Identification and Characterization of Single Strand DNA-Binding Protein That Represses Growth Hormone Receptor Gene Expression
Ram K. Menon,
Hui Cheng and
Manbir Singh
Department of Pediatrics University of Pittsburgh School of
Medicine Childrens Hospital of Pittsburgh Pittsburgh,
Pennsylvania 15213
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ABSTRACT
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The GH receptor is essential for the actions of GH
on growth and metabolism. Electromobility shift assay established that
a 42-bp enhancer element in the promoter of the L1 transcript of the
murine GH receptor bound nuclear proteins specific for the coding
strand or the DNA duplex. Using methylation interference footprinting
and electromobility shift assay with mutant oligonucleotides, the
DNA-binding sites for the single-strand DNA-binding protein (SSBP) and
the double-strand DNA-binding protein (DSBP) were mapped and shown to
be contiguous with partial overlap. Shift-Western analysis indicated
that the SSBP was a component of the DSBP complex. A functional
interaction between SSBP and DSBP was suggested by the effect of the
exclusion of SSBP on equilibrium binding and dissociation rate ("off
rate") of the DSBP-DNA complex. Experiments using the anionic
detergent deoxycholate provided evidence for a direct protein-protein
interaction between SSBP and DSBP. Using lectin-affinity
chromatography, discordance between the pattern of O-glycosylation of
SSBP and DSBP was demonstrated. Transient transfection experiments
support the role of SSBP as a repressor of DSBPs activation of
transcription of the GH receptor gene. Southwestern analysis indicated
that a protein of molecular mass 23-kDa exhibited binding activity
specific to the coding strand of the enhancer element. We conclude that
single- and double-strand DNA-binding proteins conjointly regulate the
expression of the murine GH receptor gene.
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INTRODUCTION
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Pituitary GH is essential for postnatal growth in animals. In
addition to growth, GH affects the metabolism of fat, protein, and
carbohydrate. GH exerts these actions both by its direct effect on
target organs and by stimulating the production of insulin-like growth
factor I (IGF-I). At the tissue level, these pleiotropic actions of GH
result from the interaction of GH with a specific cell surface
receptor, i.e. GH receptor. The association of GH with the
GH receptor triggers the postreceptor signaling systems, which
culminate in the biological actions of GH. GH receptors are present in
all the tissues toward which GH actions are directed. Thus the ability
of GH to exert biological effects is intimately linked to the number
and function of GH receptors in these tissues.
The GH receptor belongs to a gene family that includes the receptors
for PRL, a number of cytokines such as granulocyte colony-stimulating
factor, erythropoietin, granulocyte macrophage colony-stimulating
factor, and a wide variety of interleukins (1). The identification and
partial characterization of the promoter-regulatory regions of the
murine (2) and ovine (3) GH receptor gene were recently reported. The
organization of the 5'-region of the GH receptor gene is complex. In
the mouse, two 5'-untranslated regions (UTR), termed L1 and L2, have
been identified (2, 4). Southard et al. (4) determined that
GH receptor transcripts containing the L1 UTR are expressed
preferentially in liver compared with the placenta. In contrast, L2
UTR- containing transcripts are preferentially expressed in the
placenta. A similar scheme of multiple 5'-UTRs for the GH receptor gene
exists in the human (5).
The expression of the GHR is tissue- and development-specific. Thus
expression is highest in the liver, with lesser amounts being expressed
in heart and kidney. In the liver, expression of GH receptor
transcripts is very low in the fetus and increases postnatally to
maximal during pregnancy (1, 4). Nutritional status (1), thyroidal
status (6), and diabetes mellitus (7) are some of the states in which
expression of GH receptor is altered. Whereas detailed information
about the molecular mechanisms involved in regulating the expression of
the GH gene is available (8), much less is known about the factors
modulating the expression of the GH receptor (1). Previous reports from
this laboratory have described the identification and partial
characterization of two enhancer elements for the L1 transcript of the
murine GH receptor gene. Our analysis had indicated that the cognate
trans-acting factor for one these elements belongs to the
CTF/NF-1 family of transcription factors (9). In another report we
presented evidence that the second enhancer element, termed FP1,
defined a novel protein-DNA binding motif and is involved in the
developmental expression of the murine GH receptor gene (2).
Based on experiments detailed in the current report, we have
expanded by 12 nucleotides the enhancer element FP1 described in the
previous report (2). We now present results to support the conclusion
that the expanded 42-bp enhancer element, which we term FP42, binds
both a single strand (SSBP) and a double-strand DNA-binding protein
(DSBP). We demonstrate that the binding sites for the SSBPs and DSBPs
are distinct, although they are contiguous with partial overlap. We
provide proof that there is physical and functional interaction between
SSBP and DSBP and that these two proteins conjointly regulate the
expression of the murine GH receptor gene. Our studies indicate that
the SSBP serves as a repressor of DSBPs role in the activation of the
transcription of the GH receptor gene.
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RESULTS
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SSBP Specifically Recognizes a Site on the Coding Strand of the GH
Receptor Promoter
We had previously identified and partially characterized an
enhancer element located about 3.4 kb upstream of the major
transcription start site for the L1 transcript of the mouse GH receptor
gene (2). A double-strand oligonucleotide probe (FP42-DS; Table 1
) containing the enhancer element can form a
single sequence-specific protein-DNA complex with liver nuclear
extracts (2). Whereas the noncoding strand (FP42-LS) failed to form a
DNA-protein complex (data not shown), the coding (FP42-US) of this
oligonucleotide formed a protein-DNA complex (Fig. 1
).
To determine whether this SSBP-DNA complex was sequence specific,
we performed competition experiments in which the binding reaction was
carried out in the presence of increasing concentrations of unlabeled
FP42-US, FP42-DS, and a single-stranded oligonucleotide with random
sequence. Whereas 100-fold molar excess of FP42-US eliminated the
formation of the SSBP-DNA complex, neither FP42-DS nor the random
sequence oligonucleotide at even a 200-fold molar excess altered this
binding (Figs. 1
and 2B
). These results demonstrate that
the SSBP-DNA complex is sequence specific. This SSBP-DNA complex has an
electrophoretic mobility distinct from that observed for the
double-strand DNA-binding protein (DSBP) complex formed by the
double-strand oligonucleotide FP42-DS (Fig. 3
). SSBP was
expressed in a variety of murine tissues (Fig. 4A
); the
paucity of SSBP-DNA complex formed with fetal liver extracts suggests
that the expression of this protein is developmentally regulated (Fig. 4A
). Whereas crude nuclear extracts from HeLa, COS-7, and HepG2 cells
formed DNA-protein complexes with FP42-US, the mobility of these
complexes was distinct from those formed with murine tissues (Fig. 4
).
