Inducible Binding of Cyclic Adenosine 3',5'-Monophosphate (cAMP)-Responsive Element Binding Protein (CREB) to a cAMP-Responsive Promoter in Vivo
Stefan Wölfl,
Camilo Martinez and
Joseph A. Majzoub
Hans-Knöll-Institut für Naturstoff-Forschung (S.W.)
07745 Jena, Germany
Division of Endocrinology (C.M.,
J.A.M.) Childrens Hospital Harvard Medical School Boston,
Massachusetts 02115
 |
ABSTRACT
|
---|
In general, DNA-binding factors that activate gene
transcription are thought to do so via reversible interaction with DNA.
However, most studies, largely performed in vitro, suggest
that the transcriptional activator, cAMP response element-binding
protein (CREB), is exceptional in that it is constitutively bound to
the promoter, where its phosphorylation leads to the recruitment of
CREB-binding protein (CBP) to form a CREB/CBP/promoter complex. We have
studied how CREB interacts with DNA in vivo to
regulate the cAMP-responsive gene encoding human CRH (hCRH).
Protein-DNA complexes were cross-linked in cells expressing the
endogenous hCRH gene by exposure to a 10 nsec pulse of high-energy
UV-laser light, followed by immunoaffinity purification of CREB-DNA
complexes. Binding of CREB to a fragment of the hCRH promoter
containing a canonical, functional cAMP response element was absent in
untreated cells, but was specifically induced after activation of the
protein kinase A pathway with forskolin. These data indicate that,
in vivo, CREB, like the majority of other DNA-binding
transcriptional activators, undergoes signal-mediated promoter
interaction.
 |
INTRODUCTION
|
---|
Genes that contain a cAMP response element (CRE) within their
promoter are regulated by a pathway that includes phosphorylation of
the CRE-binding protein (CREB) at ser 133 by either the catalytic
subunit of protein kinase A (PKA) or calcium-dependent calmodulin
kinase, resulting in the formation of a transcription-activating
complex (1, 2, 3, 4, 5). One step in the formation of this complex is the
recruitment to CREB of the CREB binding protein (CBP) (6), which
functions as an activator of the RNA polymerase II/TATA binding factor
complex (7, 8). The mechanism by which phosphorylation of CREB at ser
133 leads to transcriptional activation has been the subject of much
study. Most (9, 10, 11), but not all (12, 13), in vitro studies
conclude that phosphorylation does not change CREB-CRE affinity, and
that CREB is constitutively bound to the CRE (5). Overall, evidence
favors phosphorylation of ser 133 causing a change in the electrostatic
properties rather than in the conformation of CREB (11, 14, 15). That
CBP binding to CREB requires CREB phosphorylation suggests a mechanism
whereby this phosphorylation may activate gene transcription (7).
In vivo (i.e. within intact cells), the
mechanisms by which CREB regulates gene transcription are less clear,
with forskolin treatment causing an increase in both transcriptional
activation and protein binding to a CRE, although CREB has not been
specifically identified in the latter process (16, 17). The
phosphorylation-incompetent M1-CREB mutant can activate gene
transcription in vitro (18) but not in vivo (1, 18), allowing for the possibility that CRE-CREB interactions could
differ between in vitro vs. in vivo conditions.
A major difference between these two environments is that under
in vivo conditions, DNA is organized by histones and other
proteins into highly ordered nucleosomal structures, which likely play
an important role in limiting the accessibility of transcription
factors to DNA, thereby influencing their ability to regulate gene
transcription (19). Thus, CREB-CRE binding and transcriptional
activation in vitro may not require phosphorylation or CBP
binding, whereas in vivo, CREB phosphorylation and
subsequent recruitment of CBP might be obligatory before their binding
to the CRE/promoter.
We have analyzed the modulation by PKA of CREB binding to the CRE of
the human CRH gene in vivo. CRH has an essential role in
fetal organ maturation and the postnatal response to stress (20) and is
regulated by ligands that activate the PKA pathway (21, 22, 23, 24, 25, 26, 27, 28, 29). Within
brain hypothalamic neurons, stimulation of CRH gene transcription is
associated with a parallel increase in intracellular phosphorylation of
CREB at ser 133 (30). We first further characterized the interaction
between CREB and the CRE within the CRH gene promoter and then asked
whether, in vivo, the binding of CREB to this CRE is changed
after specific signal-mediated transcriptional stimulation via the PKA
pathway.
 |
RESULTS
|
---|
A schematic diagram of the human CRH (hCRH) gene is shown in Fig. 1A
. The two exons and 800-bp intron of
the CRH gene reside within a larger 3.8-kb HindIII fragment
(31) that contains 1 kb of DNA 5' of the transcription start site and
0.9 kb 3' of the polyadenylation site. A canonical CRE (5) is centered
at -224 nucleotides (nt) upstream from the transcription start site.
