From the
The CT element of the c- myc gene is
required for promoter P1 usage and can drive expression of a
heterologous promoter. Both double strand (Sp1) and single strand
(hnRNP K) CT-binding proteins have been implicated as mediators of CT
action. Although significant levels of CT activity persisted following
Sp1 immunodepletion, EGTA totally abolished transactivation, thus
implicating another metal requiring factor in CT element activity. As
hnRNP K binds to one strand of the CT element, but has no metal
requirement, the opposite (purine-rich strand) was examined as a target
for a metal-dependent protein. A zinc-requiring purine strand binding
activity was identified as cellular nucleic acid binding protein
(CNBP), a protein previously implicated in the regulation of sterol
responsive genes. Two forms of CNBP differed in their relative binding
to the CT- or sterol-response elements. CNBP was shown to be a
bona fide regulator of the CT element by cotransfection of
a CNBP expression vector that stimulated expression of a CT-driven but
not an AP1-dependent reporter. These data suggest that hnRNP K and CNBP
bind to opposite strands and co-regulate the CT element.
Proper regulation of the c- myc gene has been shown to
be important for the execution of several aspects of cellular
metabolism (for review, see Marcu et al. (1) ).
Induction of terminal differentiation is, in general, accompanied by a
decrease in c- myc transcription
(2, 3) . Due to
its rapid induction following treatment of numerous cell lines with a
variety of mitogenic signals, c- myc has been classified as an
immediate early response gene
(4, 5) . c- myc expression may also dictate cell death as high levels of c- myc can lead to apoptosis under some conditions
(6) . Because
the physiological and pathological consequences of c- myc expression are profound, it must be tightly regulated; indeed,
simply a 2-fold decrease in c- myc mRNA levels lengthens the
time required for G
The response of c- myc to a wide variety of
growth and differentiation factors suggests that its expression
integrates stimuli from several pathways. Two features of the c- myc gene presumably enhance the cellular ability to fine tune
c- myc transcription rates. First, there are two major start
sites, P1 and P2, that are located 176 base pairs apart and can be
regulated independently
(8) . Second, nuclear run-on experiments
have recently demonstrated that there is a holdback to RNA synthesis at
the P2 promoter
(9, 10) .
Perhaps because of such
complexities, the molecular mechanisms regulating c- myc expression have remained enigmatic. Early studies of c- myc identified six candidate regulatory regions hypersensitive to
DNase I digestion
(11, 12) . In several cell lines, the
hypersensitivity to DNase I digestion within three of these regions
correlates with active transcription of the c- myc gene. One of
these three sites, termed III
Previous studies have demonstrated that the CT element confers a
5-10-fold stimulation of transcription upon a heterologous
promoter both in vivo and in vitro (13, 14, 15) . hnRNP
In Vitro Transcription and RNase Protection
Assays-HeLa cells were grown in suspension in
spinner-modified minimal essential medium supplemented with 10% fetal
calf serum and nonessential amino acids to a density of approximately 1
Templates for in vitro transcription
were constructed by inserting four repeats of the CT element
(-149 to -122) upstream of the same promoter used for the
basal transcription control (the c- fos promoter from
-56 to +109) so that any influence of the vector on
transcription would affect both activated and basal transcription
equally. A 10-base pair deletion 109 base pairs downstream of the
transcription start site was introduced into p
Elution of the purine strand affinity
column with salt concentrations as high as 2
M failed to
remove any purine oligonucleotide binding activity, indicating either
that chromatography inactivated the factor or that its interaction with
the CT element purine strand was extremely tenacious. Tightly bound
protein, eluted from the purine strand column with denaturants (urea or
guanidine HCl), displayed specific purine strand binding activity
following dialysis (data not shown). Using this data, the activity was
purified by two successive cycles of affinity chromatography in which
the 0.1
M NaCl flow-through of a nonspecific single-stranded
DNA oligonucleotide column was directly loaded on a purine
oligonucleotide column, washed, and step eluted with 0.5
M NaCl, 1.0
M NaCl, and 5
M guanidine HCl.