The binding of HepG2 nuclear proteins to FP42-US was sequence-dependent
and exhibited nucleotide specificity for DNA binding similar to that
observed with mouse liver nuclear proteins (Fig. 4B
). Proof for the
presence of a true primate homolog of the rodent FP42-US SSBP will have
to await the identification of the protein(s) that represent SSBP
activity on electrophoretic mobility shift assay (EMSA).

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Figure 1. Nuclear Proteins from Mouse Liver Bind to FP42-US
32P-labeled FP42-US was incubated with nuclear
extracts prepared from liver of adult female mice, electrophoresed, and
subjected to autoradiography as described in Materials and
Methods. Competition between labeled and unlabeled specific
(FP42-US, lanes 24) or double-strand oligonucleotide (FP42-DS, lanes
57) at molar ratios of 25 (lane 2), 50 (lanes 3 and 5), 100 (lanes 4
and 6), and 200 (lane 7) is shown. The band representing the specific
single-strand DNA-protein complex is indicated as SSBP.
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Figure 2. Effects of Mutations in the SSBP-Binding Site on
Protein-DNA Binding
32P-labeled FP42-US was incubated with nuclear
extracts prepared from liver of adult female mice, electrophoresed, and
subjected to autoradiography as described in Materials and
Methods. Panel A, Competition between labeled and unlabeled
wild type sequence (FP42-US, lanes 24) or mutant oligonucleotide
(M2FP42-US, lanes 57; M4FP42-US, lanes 810) at molar ratios of 25
(lanes 2 and 5), 50 (lanes 3, 6, and 8), 100 (lanes 4, 7, and 9) and
200 (lane 10) is shown. The band representing the specific
single-strand DNA-protein complex is indicated as SSBP. Panel B,
Competition between labeled and unlabeled wild type sequence (FP42-US,
lanes 24), mutant oligonucleotide (M3FP42-US, lanes 57), or
oligonucleotide with random sequence (lanes 810) at molar ratios of
25 (lanes 2 and 5), 50 (lanes 3, 6, and 8), 100 (lanes 4, 7, and 9) and
200 (lane 10) is shown. The band representing the specific single-strand DNA-protein complex
is indicated as SSBP. Panel C, Gels represented in panels A and B and
for mutant oligonucelotide M1FP42-US (data not shown) were subjected to
analysis using a PhosphorImager. The data from three independent EMSA
analyses are plotted as the percent change (mean ±
SEM) in the intensities of the protein-DNA complexes formed
in the presence of molar excess (open bar =
25-fold; solid bar = 50-fold; hatched
bar = 100-fold) of unlabeled competitor compared with that
formed in the absence of unlabeled competitor.\.
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Figure 3. Effect of Mutations in the SSBP-Binding Site on
DSBP Binding
With (lanes 2, 4, 6, 8, and 10) or without (lanes 1, 3, 5, 7, and 9)
the addition of the respective unlabeled complimentary oligonucleotide,
the indicated 32P-labeled oligonucleotides were incubated
with nuclear extracts prepared from liver of adult female mice. These
oligonucleotides were either with wild type sequence (FP42, lanes 12)
or with specific mutations (M1FP42, lanes 3 and 4; M2FP42, lanes 5 and
6; M4FP42, lanes 7 and 8; M3FP42, lanes 9 and 10). The sequences of the
oligonucleotides are defined in Table 1 . The reactions were
electrophoresed and subjected to autoradiography as described in
Materials and Methods. The bands representing the
specific single (SSBP) and double-strand (DSBP) binding proteins are
indicated.
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Figure 4. Tissue and Species Distribution of SSBP
Panel A, 32P-labeled FP42-US was incubated with nuclear
extracts prepared from the indicated tissues, electrophoresed, and
subjected to autoradiography as described in Materials and
Methods. Panel B, 32P-labeled FP42-US (lane 1) was
incubated with nuclear extracts prepared from HepG2 cells (lanes 27)
or liver of adult female mice (lane 8), electrophoresed, and subjected
to autoradiography as described in Materials and
Methods. Competition between labeled and unlabeled
oligonucleotides (FP42-US, lane 3; FP42-DS, lane 4; M1FP42-US, lane 5;
M2FP42-US, lane 6; M3FP42-US, lane 7) at molar ratios of 25 is shown.
The band representing the specific single-strand DNA-protein complex is
indicated as SSBP.
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Partial Overlap of Contiguous DNA-Binding Sites for SSBP and
DSBP
To define the binding sites of the SSBP and the DSBP, the contact
points with purines for both SSBP and DSBP were determined by
methylation interference footprinting. Chemical modification of the
purines present in FP42-US revealed that methylation of four G residues
(designated GI, GII, GIII, and
GIV) interfered with the binding of SSBP; these four G
residues were located at the 5'- (GI), middle
(GII), and 3'- (GIII, GIV) regions
of FP42-US (Fig. 5A
). In contrast, methylation of the
purines of FP42-DS revealed that the contact points (GIII,
GIV, and GV) of the DSBP on the coding strand
were restricted to the 3'-end and overlapped with the contact points
GIII and GIV of the SSBP (Fig. 5B
). On the
noncoding strand of FP42-DS the contact points (designated
GVI, GVII, and GVIII) flanked the
contact points of the coding strand (Fig. 5C
). These results reveal
that whereas the DNA-binding sites of SSBP and DSBP are distinct, there
is overlap of these contiguous binding sites.

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Figure 5. Methylation Interference Footprint of Single- and
Double-Strand DNA-Protein Complex
Panel A, Oligonucleotide FP42-US was end-labeled with
[ 32P]ATP and T4 polynucleotide kinase. This probe was
used in the methylation interference assay in conjunction with adult
mouse liver nuclear proteins. Either unbound (Free) DNA, or DNA
recovered from the DNA-protein complex (Bound) was isolated and the DNA
cleaved at the methylated guanine residues by piperidine.
Asterisks indicate the reduction in the intensity of
bands (numbered I-IV) that is facilitated by binding of protein(s) to a
specific sequence of DNA; ***, for >80% interference;
**, for 5080% interference; and * for
2050% interference. Maxim-Gilbert sequencing reactions (C, C/T, and
G) of FP42-US (the sequence of which is indicated) were electrophoresed
concurrently to identify the location of the footprints. Panel B, A
PCR-generated fragment containing the FP42 enhancer site was labeled on
the sense strand of the DNA duplex and methylation interference assay
was carried out as described above in panel A.