Also depicted in Fig. 1A
are the relevant
AluI restriction enzyme sites within the hCRH gene, as well
as the expected sizes of the PCR fragments (thick bars)
derived from the two AluI fragments (thin bars)
used to subsequently assess recovery of CREB-DNA complexes.

View larger version (39K):
[in this window]
[in a new window]
|
Figure 1. The CRH Promoter Contains a Functional CRE
A, Graphic representation of the hCRH gene with its two exons
(boxes; hatched area indicates the translated
region). The major transcripition start site is indicated by the
bent arrow. HindIII restriction sites are
outside the displayed area. AluI restriction
sites are marked, with the transcription start site at nt 916, and the
intron at nt 10861885. AluI restriction fragments analyzed
are indicated by thin horizontal bars, and PCR fragments
derived from these AluI regions are marked by heavy
horizontal bars, with PCR fragment sizes (bp) below. B,
Mobility-shift assay using JEG-3 cell nuclear extract and
32P-labeled CRHCRE20 probe. Labeled probe was incubated (+)
with extract, anti-CREB antiserum (CREB Ab), or increasing
concentrations of unlabeled CRHCRE20 or an unrelated double-stranded 20
mer (Linker). Unbound probe migrates at the bottom of the gel, whereas
a specific, slower-migrating bound fragment appears in the presence of
nuclear extract, which is further retarded in the presence of CREB Ab,
and attenuated in the presence of 100- to 1000-fold molar excess of
unlabeled CRHCRE20, but not Linker. C, Stimulation of luciferase
(CRH-Luc) activity in JEG-3 cells of a CRH promoter (-663 to +127
bp)-luciferase construct transiently cotransfected with the plasmid
pRSV-PKAc, which constitutively expresses the catalytic subunit of PKA.
Expression of PKAc stimulates CRH-Luc activity more than 60 fold. D,
Stimulation by forskolin (FSK), 10 µM, of luciferase
(CRH-Luc) activity of a CRH promoter (-663 to +127 bp)-luciferase
construct transiently transfected into JEG-3 cells. Forskolin
stimulates CRH-Luc activity more than 20-fold. E, Effect of mutations
within the hCRH promoter CRE on forskolin- stimulated CRH-Luc activity.
JEG-3 cells were transfected with luciferase constructs driven by
either native hCRH promoter (-247/+127), or with a 4 base deletion,
ACGT, in the center of the putative CRE (-247/+127)/ 224), or with
double-point mutations within the putative CRE
(-247/+127)C-224G/A-220T). Cells were treated with forskolin (10
µM) 6 h before harvest. Both mutations result in a
10-fold reduction in forskolin-stimulated luciferase activity.
|
|
The CRH Promoter Contains a Functional CRE That Binds CREB
To determine whether the hCRH CRE consensus sequence centered at
nt -224 is a typical CRE, we first asked whether it can bind CREB
(Fig. 1B
). When nuclear extract from JEG-3 cells was incubated with a
32P-labeled double-stranded oligonucleotide, corresponding
to the hCRH gene sequence containing the CRE plus 6 bp flanking each
end (CRHCRE20), we observed the formation of a major high molecular
weight complex. This complex was abolished by coincubation with
increasing amounts of cold hCRH CRE oligonucleotide, but not by a
1000-fold molar excess of an unrelated oligonucleotide polylinker of
the same size. The migration of the complex was supershifted by
coincubation with an antiserum directed against CREB, indicating that
it contains CREB bound to the hCRH CRE (Fig. 1B
). A nucleotide fragment
containing a deletion of the four central nucleotides of the CRE
sequence (ACGT) was unable to form a high molecular weight complex when
incubated with nuclear extract (data not shown).
We next studied the structure/function properties of the CRE centered
at nt -224. Cotransfection of a CRH-luciferase construct, extending
from -663 nt of the hCRH gene promoter to +127 bp of the
5'-untranslated region, with increasing amounts of a construct
constitutively expressing the catalytic subunit of PKA resulted in as
much as a 60-fold induction of luciferase activity (Fig. 1C
).
Luciferase activity of this construct was also stimulated 20-fold by
treatment with forskolin, 10 µM (Fig. 1D
). Using a
smaller promoter fragment extending from -247 bp to +127 bp
(-247/+127), which includes the CRE (at -227 to -220 nt), deletion
of the central four nucleotides of the CRE, ACGT (-247/+127/
224)
resulted in a 10-fold decrease in forskolin-stimulated luciferase
activity (Fig. 1E
). Site-directed mutatgenesis of two nucleotides that
are critical for CRE function, C-224G and A-220T(32), also resulted in
a 10-fold reduction in forskolin-stimulated transcription (Fig. 1E
).