Following two rounds of affinity purification, SDS-polyacrylamide gel
electrophoresis showed the prominent bands to be a doublet of
approximately 19 kDa (Fig. 6, lanes 4 and
5). EMSA analysis of the renatured proteins from the doublet
revealed an EGTA-sensitive binding activity with a gel mobility shift
identical to that seen with crude extract (data not shown).
Both CNBP-L and CNBP-S, expressed as recombinant
glutathione S-transferase-fusion proteins, bound specifically
to the CT element purine strand (Fig. 7); complex formation was
eliminated by preincubation of the recombinant protein with EGTA (data
not shown). Removal of the gst portion of the recombinant protein with
thrombin generated a purine strand binding activity with an EMSA
mobility identical to that of the major purine strand binding activity
in nuclear extract (Fig. 8, lanes 5 and
6). As further proof of identity, EMSA purine strand complexes
generated using either HeLa nuclear extract, were supershifted with
affinity-purified antibodies to recombinant CNBP (Fig. 8,
lane 2), but not with antibodies to either hnRNP K
( lane 3), or glutathione S-transferase
( lane 4). Although the purine strand was the
preferred ligand for recombinant as well as HeLa CNBP, double strand CT
element nevertheless competed effectively for binding to the CNBPs at
5-fold greater concentrations (data not shown).
CNBP was found to be the second single strand-specific DNA
binding protein interacting with the CT element, a segment of DNA that
up-regulates expression of the c- myc gene. CNBP binds to the
purine-rich strand of the CT element, opposite to hnRNP K, which can
bind to pyrimidine-rich strand. Both proteins up-regulate CT
element-driven chloramphenicol acetyltransferase expression in
transient transfection assays. These properties define a system whereby
formation of an open complex at the CT element composed of unpaired
strands, CNBP, hnRNP K, and any additional factors, could be favored by
several mechanisms. Modulation of the concentrations of either active
CNBP or active hnRNP K, in vivo, could influence the ability
of the other to find the CT element by exposing its binding site.
Because five CT elements reside in the c- myc promoter, open
complex formation would have an exponential dependence on the
concentrations of individual components, rendering the complex
sensitive to even modest fluctuation in the levels of its components.
Thus physiological conditions that alter either CNBP or hnRNP K levels,
or that sequester one or the other protein, would be expected to
modulate CT element activity. Importantly, hnRNP K has been shown to
interact with several regulators of cell growth or gene expression,
including the oncoproteins c-Src
(23, 24) and
Vav
(25) , thus highlighting potential links between the CT
element and important cellular processes. The protein neighbors of CNBP
remain to be explored. Because Sp1 can bind to the double-stranded CT
element and activate transcription, at least in vitro, a
complex equilibrium exists at this site. Cellular conditions such as
transcription driven supercoiling of upstream sequence or replication
could favor single strand CT formation with concomitant CNBP and hnRNP
K action, whereas activation of a quiescent c- myc gene might
require Sp1 binding to the duplex CT element.
If hnRNP K and CNBP
were to bind the CT element in vivo, then a complex pattern of
sensitivity to, and protection from, agents that cleave single strand
DNA might be expected. Indeed, the in vivo profile of the
reactivity of the CT region with the single strand-specific oxidizing
agent KMnO
Several features of CNBP as a
transcription factor are noteworthy. The protein exists in two forms
with different nucleic acid recognition properties. The form of CNBP
first identified, CNBP-L, was devoid of an effect on the sterol
response element in cotransfection experiments
(16) . However,
CNBP-L binds particularly poorly to the G-rich single strand of the SRE
as compared with the CT element or to CNBP-S binding to the SRE. Two
recently cloned proteins, sterol regulatory element binding proteins 1
and 2, have emerged as important up-regulators of genes in the
cholesterol biosynthetic pathway, thus suggesting that any role for
CNBP in the regulation of these genes may be a negative one
(26, 27, 28) . This would be consistent with the
increase in CNBP levels under sterol-repressed conditions
(16) .