Asterisks indicate the reduction in the intensity of bands
(underexposed gel depicted in inset) numbered (III-V)
that is facilitated by binding of protein(s) to a specific sequence of
DNA; ***, >80% interference; **, 5080%
interference. The sequence of the FP42 enhancer site is indicated.
Panel C, Sequence of both strands of the enhancer element FP42.
*, Bases whose methylation reduces the proportion of
FP42-US DNA in the bound fraction; the bases whose methylation
decreases the proportion of FP42-DS in the bound fraction are
underlined. \.
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The functional significance of the contact points identified by the
methylation interference assays was investigated by electromobility
shift assay (EMSA). Compared with the wild type sequence,
oligonucleotides (Table 1
) with mutation at the GI
(M2FP42-US), GI and GII (M3FP42-US), or
GIII and GIV (M1FP42-US) had decreased affinity
for SSBP. This was demonstrated by the decreased ability of the mutant
oligonucleotides to compete with the wild type sequence for binding to
SSBP (Fig. 2
). In contrast, an oligonucleotide (M4FP42-US; Table 1
)
with mutations of all the four Gs identified to make contact with the
SSBP completely failed, even at a 200-fold molar ratio, to compete with
the wild type oligonucleotide (Fig. 2
, A and C). These results confirm
that the Gs identified as contact points by the methylation
interference assay are essential for binding of the SSBP to FP42-US,
and that the entire DNA-binding site is needed for binding of SSBP to
DNA with full affinity.
Physical and Functional Interaction between SSBP and DSBP
The interrelationship between the binding sites for the SSBP and
the DSBP was investigated by using the wild type and mutant
oligonucleotides in EMSA. Addition of unlabeled noncoding strand
(FP42-LS) to a binding reaction containing labeled coding strand
(FP42-US) resulted in the abolishment of binding of SSBP and the
appearance of binding of DSBP (Fig. 3
, lane 2). This result is
consistent with the above discussed findings indicating specificity of
the SSBP for the coding strand and suggest that formation of the DNA
duplex excludes the binding of the SSBP. In a similar series of EMSA
using M1FP42-US, M2FP42-US, or M4FP42-US as the labeled probes, the
addition of the respective unlabeled complimentary oligonucleotide
resulted in differential effects on the formation of the DSBP-DNA
complex. Whereas the oligonucleotide mixtures containing
32P-labeled M1FP42-US or M4FP42-US oligonucleotide could
not bind DSBP, addition of the unlabeled oligonucleotide complimentary
to labeled M2FP42-US resulted in the formation of the DSBP (Fig. 3
).
These results indicate that the G residues (GIII and
GIV) at the 3'-end of the enhancer element are essential
for binding of the DSBP and that the binding of SSBP to the 5'-end of
the element alone will not result in the binding of the DSBP.
Analysis of the steady state binding of the DSBP to the DNA duplex
indicated that the binding of the DSBP to oligonucleotides with mutant
binding site that abrogated the binding of the SSBP (M2FP42-DS and
M3FP42-DS) was increased 4- to 5-fold compared with the DNA duplex with
the wild type sequence (Fig. 6
). To quantitatively
analyze the effect of the discrete mutations in the SSBP DNA-binding
site on the DSBP DNA binding, we next estimated the dissociation rates
of the mutated binding sites relative to the wild type FP42 site. We
approached this analysis by measuring the dissociation rates in terms
of the half-life, determined by estimating the amount of bound DNA
measured at time points subsequent to the addition of a vast excess of
specific competitor DNA to a binding reaction that has reached
equilibrium (10). Figure 7A
represents an example of
such an experiment performed with FP42-DS and M2FP42-DS. The data are
plotted on the accompanying graph (Fig. 7B
) as the log of the percent
change in binding from that at time zero vs. minutes after
the addition of the competitor DNA. The results of these analyses
reveal that the half-life (t1/2) of the protein-DNA complex
formed by the DNA (M2FP42-DS) with the mutant binding site that
abrogated the binding of the SSBP was significantly decreased from that
of the protein-DNA complex formed with the wild type sequence FP42-DS
(Fig. 7B
and Table 2
). Interestingly, the decrease in
the dissociation rate of the DSBP-DNA complex was roughly proportional
to the extent of the alteration of the SSBP DNA-binding site. Hence,
the half-life of the DSBP-DNA complex formed with M3FP42-DS was even
shorter than that formed with M2FP42-DS (Fig. 7B
and Table 2
).

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Figure 6. Effects of Mutations in the DNA-Binding Site or DOC
on the Relative Affinities of the DSBP-DNA Complex
To quantitate the effect of mutations in the DNA-binding site by either
alterations in the nucleotide sequence or presence of anionic detergent
(DOC), EMSA was carried out with equal amounts of liver nuclear
extracts and equimolar amounts of the indicated 32P-labeled
double-strand DNA probes in the absence of unlabeled specific
competitor. After electrophoresis, the dried gels were subjected to
analysis using a PhosphorImager, and the amount of bound DNA-protein
complex was calculated as a percent of the total amount of probe added
per reaction. The data represent mean ± SEM of three
independent experiments. *, P < 0.05 as compared
with FP42-DS by one-way ANOVA with Duncans Correction for Multiple
Comparisons.
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Figure 7. Determination of Dissociation Rates (Half-Life) of
the Cognate DNA-Binding Protein for FP42-DS, M2FP42-DS, and M3FP42-DS
Panel A, 32P-labeled FP42-DS (lanes 15) or
M2FP42-DS (lanes 610) was incubated with nuclear extracts prepared
from liver of adult female mice. After the reactions had reached
equilibrium (20 min), a 400-fold excess of homologous unlabeled
double-stranded oligonucleotide was added, and aliquots of the mixture
were loaded onto a running gel at the indicated time points. After
electrophoresis, the dried gels were subjected to analysis using a
PhosphorImager. Panel B, Data (mean ± SEM; n =
3) from the PhosphorImager analysis of gels similar to that shown in
panel A are plotted as the log of the percent bound probe relative to
probe bound at the time of addition of the unlabeled competitor (time
0), as a function of time. The half-life of the complexes were derived
from the slope of the curves (10 ); FP42-DS(t1/2) =
25 min, M2FP42-DS(t1/2) = 14 min, and
M3FP42-DS(t1/2) = 10 min.
