
transcription. Both oligonucleotides inhibited rDNA transcription as
well as transcription from the adenovirus major late promoter. Only the
oligonucleotides that contain the E-box sequence competed in the
transcription. These data indicate that the promoter sequence of
mammalian ribosomal RNA gene contains an USF-binding site, that the
44-kDa polypeptide of CPBF is related to the 44/43-kDa polypeptide of
human USF, and that USF or USF-related protein can transactivate both
pol I and pol II promoters.
Transcription of ribosomal RNA genes (rDNA) is controlled by
cis-acting elements and trans-acting factors (usually proteins) that
interact with the specific cis-elements (for reviews, see Refs.
1-3). There are three important cis-acting elements on rDNA,
namely promoters, enhancers, and terminators. The core promoter region
of most species lies between -40 and +10 with respect to the
initiation site. The enhancer element in rat rDNA consists of a repeat
element (11.5 times repeat of a 130-base pair sequence)
(4, 5) located proximal to the core promoter and a non-repeat
element far upstream (approximately 2 kilobase pairs from the +1
site) of the core promoter
(4) . The repeat element is pol
I(
Until 1990, only limited
success was achieved in the characterization of all the crucial
trans-acting factors that interact with the various cis-elements on
rDNA and regulate rDNA transcription initiation. In the past 3 years,
however, significant achievements have been made in this regard. In
addition to RNA polymerase I, initiation of rDNA transcription requires
TATA-binding factor (TBP) and TBP-associated pol-specific factors
called TAFs, present in a complex called SL1
(8) . The species
specificity of pol I transcription, which is one of the hallmarks of
pol I transcription, appears to reside in one or more of TAFs
(9) . Human or frog SL1 does not interact with respective core
promoter element unless another factor, upstream binding factor (UBF),
is also present
(10, 11) . The rodent SL1, however, can
independently bind to the core promoter
(12) .
A factor from
the rat mammary adenocarcinoma ascites cells designated E
The
oligonucleotides used as double-stranded DNA probes and competitors in
the electrophoretic mobility shift assay were ( a) rat core
promoter (rCP), ( b) mouse metallothionein-I (MT-I) MLTF,
( c) mouse MT-I MRE-d, ( d) mouse MT-I Sp1,
( e) CTF/NF1, and ( f) Oct 1. Oligonucleotides rCP,
MLTF, MRE-d, and Sp1 were synthesized in the in-house facility. The
oligonucleotides CTF/NF1 and Oct 1 were obtained from Promega (Madison,
WI).
The present study has demonstrated that the 44-kDa
polypeptide of rat CPBF is immunologically and functionally related to
the RNA polymerase II transcription factor USF/MLTF
(28) . It is
likely that CPBF is the rat homolog of human USF protein. Several lines
of evidence presented here support such a notion. First, anti-USF
antibodies cross-react with the 44-kDa subunit of CPBF. Second, binding
of CPBF to rDNA core promoter can be inhibited by the MLTF/USF
oligonucleotide in trans. Third, RNA polymerase I
transcription is competed by MLTF oligonucleotide in trans,
whereas RNA polymerase II transcription is competed by rat rDNA core
promoter oligonucleotide in trans. Fourth, preincubation with
an oligonucleotide that does not contain the E-box motif, the
USF-binding site, does not block transcription. Finally, as observed
for USF, CPBF is also thermostable; the major CPBF-DNA complex is
unaltered in extracts incubated up to 55 °C (data not shown).
USF was identified initially by its ability to bind and activate the
adenovirus major late promoter
(29, 30) . Two different
forms of USF were identified in HeLa cells with molecular masses of 44
and 43 kDa
(31) . USF can bind and activate several pol II
promoters that include those of mouse metallothionein-I
(25, 32) and p53 (Ref. 33, and references therein). It can also
stimulate pol III transcription of sea urchin U6 small nuclear RNA gene
(34) . To our knowledge, the present study is the first report
of the involvement of a USF related protein, namely CPBF, in pol I
transcription. The strong immunological cross-reactivity of the 43-kDa
(USF-1) antibody with the 44-kDa polypeptide of CPBF in fraction DE-B
and purified fraction from rodent cells suggests that either the 44-kDa
form is the predominant homolog of USF in rodent cells, or the CPBF
polypeptide represents a new member of the USF family or the USF
related-helix-loop-helix proteins (Refs. 28 and 35, and references
therein).