It is noteworthy that both of the elements recognized by CNBP in
single-stranded form are also bound by a transcription factor in
double-stranded form; Sp1 has a positive effect on transcription from
the CT element,
We thank Lance Liotta and Lou Staudt for critical
evaluation of the manuscript. We thank Kevin Gardner for helpful
discussions during the course of the work and Suzanne Sanford for
superb technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
cells to enter S phase by 3-4 h
and prolongs the cell cycle by 12% in actively dividing cells
(7) .
, is situated 125 base pairs
upstream of P1 and consists of five imperfect direct repeats of the
sequence CCCTCCCCA (termed the CT element); four repeats are in tandem,
while the fifth downstream repeat is separated by nine base pairs.
(
)
K, through binding to single-stranded CT DNA
(13) ,
and Sp1, through binding to the double-stranded CT element
(15) , have each been proposed to be regulators of promoter
activity. If hnRNP K binds to the pyrimidine-rich strand, are there
factors that bind to the purine-rich strand? We present here the
purification and cloning of such a binding activity, and identify it as
the previously studied cellular nucleic acid binding protein
(16) .
10
/ml. Nuclear extracts were prepared as described
previously
(17) . In vitro transcription reactions were
performed in 50 µl and contained 500 µ
M of each rNTP,
50 µ
M NaCl, 1 m
M dithiothreitol, 1.5 m
M MgCl
, 20 m
M Tris 8.0, 2 m
M spermidine, and 100 ng of each template. The amount of nuclear
extract given in the figure legends is in micrograms of protein.
Reactions were incubated at room temperature for 1.5-2 h,
terminated by the addition of 50 µl of 10% SDS, phenol extracted,
and precipitated. In vitro synthesized RNA was hybridized with
P-labeled antisense RNA in 30 ml of 0.5
M NaCl,
20 m
M Tris 7.5, and 1 m
M EDTA at 65 °C for 3 h.
RNA probes were made according to SP6 RNA polymerase
manufacturer's (Promega Biotec) specifications. RNase T
digestion was performed by addition of 300 ml of 0.3
M NaCl, 20 m
M Tris 7.5, 7% formamide, and 1000 units of
T
(Life Technologies Inc.). After 1 h, digestions were
extracted with a 1:1 (v/v) mixture of phenol-chloroform and then
ethanol precipitated. Products were separated on 6% denaturing
polyacrylamide gels.
56CT so that RNase
protection assay of the CT-activated transcription products yielded a
molecule that was 10 bases smaller than the protected transcription
product of the basal promoter. Details of plasmid construction will be
given in a subsequent article.
(
)
Chromatography and Protein
Purification
Oligonucleotide was coupled to CNBr-Sepharose
according to manufacturer's (Pharmacia Biotech Inc.)
specifications at 0.5 mg/ml. Typically, 1.5 column volumes of extract
was loaded on the affinity column at 1 column volume/h in 20 m
M Tris, pH 8.0, 0.05% Tween 20 (Bio-Rad), 50 µ
M ZnCl, 50 µ
M EDTA, 20% glycerol, plus the
indicated concentration of NaCl. For purification of CNBP, extracts
were loaded onto the purine oligonucleotide column at 0.1
M NaCl and step eluted with the same buffer containing 0.5
M NaCl, 1.0
M NaCl, and finally 5
M guanidine HCl.
For the second round of affinity purification, the guanidine eluate was
dialyzed against 0.1
M NaCl buffer and fractionated again on a
column one-fifth the size of the first affinity column. Prior to each
cycle of affinity purification, extract was first passed through a
nonspecific single strand DNA oligonucleotide column at 0.1
M NaCl to remove nonspecific single strand binding proteins.
EMSA
One nanogram of oligonucleotides
phosphorylated with P-gamma-ATP (10,000 cpm) were
incubated with the indicated proteins in a final volume of 7 µl
containing 50-70 m
M NaCl, 20 m
M Tris, pH 8.0,
0.25 mg/ml bovine serum albumin, and 0.02% Tween 20. Binding reactions
proceeded for 30 min on ice and were loaded on prerun 4% nondenaturing
polyacrylamide gels and then run at 15-20 V/cm, dried, and
exposed to autoradiography.