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The experiments analyzing the effect of the mutated DNA-binding site on
the dissociation rate of the DSBP-DNA complex suggested an interaction
between the SSBP and DSBP. To determine whether this interaction
involved direct physical interaction between SSBP and DSBP, we
exploited the property of the mild anionic detergent deoxycholate (DOC)
to inhibit protein-protein interactions without ordinarily altering
protein-DNA interaction (11, 12, 13). We reasoned that if the increase in
steady state binding and the shortening of the half-life of the
DSBP-DNA complex observed with M2FP42-DS and M3FP42-DS resulted from
the lack of interaction between SSBP and DSBP, then perturbation of the
putative protein-protein interaction between SSBP and DSBP by DOC
should also result in similar alterations in the binding
characteristics of the DSBP complex formed by the wild type DNA-binding
site, i.e. FP42-DS. Therefore, 32P-labeled
FP42-DS was incubated with liver nuclear extracts in the presence or
absence of varying (0.09% and 0.15%) DOC and the steady state binding
and dissociation rate in terms of half-life of the DNA-protein complex
estimated as described above. Whereas DOC did not significantly alter
the DNA binding of SSBP (data not shown), there was a dose-dependent
increase in the steady-state binding of the DSBP to the wild type FP42
(Fig. 6
). Furthermore, Fig. 8
illustrates that in the
presence of DOC there was a decrease in the half-life of the
protein-DNA complex formed by FP42-DS. This decrease in the half-life
of the DSBP-DNA complex was roughly proportional to the concentration
of DOC (Table 2
). To exclude the possibility that this effect of DOC
was due to the dissociation of an oligomeric DSBP-DNA complex that did
not involve SSBP, we studied the effect of DOC on the half-life
of the DSBP-DNA complex formed with M3FP42-DS (Table 2
). Since
M3FP42-DS contains a mutant binding site that abrogates binding of SSBP
but not of DSBP, the lack of effect of DOC on the half-life of the
DSBP-complex formed by M3FP42-DS indicates that the effect of DOC on
FP42-DS binding was due to perturbation of protein-protein
interaction(s) mediated by the SSBP-binding site. We conclude from
these experiments that there is a functional association between the
SSBP and the DSBP and that SSBP serves to stabilize the DSBP complex as
evidenced by the decrease in the half-life of the DSBP complex when the
SSBP is excluded from this complex.

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Figure 8. Effect of DOC on Dissociation Rate of the Cognate
DNA-Binding Protein for FP42-DS
In the presence of the indicated concentration of DOC,
32P-labeled FP42-DS was incubated with nuclear extracts
prepared from liver of adult female mice. After the reactions had
reached equilibrium (20 min), a 400-fold excess of unlabeled FP42-DS
was added, and aliquots of the mixture were loaded onto a running gel
at the indicated time points. After electrophoresis, the dried gels
were subjected to analysis using a PhosphorImager, and the data
(mean ± SEM; n = 3) plotted as the log of the
percent bound probe relative to probe bound at the time of addition of
the unlabeled competitor (time 0), as a function of time. The half-life
of the complexes was derived from the slope of the curves (10 );
t1/2 (min) = 25 (0% DOC), 21 (0.09% DOC) and 14 (0.15%
DOC).
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A limitation of the experiments described above is that they relied on
an in vitro assay, i.e. EMSA, to analyze the
putative interaction between the SSBP and DSBP. We next studied the
in vivo functional significance of the SSBP-DSBP interaction
by testing the ability of the enhancer element with the respective
mutations to exhibit activity in the context of an heterologous
thymidine kinase promoter contained in the luciferase vector pTK81. In
transient transfection experiments using COS-7 cells, consistent with
our previous findings, the wild type enhancer sequence pTKFP42
exhibited 2- to 3-fold activity over the activity of the vector alone
(Fig. 9
and Ref. 2). Whereas the plasmid pTK-M4FP42,
containing the enhancer sequence with mutations that abolished both
SSBP and DSBP DNA-protein binding in EMSA, did not exhibit significant
activity compared with the vector alone, the plasmid pTK-M2FP42
containing mutations that selectively abrogated the binding of the
SSBP, but retained DSBP binding, exhibited a 6- to 7-fold increased
luciferase activity compared with the vector alone and 2- to 3-fold
increased activity compared with pTK-FP42. These results support the
conclusions that the SSBP-DSBP interaction is not essential for the
transcriptional activity of DSBP and that SSBP acts as a repressor of
activity of the DSBP in vivo. Since it was not possible to
selectively abolish SSBP while retaining DSBP binding, we cannot
discern whether SSBP has transcriptional regulatory activity
independent of DSBP.

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Figure 9. Transient Expression Analysis of Enhancer Element
FP42 in an Heterologous Promoter
Expression plasmids were generated by inserting the wild sequence GH
receptor enhancer FP42 or oligonucleotide with mutations in the binding
sites of the SSBP and DSBP (open bars) into the plasmid
pTK81 containing the luciferase gene (shaded bars)
driven by the thymidine kinase (TK) promoter (solid
bars). Equimolar amounts of the vector pTK81 and luciferase
fusion plasmids were transfected into COS-7 cells and luciferase
activity measured as described in Materials and Methods.
Luciferase specific activity in cell homogenates is expressed as
relative light units equalized for transfection efficiency monitored by
cotransfection of a plasmid expressing ß-galactosidase. Results
represent the mean ± SEM of four independent
transfections performed in quadruplicate. Solid
triangles indicate the location of the mutated nucleotides; the
sequence of the oligonucleotides are defined in Table 1 . Using Student
t test, * = P < 0.0001 compared
with pTK81; ** = P < 0.0001 compared with
FP42.
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SSBP is a Component of the DSBP-DNA Complex
The results of the EMSA and methylation interference
assays suggested an interrelationship between the SSBP and the DSBP. To
investigate the possibility that the SSBP is a component of the
DSBP-DNA complex, we used the technique of Shift-Western analysis.
These experiments established that DSBP complex, transferred to
nitrocellulose, exhibited binding to FP42-US (Fig. 10
, panel 1) and FP42-DS (Fig. 10
, panel 5) but not to FP42-LS (Fig. 10
, panel 4). In competition experiments a 50-fold molar excess of
unlabeled FP42-US (Fig. 10
, panel 2) or FP42-DS (Fig. 10
, panel 6)
significantly decreased the binding of the respective labeled probe to
the DSBP on the nitrocellulose membrane. In contrast, a similar 50-fold
molar excess of an oligonucleotide (M4FP42-US; Table 1
), with mutations
of all the four Gs identified to make contact with the SSBP, did not
significantly effect binding of labeled FP42-US (Fig. 10
, panel 3).
These results demonstrate that the binding of FP42-US and FP42-DS to
the DSBP protein complex on the nitrocellulose membrane is sequence
specific and provide evidence that the SSBP is a component of the DSBP
complex.