Although CPBF from the rat cells contains a 39-kDa
polypeptide, this polypeptide is not immunologically related to the
larger polypeptide. The two CPBF polypeptides can bind to the core
promoter
(16) . It is, however, not certain whether both
polypeptides are required for trans-activation of the pol I promoter.
The 39-kDa polypeptide does not appear to be a degradation product of
the larger polypeptide, as incubation of CPBF at 37 °C did not
result in increased level of the smaller polypeptide (data not shown).
The smaller polypeptide may be closely associated with CPBF only in rat
or mouse. On the other hand, it may have a specific role in rDNA
transcription, which is yet to be elucidated.
It is noteworthy that
HeLa USF consists of 43- and 44-kDa polypeptides which interacts
independently with a specific DNA element
(31) . Using a
recombinant 43-kDa USF, Pognonec and Roeder
(36) have
demonstrated that the 43-kDa component exhibits an intrinsic DNA
binding and transcriptional activation, and suggested that the tightly
controlled interaction between both components may be necessary for the
activation of adenovirus major late promoter. Interestingly, CPBF
purified from HeLa cells consists of a single 44-kDa polypeptide, which
is identical to the molecular size of human USF-2; this protein by
itself could stimulate human and rat rDNA transcription as much as
5-fold.(
The present data demonstrate that the 44-kDa polypeptide of CPBF or
USF/MLTF is involved in the initiation of pol I transcription. By
contrast, the 43-kDa polypeptide along with the 44-kDa polypeptide is
responsible for the trans-activation of pol II promoters. USF has been
shown to interact with RNA polymerase II transcription factor complex,
TFIID
(30, 31) , which is composed of TATA box-binding
protein, TBP and TAFs. Interaction of CPBF, the USF-related protein,
with the E-box-like sequence in the rDNA core promoter may help recruit
TBP and TAF(s). Testing this possibility requires further study.
Nevertheless, this study has clearly shown for the first time that a
USF-like protein can bind to the pol I core promoter sequences and
trans-activate pol I promoter.
We thank Dr. Robert G. Roeder for providing the
adenovirus major late promoter (pAdML(C
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
-specific, whereas the non-repeat element can
enhance transcription from both pol I and pol II promoters in vitro and in vivo (5, 6) . A third cis-acting
element, which is rather unique to ribosomal RNA gene, is a terminator
sequence located at the 3` end of pre-ribosomal RNA coding region and
just upstream of the promoter
(7) .
BF
(enhancer 1-binding factor) that binds to the enhancer and promoters of
rat rDNA was characterized in our laboratory
(13) . Subsequent
study showed that this protein is related to the human Ku autoantigen
(14) and that it is involved in the initiation of rDNA
transcription, particularly in the preinitiation complex formation
(15) . Recently, we have characterized another protein from the
rat cells, called core promoter-binding factor (CPBF)
(16) .
This protein consists of two polypeptides with molecular masses of 44
and 39 kDa. Reconstitution transcription assay showed that it is
essential for rDNA transcription. Immunoprecipitation experiments using
antibodies against the human Ku protein revealed that rat
E
BF can cross-react with these antibodies and that it can
interact physically and functionally with CPBF
(17) . Because
association between E
BF and CPBF enhances DNase I
footprinting of the core promoter(
)
and
stimulates rDNA transcription in a synergistic manner, it was of
interest to characterize CPBF further with respect to the nature of its
functional interaction with E
BF/Ku. In the course of this
study, we found that CPBF, the core promoter-binding factor, is
immunologically and functionally related to USF, the upstream
stimulatory factor.