Protein Sequencing: Preparation of Fragments for Internal
Sequence Analysis
To generate peptides for internal sequence
analysis, the protein was first reduced and alkylated with
N-isopropyliodoacetamide and then cleaved with cyanogen
bromide and digested with trypsin; the resulting peptides were
separated by HPLC chromatography, all as described previously
(18) . The sequences of purified peptides were determined with a
Porton/Beckman 2090 online sequencer using standard program 1.
Phenylthiohydantoin amino acid identification was accomplished with a
Beckman System Gold system with a modified sodium acetate gradient
program and a Hewlett-Packard narrow bore C-18 column.
Transfection and Chloramphenicol Acetyltransferase
Assays
Hela cells were cultured in Dulbecco's modified
minimal essential medium supplemented with 10% fetal calf serum. 5
10
cells were resuspended in 250 µl of
Dulbecco's modified minimal essential medium, and incubated on
ice for 10 min with plasmid DNA. Electroporation was performed with
Cellporator (Life Technologies, Inc.) at 230 V, 1180 millifarads
setting. After electroshock, cells were incubated on ice for an
additional 10 min. Transfected cells were added to 10 ml of medium and
incubated for 48 h before harvesting for chloramphenicol
acetyltransferase assays
(19) . Expression constructs were
generated via PCR using the following oligonucleotides: amino-terminal
CNBP TGA GAT GGA TCC ATG AGC AGC AAT GAG TGC TTC AAG TGT GGA and
carboxyl-terminal CNBP TAA AGC GGA TCC TAA GGC TGT AGC CTC AAT TGT GCA
TTC CCG. PCR products were digested with BamHI and cloned into
the BamHI site of PC Amp DNA I (Invitrogen).
A Purine Strand-specific Binding Factor Requires
Zinc
hnRNP K binds the pyrimidine-rich strand of the CT element.
What is the state of its partner purine strand? The displaced strand
might be naked; alternatively it might be complexed with other
macromolecules. Examination of the native c- myc CT element
in vivo using potassium permanganante modification as a single
strand-specific probe revealed prominent reactivity at the margins of
the CT region (data not shown). The relative lack of reactivity within
the CT repeats suggested that bound factors were protecting this region
from permanganate reactivity, which predicts the existence of a purine
strand-specific binding factor. To test this, EMSAs were performed
using HeLa cell nuclear extracts and the purine single strand as probe.
An abundant purine strand binding activity was discovered
(Fig. 1, lane 1). This binding activity was
specific since increasing amounts of the purine strand effectively
competed the probe, whereas two nonspecific oligonucleotides at
identical concentrations had no effect (Fig. 1, lanes 2-13). Additional characterization revealed that
the purine strand binding activity was dependent upon divalent cations,
since binding was abolished by EGTA (Fig. 2, compare lanes 2 and 3). Zinc, and to a lesser extent
manganese, conferred resistance to EGTA inhibition (compare lanes 5 and 6). The addition of magnesium, zinc,
manganese, or calcium in the absence of EGTA had no effect on binding
( lanes 8-11). It seemed that this
zinc-dependent purine strand binding activity might contribute to
CT-mediated transcription activation.