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Figure 10. Detection of SSBP in DSBP-DNA Complex by
Shift-Western Blot Analysis
Unlabeled FP42-DS was incubated with liver nuclear extracts, and the
resultant protein-DNA complexes were separated by electrophoresis
through a 6% nondenaturing gel. After localization of the protein-DNA
complex by alignment with a concurrently electrophoresis reaction with
32P-labeled FP42-DS, the proteins were electro-transferred
onto nitrocellulose membrane. The nitrocellulose membrane was then
probed with 32P-labeled FP42-US (lanes 13), FP42-LS (lane
4) or FP42-DS (lanes 56). Competition between labeled and unlabeled
specific (FP42-US, lane 2; FP42DS, lane 6) or mutant (M4FP42-US, lane
3) oligonucleotides at molar ratio of 50 is shown; the sequences of
these oligonucleotides are specified in Table 1 .
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Posttranslational Modifications of SSBP and DSBP
Many transcription factors have their activity regulated by
posttranslational modifications, such as phosphorylation (14, 15) and
O-glycosylation (16). In an attempt to define the relationship between
SSBP and DSBP, we investigated the status of these common
posttranslational modifications for these two proteins. We first
examined whether phosphorylation of SSBP and DSBP was important for
their DNA-binding activity. The inability of either calf intestinal
alkaline phosphatase or antiphosphotyrosine antibodies to alter the
binding of SSBP or DSBP indicates that the DNA-binding activities of
SSBP and DSBP are not significantly modulated by their phosphorylation
status (data not shown).
Glycosylation of transcription factors is a well established
posttranslational modification. In all characterized examples, this
involves N-acetlyglucosamine (GlcNAc) residues attached to
serine and/or threonine residues of the protein (16). Since proteins
bearing GlcNAc residues are often bound by the lectin wheat-germ
agglutinin (WGA) (17, 18), we used WGA affinity chromatography and EMSA
to test for O-glycosylation of the SSBP and the DSBP. DSBP binding
activity could be recovered in the eluate ensuing after the application
of the competitor sugar N-acetylglucosamine to the WGA
column equilibrated with an aliquot of liver nuclear extract (Fig. 11
). In contrast, no SSBP-binding activity could be
demonstrated in the same eluate (Fig. 11
). These results suggest that
the DSBP is significantly glycosylated, while the SSBP does not undergo
a similar posttranslational modification. Since O-glycosylation of
DNA-binding proteins does not affect the DNA-binding properties of
proteins (19), this discordance in the glycosylation status cannot be
implicated for the differential DNA-binding properties of the SSBP and
the DSBP and would support the possibility that the SSBP and DSBP
activities represent two distinct proteins and not modifications of the
same protein.

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Figure 11. WGA Affinity Chromatography of DSBP and SSBP
Binding Activities
Autoradiography of EMSA with 32P-labeled FP42-DS (lanes
15) or FP42-US (lanes 610) of either crude liver nuclear extracts
(lanes 1 and 6) or proteins eluted by the application of
n-acetylglucosamine to a WGA column that had been allowed to
equilibrate with crude liver nuclear extracts (lanes 35 and 810).
The binding activities of the proteins eluted in the wash solution
immediately before the application of the n-acetlyglucosamine is also
shown (lanes 2 and 7). The bands representing specific double (A) or
single (B) strand DNA-binding proteins are indicated.
|
|
Single-Strand DNA Binds to a 23-kDa Protein
To identify the molecular size of the protein interacting with the
coding strand of the GH receptor enhancer (FP42-US), Southwestern
analysis was performed. These experiments established that, after
SDS-PAGE and transfer to nitrocellulose membrane, a 23-kDa liver
nuclear protein exhibited binding to FP42-US (Fig. 12
).
To determine whether the binding of FP42-US to the 23- kDa protein was
sequence specific, we performed competition experiments in which the
binding reaction was carried out in the presence of unlabeled FP42-US,
M4FP42-US, or an oligonucleotide with random sequence (Table 1
).
Whereas a 50-fold molar excess of unlabeled FP42-US significantly
abrogated the binding of the labeled FP42-US to the 23-kDa protein, a
similar 50-fold molar excess of M4FP42-US or the random sequence
oligonucleotide did not significantly alter this binding (Fig. 12
).
These results demonstrate that the binding of the 23-kDa protein in
liver nuclear extracts to FP42-US is sequence specific.

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|
Figure 12. Determination of Molecular Weight of SSBP by
Southwestern Blot Analysis
Fifteen-microgram aliquots of nuclear extracts from mouse liver were
electrophoresed through a 12% SDS-polyacrylamide gel and transferred
onto nitrocellulose. The nitrocellulose membrane was then probed with
32P-labeled FP42-US in the absence (lane 4) or presence of
50-fold molar excess of the wild type sequence FP42-US (lane 3),
M4FP42-US (lane 2), or an oligonucleotide with random sequence (lane
1); the sequences of these oligonucleotides are specified in Table 1 .
Position of concurrently electrophoresed protein size markers are
indicated.
|
|
 |
DISCUSSION
|
---|
We have demonstrated that the coding strand of an enhancer region
regulating the expression of the murine GH receptor gene binds to a
sequence-specific DNA-binding protein. Although this is the first
example of a SSBP modulating the transcription of the GH receptor gene,
there is increasing evidence that sequence-specific, SSBPs are involved
in the regulation of gene expression (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Thus, recent reports
have provided evidence for the role of SSBPs in the transcriptional
regulation of genes such as the TSH receptor (23, 24),
3-hydroxy-3-methylglutaryl enzyme A reductase (25), tyrosine
aminotransferase (26), adipsin (27), ß-casein (28, 29), actin (30, 31), platelet-derived growth factor A-chain (32), estrogen receptor
(33), and the GH (34) genes. DNA is a structurally dynamic
macromolecule that is able to adopt a number of alternative
conformations. This conformational plasticity is believed to play an
important role in the regulation of DNA replication, recombination, and
transcription. Although the precise mechanisms by which the local
topological features of these cis-acting elements may
regulate gene transcription is unclear, one of the possibilities is
that conformational changes of these DNA elements may alter the
characteristics of the interaction between trans-acting
factors and their cognate DNA elements. Whereas most eukaryotic
sequence-specific DNA-binding proteins exclusively recognize native
double-strand sequences, Larsen and Weintraub (35) originally proposed
the existence of a class of regulatory proteins that do not recognize
the classic B form DNA but specifically recognize altered structures of
DNA. These altered forms of DNA include H-DNA form, an intramolecular
triplex/single-strand structure, and an alternative intramolecular
triplex structure that can form with a G-G-C composition (36, 37, 38). Some
sequence-specific SSBPs recognize double-stranded DNA (21, 39), whereas
others (32), such as the SSBP described in the current report, bind
almost exclusively to single-strand DNA.