Plasmids and Oligodeoxynucleotides
The following
plasmids were used for transcriptional analysis: ( a)
pB7-2.0 plasmid, which contains rat rDNA region spanning from
-167 to +2000; and ( b) pAdML(CAT)
plasmid, which contains adenovirus major late promoter spanning
-400 to +10 linked to a 390-base pair synthetic DNA fragment
containing C, A, and T residues (for details, see Ref. 18).
Preparation of CPBF and Extracts for
Transcription
Whole cell extract was prepared from rat mammary
adenocarcinoma ascites cells and rat hepatoma N1-S1 cells essentially
as described by Zhang and Jacob
(13) and was fractionated by
DEAE-Sephadex chromatography. The fraction eluting with 175 m
M
(NH)
SO
, designated DE-B, was used
for rDNA transcription
(19) . HeLa nuclear extract was prepared
following the protocol of Shapiro et al. (20) . CPBF
was purified to homogeneity from rat mammary adenocarcinoma ascites
cells according to the procedure of Liu and Jacob
(16) . The 44-
and 39-kDa polypeptides comprising CPBF were resolved on
SDS-polyacrylamide gel electrophoresis and detected by silver staining.
Electrophoretic Mobility Shift Assay
(EMSA)
Electrophoretic mobility shift assay using labeled rat
rDNA core promoter, MLTF, and fraction DE-B was performed as described
by Liu and Jacob
(16) .
In Vitro Transcription Analysis
In vitro transcription of rat rDNA was performed essentially as described
by Kurl et al. (19) . Plasmid pB7-2.0 linearized
with XhoI was used as the template. The size of the correct
run-off transcript (initiated at the +1 site) must be 635
nucleotides. In vitro transcription of the
pAdML(CAT) template was carried out essentially as
described by Sawadogo and Roeder
(18) . In competition
transcription assays, the extracts were preincubated with competitor
oligonucleotides for 10 min on ice prior to addition of the templates.
RNA was extracted and electrophoresed on 4% polyacrylamide, 7
M urea gels. The transcripts were visualized by
autoradiography and quantitated by densitometric scanning using One
scanner (Apple Computer, Inc.) and the Scan Analysis Program.
Western Blot Analysis
HeLa nuclear extract,
fraction DE-B from rat ascites cell extract, and purified CPBF were
subjected to electrophoresis in 10% SDS-polyacrylamide gels. For
immunodetection, proteins were transferred to nitrocellulose membranes
and blocked with 10% nonfat dry milk in Tris-buffered saline containing
0.05% Tween 20 (TBST). The membranes were probed with anti-USF antibody
(1:1000 dilution) and washed three times with TBST. The bound primary
antibody was detected using alkaline phosphatase-conjugated anti-rabbit
IgG (Promega), and the color was developed using
5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium
solution.
The Core Promoter Regions of Mammalian Ribosomal RNA
Gene Contain an E-box-like Sequence
The similarities in the
molecular size and other characteristics of the human gene-specific USF
and the larger polypeptide of CPBF prompted us to examine closely the
rat ribosomal RNA gene promoter for a potential USF-binding site.
Analysis of the core promoter sequence of rat, mouse, hamster, and
human ribosomal RNA gene revealed the presence of a sequence CACGCTG
(located between +6 and +12 with respect to the initiation
site) that was similar to the E-box motif (CACGTG)
(24) to
which USF (upstream stimulatory factor) binds (see Fig. 1). Although
the rDNA promoter sequence contained an additional C in the potential
USF binding site, it was logical to investigate further the structural
and functional relationship between CPBF and USF.