Figure 1:
A purine
strand binding activity is sequence-specific. Three micrograms of
nuclear extract was incubated with 1 ng of P-labeled
purine strand probe alone ( lane 1) or with 1, 5, 25,
or 125 ng of specific competitor ( lanes 2-5) or
with two different nonspecific competitors ( lanes 6-9 and 10-13). Arrow to
the right indicates the specific mobility
shift
Figure 2:
Purine
strand binding activity is EGTA sensitive. Nuclear extract was
preincubated either without ( lanes 1, 2,
8-11), or with ( lanes 3-7) 4
m
M EGTA. To block EGTA inhibition, some preincubations
included 100 µ
M MgCl( lane 4), ZnCl
( lane 5),
MnCl
( lane 6), or CaCl
( lane 7). The effects of each of these metals
alone is shown in lanes 8-11. Samples were then
assayed by EMSA as described under ``Materials and Methods''
using the purine-rich single strand as probe. (Following EGTA
treatment, probe was bound by EGTA-insensitive factors, which yielded
complexes of slower electrophoretic mobility and are not shown; see
lanes 3, 4, and
7)
Zinc-requiring Factors Mediate CT Element
Transactivation
When located upstream of the c- fos minimal promoter, four tandem CT repeats drove increased levels of
specific transcription in vitro using HeLa cell nuclear
extracts (Fig. 3 a, lane 1, compare
basal- and CT-mediated bands). Chelation of HeLa
extracts with EGTA totally eliminated CT element-stimulated
transcription ( lane 2), implicating a divalent cation
requiring protein in CT-mediated transactivation. The level of RNA
synthesis supported by the minimal fos promoter was
unperturbed by EGTA, indicating that the affected proteins were not
required for basal transcription. Zinc was the only divalent cation
that protected extracts from the inhibitory effects of EGTA
(Fig. 3 b), implicating a zinc metalloprotein in CT
element activity. Although the CT element has been considered simply as
a noncanonical Sp1 site, extracts completely immunodepleted of Sp1
retain considerable ability to transactivate through CT repeats in
vitro, revealing the contribution of additional factors to
CT-activated transcription (data not shown). It therefore seemed that a
second zinc metalloprotein, in addition to Sp1, contributed to
up-regulation through this element.
Figure 3:a, CT-activated transcription
in vitro is sensitive to EGTA. Nuclear extract was
preincubated either without ( lane 1) or with
( lane 2) 4 m
M EGTA at 30 °C for 10 min.
Remaining components required for in vitro transcription were
then added as described under ``Materials and Methods,'' and
transcripts were quantitated by RNase protection. Bands corresponding
to basal and CT-activated transcripts are indicated to the
right. b, CT-mediated transcription requires zinc.
Nuclear extract was preincubated with 4 m
M EGTA ( lanes 1-4). In order to block EGTA inhibition, either 100
µ
M of CaCl( lane 2),
MgCl
( lane 3), or ZnCl
( lane 4) were included in the preincubation.
In vitro transcription products directed by basal or
CT-bearing templates were assayed by RNase protection and are indicated
to the right. (Less EGTA inhibition was noted than in Fig. 1,
because reactions were preincubated at 4 instead 30 °C; the metals
alone had no effect on in vitro transcription, data not
shown).
Purine Single Strand Binding Factors Are Involved in
CT-mediated Transcription Activation
Was this purine strand
binding activity a player in CT element transcriptional stimulation? If
so, then the same purine-rich oligonucleotide that effectively competed
the zinc-dependent binding activity (see Fig. 1) would be
predicted to antagonize CT-dependent activation by titrating the same
binding activity in in vitro transcription reactions. To test
this, increasing amounts of either the purine oligonucleotide or a
nonspecific oligonucleotide were added to in vitro transcription assays. Indeed, 90% of CT element-mediated
stimulation was eliminated at the highest purine oligonucleotide
concentration, whereas a nonspecific oligonucleotide at the same
concentration inhibited transcription activation only 10%
(Fig. 4). The concentration of oligonucleotide required to
compete CT transactivation correlated with the concentration required
to compete the EGTA-sensitive mobility shift. Although the simplest
interpretation of this result is titration of a trans-acting factor,
qualitatively similar results, previously reported by Cooney et al. (20) , were attributed to triplex formation between the
purine oligonucleotide and the CT element in the template. Formation of
this triplex was hypothesized to block binding of any positively acting
transcription factors at the CT element.
Figure 4:
The
purine single strand inhibits CT-mediated transcription activation.
0.25, 0.5, 1.0, or 2.0 µg of either the purine single strand
( lanes 2-5) or a nonspecific single-stranded
oligonucleotide ( lanes 6-9) were added to
in vitro transcription reactions utilizing 60 µg of
nuclear extract. Reaction products were analyzed by RNase protection.