The identity of the SSBP characterized in this report remains to be
defined. Computer analysis of the nucleotide sequence of the enhancer
element (FP42) did not reveal any potential DNA-binding sites for
proteins binding to either single-strand or double-strand DNA.
Furthermore, the inability of an oligonucleotide incorporating the
consensus binding motif for the single-strand DNA-binding protein,
Y-box protein (24), to compete for binding of SSBP to FP42-US (data not
shown) suggests that these two proteins are not similar. Many of the
reported SSBPs have been shown to bind to cis-acting
promoter elements that are hypersensitive to DNase I and/or
single-strand-specific nucleases (32, 39). The intracellular milieu
strongly favors the maintenance of a double-stranded DNA configuration,
thereby restricting access to SSBPs. The current hypothesis is that the
sensitivity of these regions to nucleases, resulting from alterations
in the structure of active chromatin with differential dissociation of
nucelosomes and/or local alteration in the conformation of the DNA,
facilitates the interaction of the SSBP with DNA. In addition, some of
these regions exhibit a strong purine/pyrimidine asymmetry and have
been termed CT elements (39, 40). Inspection of the nucleotide
composition of the FP42 enhancer element does not reveal a
purine/pyrimidine asymmetry; whether this region exhibits nuclease
hypersensitivity remains to be determined.
The DNA-binding site of the SSBP characterized in this report is
contiguous with and partially overlaps the binding site of a DSBP.
Previous reports have also described the proximity of binding sites for
SSBPs and DSBPs on the cis-acting promoter elements of other
genes (23, 34, 41), suggesting that the coexistence of the binding
sites for SSBPs and DSBPs may not be a random association and may be
necessary for the function of these trans-acting factors. At
least two lines of evidence suggest that there is direct physical
interaction between the SSBP and DSBP described in this report. First,
the experiments using the Shift-Western technique demonstrated that the
SSBP is present within the DSBP-DNA complex. Second, DOC, an agent that
disrupts protein-protein interaction (13), alters the dissociation
rates of the DSBP-DNA complex. The claim that the effect of DOC on the
dissociation rate is because of inhibition of a putative interaction
between SSBP and DSBP is substantiated by the finding that selective
mutation of the SSBP DNA-binding site to exclude SSBP from the complex
also results in a similar effect on the dissociation rate of the
DSBP-DNA complex. Hence, the congruity of the results from these two
approaches indicate that, at least in vitro, SSBP serves to
stabilize the DSBP complex and that this function involves physical
interaction between the SSBP and DSBP.
Some sequence-specific SSBPs can exhibit enhancer (26) and others
can exhibit repressor (24, 25, 34) activities. The results of the
transient transfection experiments in our study indicate that the SSBP
regulating the activity of the GH receptor gene promoter represses the
activity of the cis-element with which it interacts. Our
results suggest that SSBP serves to repress this activity of the
enhancer element by interfering with the function of the cognate DSBP.
This model is supported by the following results. First, delineation of
the DNA-binding sites established that there is partial overlap of the
binding sites for the SSBP and DSBP. Whereas the binding site of the
DSBP on the coding strand is narrowly restricted to three contiguous
nucleotides, the binding site of the SSBP is spread over the entire
42-bp enhancer element. It is of interest to note that this broad
distribution of the DNA-protein contact points is a feature common to
many previously characterized SSBPs (22, 34, 38, 41). Second, the
site-directed mutations in the enhancer element that selectively
disrupted the binding sites unique to the SSBP, while retaining the
binding sites common to the SSBP and DSBP, resulted in an increase in
the binding of DSBP to the enhancer element. This finding suggests that
SSBP restricts the access of the DSBP to the cognate DNA-binding site.
This interaction could result from a variety of mechanisms including
steric hinderance or alteration in the conformation of the DSBP
molecule. Due to the fact that it was not possible to abolish binding
of DSBP without concomitantly abrogating binding of the SSBP, we are at
the present time unable to discern whether SSBP has transcriptional
activity independent of the cognate DSBP.
The DSBP and SSBP present in adult female liver are differentially
O-glycosylated. Alteration in the O-glycosylation status has been
implicated in the modulation of trans-activation properties
of transcription factors (19, 42). The current hypothesis for the
role of O-glycosylation is based on the observation that in well
characterized examples, the O-glycosylated amino acid residue on the
transcription factor is adjacent to a phosphorylated amino acid
residue. Thus the current model envisages a reciprocal relationship
between O-glycosylation and phosphorylation, with O-glycosylation
indirectly modulating DNA binding and/or transactivation by regulating
the phosphorylation status of the trans-acting factor (19).
Due to constraints on the choice of experiments because of lack of
knowledge of their identity, the phosphorylation status of SSBP and
DSBP could only be assayed by DNA binding. Our experiments indicate
that phosphorylation does not significantly alter the DNA-binding
properties of either SSBP or DSBP. However, the significant
O-glycosylation of the DSBP predicts a role for phosphorylation in the
function of DSBP. We speculate that the ability of DSBP to
trans-activate the GH receptor gene, a property we could not
assess directly, could be regulated by DSBPs phosphorylation status.
It is noteworthy that the demonstration of physical interaction between
SSBP and DSBP is compatible with the observation that all
O-glycosylated proteins, thus far identified, form reversible
multimeric complexes (19).
The in vivo biological role of the SSBP identified in
this report is not clear. Our previous work indicated that the cognate
enhancer element FP42 and the DSBP play a role in the
development-specific expression of the GH receptor gene (2). Because
the levels of the SSBP as estimated by EMSA are actually significantly
lower in the fetal liver, the demonstration of a repressor role for
this protein in transient transfection experiments suggests that lack
of the SSBP in the fetal tissue cannot be implicated in the decreased
expression of the GH receptor in the fetal liver. The L1 transcript is
selectively up-regulated in the liver during pregnancy (4). Since there
was no significant change in the level of the SSBP in nuclear extracts
from the liver of pregnant mice, we cannot ascribe a role for this
protein in the increased expression of the GH receptor during
pregnancy. However, the complex nature of the interaction between the
enhancer element FP42 and its cognate DSBP and SSBP proteins leaves
open the possibility that the modulation of the status of one of them
could have a significant effect on the activity of the complex. Hence
it is possible that a change in the topology of the enhancer element
FP42 could restrict the interaction of the SSBP with the DSBP and FP42
and result in derepression of the activity of the enhancer element and
increased expression of the GH receptor gene. Similarly, the
posttranslational status (e.g. phosphorylation) of the DSBP
could affect the interaction with SSBP and thereby modulate the
transactivation potential of DSBP. The verification of these complex
hypothetical models would necessarily involve the identification and
further characterization of the proteins that represent the SSBP and
DSBP activities on EMSA.