CPBF and USF, the Basic Helix-Loop-Helix-Zipper
DNA-binding Protein, Bind to the Same Sequence within the Rat rDNA Core
Promoter
To prove the potential relationship between CPBF and
USF, we studied the specificity of the CPBF binding to the rDNA
promoter sequence by a competition EMSA. Fraction DE-B prepared from
N1-S1 cells was preincubated with unlabeled competitor oligonucleotides
before addition of the labeled core promoter. In the absence of cold
competitor oligonucleotide, a doublet (indicated by arrows) of
CPBF-DNA complex ( C) was observed (Fig. 2 A, lane 2). An oligonucleotide representing the rat core promoter
region with a E-box motif (see Fig. 1) efficiently competed for
the CPBF binding activity (Fig. 2 A, lane 3). To confirm that CPBF binding activity represents USF
or USF-related protein, unlabeled oligonucleotide corresponding to the
MLTF/USF binding site on the mouse metallothionein-I gene promoter was
used in the EMSA. This oligonucleotide containing the E-box motif also
competed as efficiently as the rat rDNA promoter oligonucleotide for
the CPBF binding activity (Fig. 2 A, lane 4). The specificity of the competition was assessed by
using oligonucleotides that do not contain E-box motif, namely Sp1,
MRE-d (derived from the mouse MT-I gene promoter), NF1, and Oct 1. The
oligonucleotide Sp1 that binds Sp1
(25) did not compete for
CPBF binding activity (Fig. 2 A, lane 5). Similarly, the metal-responsive element MRE-d that
binds another transcription factor
(26) did not compete for the
CPBF binding activity (Fig. 2 A, lane 6). The
other two competitor oligonucleotides, NF1 and Oct 1, which bind the
transcription factors CTF/NF1 and Oct 1, respectively, also did not
compete for CPBF (Fig. 2 A, lanes 7 and
8). The slower migrating bands probably represent the complex
between the core promoter and the RNA polymerase I transcription factor
EBF
(13) .
Figure 1:
Nucleotide sequence of rat (21), mouse
(22), hamster (23), and human rDNA (22) core promoters (-36 to
+14). The boldface letters indicate the E-box consensus
sequence (CACGTG).
Figure 2:
A, electrophoretic mobility shift assay
using rat core promoter oligonucleotide (rCP) and fraction DE-B (4
µg) from rat N1-S1 cells. Lane 1,
P-labeled 58-base pair rCP alone; lane 2, rCP and fraction DE-B. In competition assay, fraction
DE-B was preincubated with 100 ng each of the oligodeoxynucleotide
corresponding to rCP ( lane 3), MLTF ( lane 4), Sp1 ( lane 5), MRE-d ( lane 6), NF1 ( lane 7), and Oct 1 ( lane 8) (see ``ExperimentalProcedures''
for details on the competitors). Each reaction contained 40 ng of
poly(dI
dC) as nonspecific competitor. F designates the
position of the free probe. Arrow indicates the CPBF-DNA
complex ( C) and arrowheads E
BF-DNA
complexes. B, electrophoretic mobility shift assay using mouse
MT-I MLTF oligonucleotide probe and fraction DE-B (4 µg) from rat
hepatoma N1-S1 cells. Lane 1,
P-labeled
MLTF alone; lane 2, fraction DE-B incubated with
labeled MLTF probe. In competition assay fraction DE-B was preincubated
for 10 min. with 100 ng each of unlabeled oligonucleotides
corresponding to the binding sequences for rCP ( lane 3), MLTF ( lane 4), MRE-d ( lane 5), Sp1 ( lane 6), and NF1 ( lane 7). Each reaction contained 100 ng of poly(dI
dC) as
nonspecific competitor. F designates the free probe, C represents the USF protein-MLTF complex, and C1 represents possibly E
BF protein
interaction.