Bands corresponding to basal and CT-mediated transcription are
indicated to the right.
If a trans-acting factor
necessary for CT element function bound to the purine strand, then
passage of extracts through a purine strand affinity column should
subtract this factor, inactivating CT-stimulated but not basal
transcription. Alternatively, if the purine strand oligonucleotide
directly inactivated the CT element through triplex formation with the
template, then extracts should be inert to purine strand affinity
chromatography. The purine oligonucleotide was coupled to Sepharose,
and nuclear extract was passed through the column at 0.1
M NaCl. The column was washed and sequentially step eluted with 0.5
M NaCl and 5
M guanidine HCl. The flow-through of the
column was devoid of CT-mediated transcription activation, but still
supported basal transcription (Fig. 5), proving that the mechanism of
purine strand inhibition of transcription was due to interaction with
trans-acting factors and did not require triplex formation. Passage of
nuclear extract through a nonspecific oligonucleotide column had no
effect on transcription activation (data not shown). Elution of the
zinc-dependent purine strand binding activity from the affinity column
required denaturation (see below), therefore it was not surprising that
the high salt- and guanidine-eluted fractions failed to restore CT
activation when added to the flow-through of the purine oligonucleotide
column (data not shown).
Figure 6:
Purification of the major purine strand
binding activity. Nuclear extract was fractionated over a purine strand
affinity column at 0.1
M NaCl, washed with 1.0
M NaCl, and eluted with guanidine HCl. The guanidine HCl eluate was
dialyzed to 0.1
M NaCl and fractionated again on the affinity
column. Sixty micrograms of nuclear extract ( lane 2),
10 µl of first round affinity-purified protein ( lane 3),
and 2.5 ( lane 5) or 10 µl ( lane 4) of
second-cycle affinity-purified protein were visualized by gold-blot
analysis after electrophoresis (SDS-polyacrylamide gel electrophoresis,
15%). The molecular weights of marker protein ( lanes 1 and 6) are indicated to the right. The doublet
that was electroeluted and sequenced is indicated by an arrow ( right).
CNBP Is the Major Purine Strand Binding
Activity
Two cycles of purine strand affinity chromatography,
each requiring elution with denaturants followed by renaturation,
yielded the zinc-dependent purine strand binding activity copurifying
with a doublet of polypeptides at approximately 19 kDa (Fig. 6,
lanes 4 and 5); this doublet comprised the
vast majority of the protein in this sample. To identify and
characterize the polypeptides binding to the affinity column, the
eluted fractions were digested with trypsin and the resulting peptides
were separated by HPLC and sequenced. Six unambiguous sequences were
obtained (), all identical to predicted tryptic fragments
of CNBP, a zinc finger protein previously shown to bind the purine-rich
single strand of the sterol response element (SRE) and postulated to
play a role in sterol metabolism
(16) . Several possibilities
could account for the appearance of a doublet, including proteolysis,
posttranslational modification, or forms of CNBP specified by
alternately spliced mRNAs. The existence of another protein comigrating
with, but unrelated to CNBP, was also considered. In order to help
distinguish between these possibilities, reverse transcription PCR was
performed using primers derived from extreme amino- and
carboxyl-terminal CNBP sequence; this yielded a doublet at 550 base
pairs (data not shown). Cloning and sequencing of these PCR products
revealed DNA encoding the previously described CNBP (referred to
hereafter as CNBP-L), as well as an alternately spliced cDNA in which 7
amino acids (residues 36-42) were deleted (CNBP-S). These
alternately spliced RNAs most likely encode the purified protein
doublet.
Figure 7:
Recombinant CNBP-L and CNBP-S account for
the major purine strand activity. 1, 5, 25, or 125 ng of either
specific ( lanes 2-5) or nonspecific
oligonucleotides ( lanes 6-9 and
10-13) were incubated with 30 ng of recombinant
gst-CNBP-L ( top), or gst-CNBP-S ( bottom) using the
purine single strand as probe. Complexes were resolved by nondenaturing
polyacrylamide electrophoresis.