In summary, we have identified and partially characterized the
DNA-protein interactions between a SSBP and its cognate
cis-element in the promoter of the murine GH receptor gene.
We have further demonstrated a physical and a functional interaction
between this newly identified SSBP and a previously identified DSBP
that also interacts with this cis-element. Our experiments
suggest that, in vivo, SSBP decreases the activity of the GH
receptor gene promoter by modulating the ability of the DSBP to
trans-activate GH receptor gene expression.
 |
MATERIALS AND METHODS
|
---|
EMSA
Nuclear extracts from mouse liver were prepared as described by
Gorski et al. (43). Protease inhibitors (leupeptin, 2
µg/ml; pepstatin, 1 µg/ml; and aprotinin 1%) were included in the
buffers used to prepare the nuclear extracts. Custom synthesized
oligonucleotides were used for single-stranded DNA probes and
double-stranded DNA fragments used as probes were obtained by annealing
complementary single-stranded oligonucleotides. The DNA was end-labeled
with [
32P]ATP and T4 polynucleotide kinase.
Approximately 6 fmol DNA were added to 14 µg nuclear extract in a
final volume of 50 µl containing either 2 µg
poly(deoxyadenylic-deoxythymidylic acid), 25 mM HEPES (pH
7.2), 75 mM KCl, 25 mM NaCl, 2.5 mM
MgCl2, BSA (250 µg/ml), 10% glycerol, 0.025% NP-40, and
1 mM dithiothreitol (DTT) (for single-strand DNA probes) or
2 µg poly(deoxyadenylic-deoxythymidylic acid), 20 mM
Tris-HCl (pH 8.0), 50 mM NaCl, BSA (50 µg/ml), 1% NP-40,
1 mM EDTA, 10% glycerol, and 1 mM DTT (for
double-strand DNA probes). After incubation at room temperature for 30
min, DNA-protein complexes were resolved on a 6% nondenaturing
polyacrylamide gel with 90 mM Tris-borate, 2 mM
EDTA buffer. Where indicated, the gels were dried and sequentially
subjected to autoradiography with intensifying screens (DuPont,
Wilmington, DE) at -80 C and analysis via PhosphorImager (Molecular
Dynamics, Sunnyvale, CA). Competition experiments included the addition
of excess unlabeled DNA fragments to the reaction mix. In some
experiments nuclear extracts were incubated with the indicated amounts
of antiphosphotyrosine antibodies for 120 min at 4 C before addition to
the binding reactions. The antiphosphotyrosine mouse monoclonal
antibodies tested were PY20 (ICN Biomedicals,, Inc., Costa Mesa, CA)
and 4G10 (Upstate Biotechnology Inc., Lake Placid, NY).
Methylation Interference Assay
Gel-purified single-stranded oligonucleotide was
end-labeled with [
32P]ATP and T4 polynucleotide
kinase. Double-stranded 100-bp DNA probes that contained the FP42
enhancer element and labeled either on the coding or noncoding strand
were generated using end-labeled oligonucleotides in PCR. The single-
or double-stranded probes were modified with dimethyl sulfate for 40
min on ice (44). For the preparative EMSA, protein-DNA complexes were
formed by incubating
32P-labeled or unlabeled double-
stranded FP42 DNA duplex liver nuclear extract as described above for
EMSA. After electrophoresis the undried gel was analyzed by
PhosphorImager, and the regions corresponding to the protein-DNA
complex and unbound probe were excised, eluted, and then precipitated.
Base elimination and strand scission reactions at adenine and guanine
(A < G) were performed as described (44). The samples were then
lyophilized, resuspended in water, relyophilized (three times), and
analyzed by electrophoresis through a 12% sequencing gel. The dried
gel was sequentially subjected to autoradiography and analysis via
PhosphorImager (Molecular Dynamics).
DNA Sequencing
Sequencing was carried out by the dideoxynucleotide chain
termination method of Sanger et al. (45) using the Sequenase
2.0 kit (U.S. Biochemical Corp., Cleveland, OH). Sequencing primers
were either complementary to the canonical T3, T7, or SP6 sites
flanking the multiple cloning site of the vector or were complementary
to experimentally established sequences. The sequence data were managed
using the sequence analysis program MacVector5.0 (Oxford
Molecular Group, Campbell, CA).
Reporter Gene Constructs
The activity of the enhancer element FP42 was tested via the
ability to exhibit activity in the context of an heterologous thymidine
kinase promoter contained in the luciferase vector pTK81 (ATCC,
Rockville, MD). Double-stranded oligonucleotides with either the wild
type enhancer element sequence (pTK-FP42) or enhancer element with
mutations (pTK-M2FP42, pTK-M4FP42) were ligated into the vector pTK81
by exploiting convenient restriction sites in the vector. All
constructs were sequenced through the vector-insert junctions to ensure
nucleotide fidelity and verify directionality.
Transient Expression of Reporter Gene
The culture media used for tissue culture experiments were
obtained from Life Technologies (Gaithersburg, MD) unless otherwise
stated. COS-7 cells (ATCC) were maintained in Eagles MEM (with
nonessential amino acids, sodium pyruvate, and Earles balanced salt
solution), 10% FBS and penicillin G (100 U/ml), and streptomycin (100
µg/ml). Cells (1 x 106) were plated on 60- mm
plates 24 h prior to transfection. Fifteen micrograms of plasmid
DNA were transfected per plate using the calcium phosphate transfection
method (Life Technologies, Gaithersburg, MD). After 6 h incubation
the cells were washed with PBS and then supplemented with medium for
40 h before harvest for luciferase assay. For estimation of
luciferase activity the plates were rinsed twice with PBS, and the
cells were harvested by the addition of 200 µl lysis buffer (25
mM Tris-phosphate, pH 7.8, 2 mM DTT, 2
mM 1, 2
diaminocyclohexane-N,N,N',N-tetraacetic acid,
10% glycerol, 1% Triton X-100). After a brief freeze-thaw cycle, the
insoluble debris was removed by centrifugation at 4 C for 23 min at
12000 x g. The supernatant was then immediately
assayed for luciferase activity. All transfections were performed at
least in quadruplicate. Transfection efficiency was monitored by
cotransfection of 1 µg of the plasmid, pßgal-Control (CLONTECH Lab,
Palo Alto CA). Protein concentration of the supernatant was determined
using the Bradford protein assay (Bio-Rad Laboratories, Hercules,
CA).
Luciferase Activity Assay
Luciferase activity was measured in the cell lysates using
reagents from Analytical Luminescence Laboratory (San Diego, CA).