To test further the potential
relationship of CPBF and USF, an electrophoretic mobility shift assay
was performed using labeled major late transcription factor (MLTF),
which is known to bind USF. Fraction DE-B from N1-S1 cells was
preincubated with unlabeled competitor oligonucleotides before the
addition of labeled MLTF oligonucleotide. In absence of the cold
competitor, a doublet ( C) of USF-DNA complex was observed
(Fig. 2 B, lane 2), which is similar to
the doublet of CPBF-DNA complex (Fig. 2 A, lane 2). The most noteworthy observation was that the USF-DNA
complex formation was efficiently competed by cold oligonucleotide
corresponding to the rat rDNA core promoter oligonucleotide (rCP)
(Fig. 2 B, lane 3). As expected, MLTF
oligonucleotide also competed for this complex formation
(Fig. 2 B, lane 4), whereas MRE-d, Sp1,
and NF1 oligonucleotides that do not contain E-box motif did not
compete for the USF-DNA interaction (Fig. 2 B, lanes
5-7). The complex C1 (Fig. 2 B)
probably represents EBF-DNA interaction, which is competed
by cold rCP, MLTF, and to some extent by SP1 and NF1 but not by MRE-d.
CPBF Is Immunologically Related to USF
To
substantiate further that CPBF is related to USF, an immunoblot
analysis was performed using fraction DE-B from rat cells, HeLa nuclear
extract, and purified CPBF, and polyclonal antibodies against the
43-kDa USF component. A strong reactivity of a 43-kDa protein and a
weak reactivity to the 44-kDa protein was observed in HeLa nuclear
extract (Fig. 3 A, lane 2), whereas a strong
reactivity to a 44-kDa protein and a weak reactivity to a 43-kDa
protein were observed with fraction DE-B from the rat cells
(Fig. 3 A, lane 1). The antibody
cross-reacted exclusively to the 44-kDa polypeptide of CPBF but not to
the 39-kDa polypeptide in the purified preparation (Fig. 3, B and C). The large polypeptide with a molecular mass of
approximately 116 kDa that cross-reacts with the anti-USF antibodies in
both fraction DE-B and HeLa nuclear extract probably represents TFII-I,
which is known to cross-react with the anti-USF antibodies
(27) . These results indicate that the 44-kDa polypeptide of
CPBF is immunologically related to USF or that rat CPBF is the rat
homolog of human USF.
Figure 3:
Immunological relationship of rat CPBF to
USF. Western blot analysis was performed using anti-HeLa 43-kDa USF-1
antibodies (see ``Experimental Procedures''). Panel A, lane 1, fraction DE-B (100 µg) from rat
ascites cells; lane 2, HeLa nuclear extract (100
µg). The solid arrowhead indicates the immune
complex formed between the 44-kDa polypeptide of CPBF and anti-USF
antibodies. The open arrowhead indicates the immune
complex formed between the 43-kDa human USF1 and anti-USF antibodies.
Panel B, purified CPBF (100 ng) was electrophoresed
on 10% SDS-polyacrylamide gel electrophoresis and silver-stained. The
44- and 39-kDa polypeptides are indicated by arrowheads.
Panel C, immune complex formed between the 44-kDa
polypeptide of CPBF and anti-USF antibodies. The solid arrowhead indicates the immune complex formed between the
44-kDa polypeptide of purified rat CPBF and anti-USF antibodies. The
markers (in kDa) used were Sigma prestained proteins ( panel A) and Amersham prestained proteins ( panels B and C).
RNA Polymerase I-directed Transcription of Rat Ribosomal
Gene Can Be Competed by the USF/MLTF Oligonucleotide in Trans
We
reasoned that if the ribosomal gene promoter contains USF/MLTF binding
site, an oligonucleotide corresponding to this sequence should inhibit
pol I transcription. To test this possibility, the plasmid
(pB7-2.0) containing the rat rDNA was transcribed in fraction
DE-B prepared from the rat hepatoma N1-S1 cells (see
``Experimental Procedures''). This fraction supported
efficient and correct transcription of rat rDNA in absence of
competitor oligonucleotides (Fig. 4 A, lane 1). Preincubation with increasing amounts of cold rat
core promoter (rCP) resulted in drastic reduction (90%) of
transcription (Fig. 4 A, lanes 2 and
3). As anticipated, cold MLTF oligonucleotide also inhibited
transcription to similar extent (Fig. 4 A, lane 4). By contrast, MRE-d, which does not bind or compete
for CPBF or EBF (see Fig. 2 A), did not
affect the pol I transcription (Fig. 4 A, lane 5).