Figure 8:
CNBP comprises the major purine single
strand mobility shift. Left, affinity-purified antibodies to
either CNBP ( lane 2), hnRNP K ( lane 3), or glutathione S-transferase ( lane 4), were added to a mixture of P-labeled
purine single-stranded oligonucleotide and 3 µg of HeLa cell
nuclear extract. Complexes were resolved by EMSA. Arrows indicate the two complexes that contain CNBP. Asterisk to
the left indicates supershifted CNBP complex. Right,
purified recombinant CNBP-L ( lane 5) and recombinant
CNBP-S ( lane 6) were digested with thrombin according
to manufacturer's (Sigma) specifications and incubated with
labeled purine single-stranded oligonucleotide. The upper shift most
likely contains multiple CNBP molecules/DNA strand (probe contains 3 CT
repeats) and is quantitatively variable (compare lanes 1 and 7). Free probe (probe alone, lane 8) is not shown in lanes 1-4.
CNBP-L and CNBP-S Can Be Distinguished by DNA
Binding
The alternately spliced mRNA encoding CNBP-S disrupts
the sequence GRGGF found in CNBP-L. As GRGGF constitutes a motif found
most frequently in a variety of proteins that interact with
single-stranded nucleic acid
(21) , it seemed possible that
CNBP-L and -S might not interact with all nucleic acids equivalently.
CNBP-L was originally identified by its binding to the purine-rich
single strand of the sterol response element upstream of the low
density lipoprotein receptor gene. Therefore, recombinant CNBP-L and -S
were compared quantitatively for their ability to bind to the SRE and
the CT element purine-rich strands. Although the 7-amino acid
difference between the two spliced forms did not significantly affect
binding to the CT oligonucleotide (Fig. 9 a), it caused CNBP-S
to have an approximately 27-fold higher affinity for the SRE
oligonucleotide relative to CNBP-L (Fig. 9 b, compare
lanes 2 and 13).
Figure 9:
Differential binding of CNBP alternately
spliced forms to the sterol response elements. Crude extract (3 µg,
lane 1), or decreasing concentrations of gst-CNBP-L
( lanes 2-9) or gst-CNBP-S ( lanes 10-17), were added to 1 ng of
P-labeled CT ( panel a) or SRE ( panel b) purine-rich oligonucleotides and subjected to EMSA.
Recombinant CNBP shifts are indicated by the arrows at the
right. Recombinant protein amounts are as follows: lanes 2 and 10, 750 ng; lanes 3 and
11, 250 ng; lanes 4 and 12, 80 ng;
lanes 5 and 13, 27 ng; lanes 6 and 14, 9 ng; lanes 7 and 15,
3 ng; lanes 8 and 16, 0.9 ng; and lanes 9 and 17, 0.3 ng. Probe alone is shown in
lane 18.
CNBP Can Stimulate CT Activation in Vivo
Does CNBP
participate directly in the regulation of CT-mediated expression? To
address this question, a genomic CNBP expression vector encoding both
the S and L forms was cotransfected with either a CT- or AP1-dependent
reporter construct into HeLa cells. CT-dependent expression was
stimulated 5-fold by the CNBP expression vector (Fig. 10). In contrast,
AP1-driven chloramphenicol acetyltransferase activity was not altered
by CNBP expression. Thus CNBP behaves as a transactivator of the CT
element in vivo.
reveals hypersensitivity on each strand both at
the extreme boundaries of the element and between the two most 3`
repeats (data not shown).
and SREBP-1 and -2 have been shown to
stimulate transcription through the SRE
(26, 27) .
Whether the binding of these factors is regulated by modulation of the
single-stranded character of the loci will require additional
experiments. The array of regulatory factors displayed along the
c- myc gene, the complicated interweaving of DNA topology, and
protein-protein interaction, and the control of transcription by
promoter utilization, initiation, and elongation, all serve to indicate
that the inputs of many pathways are integrated to control
transcription of this important protooncogene.
Table:
Amino acid sequences of CNBr fragments obtained
from purine strand affinity purified protein
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.