Briefly, using the automatic injectors of the Monolight 2010
Luminometer (Analytical Luminescence), 100 µl (
200 µg) of cell
extract were mixed sequentially with 100 µl buffer (3 mM
ATP, 15 mM MgSO4, 30 mM Tricine, 10
mM DTT, pH 7.8) and 100 µl 1 mM luciferin,
and the light output was measured for 30 sec. For all the samples,
determination of the ß-galactosidase activity (see below) was used to
equalize the luciferase raw activity values for transfection
efficiency. The results of the luciferase assay are expressed in
relative light units equalized for transfection efficiency.
ß-Galactosidase Activity Assay
ß-Galactosidase activity was estimated by the Luminescent
ß-Galactosidase Detection Kit II (CLONTECH Lab). Briefly, 50 µl of
cell lysate were mixed with 100 µl of the reaction buffer, and, after
an 60-min incubation period, the light output was measured for 5 sec in
a Monolight 2010 Luminometer (Analytical Luminescence).
Shift-Western Analysis
To investigate whether the single-strand binding protein
is a constituent of the double-strand DNA-protein complex, a
modification of the Shift-Western blotting method was employed (46).
Briefly, for the preparative EMSA, protein-DNA complexes were formed by
incubating
32P-labeled or unlabeled double-stranded FP42
DNA duplex liver nuclear extract as described above for EMSA. After
electrophoresis through a 6% nondenaturing gel, the localization of
the double-strand DNA-protein complex was achieved by analyzing the
undried gel on a PhosphorImager. The proteins were then transferred
electrophorectically onto nitrocellulose membranes using a Mini-Protein
Blotting system (Bio-Rad Laboratories). Blotting was done at 4 C for
1 h at 100 V in 25 mM Tris, 192 mM
glycine, 20% (vol/vol) methanol, pH 8.3. After transfer, the portion
of the nitrocellulose membrane corresponding to the different lanes was
excised. The nitrocellulose filters were then soaked overnight at 4 C
in 5% nonfat dry milk, 50 mM Tris (pH 7.5), 50
mM NaCl, 1 mM EDTA, and 1 mM DTT.
The filters were then hybridized for 1 h at room temperature with
binding buffer (20 mM HEPES, pH 7.9, 100 mM
KCl, 5% glycerol, 1 mM EDTA, 1 mM DTT, and 15
µg/ml salmon sperm DNA) containing 106 cpm/ml of the
32P-labeled FP42-US probe, after which the filters were
washed in the binding buffer at room temperature for 20 min. The
filters were autoradiographed with an intensifying screen at - 80
C; in some instances, for purposes of quantification, the filters were
analyzed using a PhosphorImager (Molecular Dynamics, CA). Competition
experiments included the addition of molar excess of unlabeled DNA
fragments to the hybridization mixture.
Effect of Alkaline Phosphatase on Single- and Double-Strand
DNA-Binding Activities
Aliquots of nuclear extracts were incubated for 30 min at 25 C
with the addition of buffer alone or the indicated amounts of calf
intestinal phosphatase (New England Biolabs, Beverly, MA) and then
subjected to EMSA with DNA labeled with [32P]dATP using
terminal deoxynucleotide transferase (Promega, Madison, WI). Heat (80 C
for 10 min)-inactivated alkaline phosphatase served as control for
these experiments.
Lectin Affinity Chromatography
To test for O-glycosylation of the DNA-binding proteins, an
aliquot of liver nuclear extracts (0.81 mg) was applied at a
rate of 3 ml/h to a 1-cm Wheat Germ-Sepharose (Sigma, St. Louis,
MO) column equilibrated in wash buffer (20 mM HEPES-KOH,
0.1 M KCl, 0.2 mM EDTA, 1 mM
phenylmethylsulfonylfluoride, and 20% glycerol). After allowing the
proteins to interact with the WGA matrix for 3 h, the column was
washed with 100 ml of wash buffer. The proteins bound to the column
were then eluted with buffer (25 mM HEPES-KOH, 0.1
M KCl, 12.5 mM MgCl2, 0.1% NP40, 1
mM DTT, 0.1 µM ZnSO4, 1
mM phenyl-methylsulfonylfluoride) containing 0.3
M N-acetylglucosamine. The eluate was assayed
for the presence of the SSBPs and DSBPs by EMSA with the respective
probe.
Southwestern Blot Analysis
Fifteen-microgram aliquots of nuclear protein from mouse
liver were heated for 3 min at 90 C in 62.5 mM Tris-HCl,
10% (vol/vol) glycerol, 5% (vol/vol) 2-mercaptoethanol, 1.05% SDS,
and 0.004% bromophenol blue. The protein samples were then
electrophoresed through a 4% stacking, 12% resolving, discontinuous
SDS-polyacrylamide gel in 25 mM Tris-HCl, 192
mM glycine, and 0.1% SDS buffer. High and low molecular
weight markers (Bio-Rad Laboratories) were also concurrently
electrophoresed. After electrophoresis, the proteins were transferred
to nitrocellulose by electroblotting (SemiPhor, Hoefer Scientific, San
Francisco, CA) for 2 h. The nitrocellulose filters were then
soaked overnight at 4 C in 5% nonfat dry milk, 50 mM Tris
(pH 7.5), 50 mM NaCl, 1 mM EDTA, and 1
mM DTT. The filters were then hybridized for 1 h at
room temperature with binding buffer (20 mM HEPES, pH 7.9,
100 mM KCl, 5% glycerol, 1 mM EDTA, 1
mM DTT, and 15 µg/ml salmon sperm DNA) containing
106 cpm/ml of the radiolabeled probe, after which the
filters were washed in the binding buffer at room temperature for 20
min. The filters were autoradiographed with an intensifying screen at
-80 C; in some instances for purposes of quantification the filters
were analyzed using a PhosphorImager (Molecular Dynamics). Competition
experiments included the addition of molar excess of unlabeled DNA
fragments to the hybridization mixture.
 |
ACKNOWLEDGMENTS
|
---|
The generosity of Dr. Mark A. Sperling in providing support and
encouragement is gratefully acknowledged. The technical suggestions
given by Drs. Rudert, Anand, and Zou are greatly appreciated.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Ram K. Menon, M.D., Division of Endocrinology, Department of Pediatrics, Childrens Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, Pennsylvania 15213.
Supported by NIH Grants K08-HD-000986 and R29-DK-49845, Genentech
Foundation for Growth and Development, Childrens Hospital of
Pittsburgh, and the Vira I. Heinz Foundation.
Received for publication December 30, 1996.
Revision received April 18, 1997.
Accepted for publication April 26, 1997.
 |
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