Figure 4:
A, inhibition of rat rRNA gene
transcription by MLTF oligonucleotide in trans. Lane 1, rat rDNA (pB7-2.0) (100 ng) was transcribed in
fraction DE-B (25 µg) from rat hepatoma N1-S1 cells (see
``Experimental Procedures'' for details). Lanes 2 and 3 represent the transcripts synthesized in
the extract that was preincubated with 100 and 200 ng of rCP,
respectively. Lanes 4 and 5, transcription
after preincubation of fraction DE-B with 200 ng each of MLTF and MRE-d
oligonucleotides, respectively, and then pB7-2.0 template was
added to the reactions. Arrow indicates the specific
635-nucleotide run-off transcripts. B, inhibition of RNA
polymerase II transcription from the adenovirus major late promoter by
rat rRNA gene core promoter (rCP) in trans. AdML G-less
cassette was transcribed in HeLa nuclear extract (72 µg) in the
absence or presence of competitors (for details, see
``Experimental Procedures''). Lane 1,
control transcription in absence of competitors; lane 2, HeLa nuclear extract was preincubated with 100 ng of
rat rCP oligonucleotide and pAdMLP(CAT) (250 ng) was added
to the reaction. HeLa nuclear extract was preincubated with 100 ng each
of MLTF oligonucleotide ( lane 3), NF1 oligonucleotide
( lane 4), MRE-d oligonucleotide ( lane 5) before addition of template and then
pAdMLP(C
AT) was added and transcription was carried out
(see ``Experimental Procedures'' for details of transcription
assay). Arrow indicates the 400-nucleotide specific
transcript.
RNA Polymerase II-directed Transcription from the
Adenovirus Major Late Promoter Can Be Competed by Rat Core Promoter
Oligonucleotide in Trans
To establish further whether rat
ribosomal gene core promoter binds USF or USF-like factors and whether
such binding blocks pol II transcription, transcription of AdML
promoter G-less cassette was performed in a transcriptionally competent
HeLa nuclear extract in presence or absence of competitor
oligonucleotides. This extract supported transcription from the AdML
promoter and produced the correct 400-nucleotide transcript
(Fig. 4 B, lane 1). Addition of
competitor oligonucleotides corresponding to rat core promoter (rCP)
and major late transcription factor (MLTF) binding site reduced
transcription by 50% (Fig. 4 B, lanes 2 and
3, respectively). Addition of competitor oligonucleotide NF1
also reduced transcription (Fig. 4 B, lane 4), as the AdML promoter contains the binding site for
CTF/NF1. On the contrary, addition of cold oligonucleotide MRE-d, which
binds the transcription factor MTF-1
(26) , failed to inhibit
transcription from the AdML promoter (Fig. 4 B, lane 5). To prove further the relationship between the two
factors, the effect of exogenous CPBF on transcription from the AdML
promoter was studied. Addition of purified CPBF to the cell-free
transcription reaction of AdML G-less cassette resulted in stimulation
of transcription (data not shown). These results indicate that the rat
core promoter region of rat rDNA can compete for USF or MLTF binding
activity, which is known to participate in transcriptional activation
of the AdML promoter by RNA polymerase II, and are consistent with the
existence of a USF/MLTF recognition sequence in the ribosomal gene
promoter.
)
The purification protocol used in our
laboratory may have preferentially selected the 44-kDa polypeptide.
BF, enhancer-1 binding factor;
TFII-I, transcription factor II-I; USF, upstream stimulatory factor;
rCP, rat core promoter; MLTF, major late transcription factor; MRE-d,
metal regulatory element d; AdML, adenovirus major late; EMSA,
electrophoretic mobility shift assay
AT)) construct used
in the transcription studies and the antiserum against human USF.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.