These findings indicate that the CRE centered at -224 nt binds CREB
and confers PKA-inducible transcriptional activation to the hCRH
promoter.
CREB Binding in Vivo Is Dependent upon cAMP
Stimulation
To examine the effect of PKA stimulation upon CREB binding to the
CRE region of the hCRH gene promoter in vivo, we used the
human NPLC cell line (33), in which the endogenous hCRH gene is
positively regulated by the PKA pathway (28). We treated NPLC cells
with 10 µM forskolin for 1 h, which we have
previously shown stimulates CRH mRNA expression 5-fold in this cell
line (28). To detect CREB binding to the CRE, we used UV laser-induced
protein-DNA cross-linking in forskolin-treated NPLC cells followed by
immunoisolation of the CREB-CRE complex with a biotinylated CREB
antibody pulled down by streptavidin-coated magnetic particles,
followed by DNA sequence-specific PCR (Fig. 2
). We have previously described this
procedure (34), which is similar to chromatin
immunoprecipitation methods recently developed for the analysis
of chromatin-protein interactions (35, 36, 37).

View larger version (30K):
[in this window]
[in a new window]
|
Figure 2. Schematic Diagram of the CREB-DNA Cross-linking and
Purification Procedure
Protocols 1 and 2 differ only by UV laser exposure before (protocol 1)
or after (protocol 2) nuclear preparation.
|
|
Whether UV laser cross-linking was performed on intact cells (protocol
1) or on isolated nuclei (protocol 2), identical results were obtained
(Figs. 2
and 3
). In both cases, the
352-bp CRH fragment containing the CRE (Fig. 1A
) was detected only when
cells had been initially treated with forskolin (lanes F, Fig. 3
, A and
B), and never in control, untreated cells (lanes C, Fig. 3
, A and B).
This is evident when PCR products were visualized by either ethidium
bromide staining (Fig. 3A
) or after more sensitive Southern blot
hybridization of the electrophoresis products to a radioactively
labeled hCRH probe (Fig. 3B
). Thus, UV laser cross-linking of CREB to
the CRH gene occurred only after treatment of cells with forskolin, and
therefore presumably only after phosphorylation of CREB at ser 133 had
occurred (1).

View larger version (56K):
[in this window]
[in a new window]
|
Figure 3. PCR Analysis of DNA Fractions Obtained
after the CREB-DNA Cross-linking and Purification Procedure
UV laser cross-linking was performed either on intact NPLC cells
(protocol 1) or on isolated nuclei (protocol 2). Bound, Fraction
remaining on the magnetic particles after AluI
digestion; Unbound, fraction released from the magnetic particles after
AluI digestion (AluI-released fraction,
see Fig. 2 ). A, Ethidium bromide-stained agarose gel of PCR products.
The expected sizes of the CRE-specific and intron-specific PCR products
are 352 bp and 163 bp, respectively (upper and lower
arrows). C, Vehicle (ethanol)-treated cells; F,
forskolin-treated cells (10 µM for 1 h). B, Southern
blot of gel in panel A, using CRH-specific 32P-labeled CRH
probe spanning both CRH PCR fragments (nt 315-1316). DNA size markers
(bp) are noted to the right of the gels. In both panels
A and B, CRH-specific PCR fragments are detected only in samples
derived from forskolin-treated cells. C, PCR of initial
HindIII-digested samples from control (C) and forskolin
(F)-treated cells discloses equal recovery of DNA from both groups of
agarose-encapsulated cells. Input DNA was serially diluted in 5-fold
steps. Equivalent amounts of PCR products from control and
forskolin-treated cells are seen at each dilution. The 937-bp product
spanning the CRE, exon 1, and parts of the intron is marked by the
arrow. The two lanes, M, contain the size standard,
 , cut with HaeIII (sizes: 1353, 1078, 872, and 63
bp). D, Anti-CREB antiserum detects CREB by Western blot of protein
extracts from vehicle control (C)- and forskolin (F)-treated NPLC
cells. The solid arrow marks the 43-kDa CREB protein,
and the dashed arrow marks a smaller fragment that
reacts with the anti-CREB antiserum. M, Protein molecular mass markers
(95, 67, 44, and 30 kDa).
|
|
Because other data have suggested that CREB binds to CRE sequences in a
phosphorylation-independent manner (9, 10, 11), we performed several
control experiments to test the validity of our findings. First, as
noted above, Southern blot hybridization of the gel in Fig. 3A
, using a
CRH-specific probe and CRH-specific primers, confirmed that the 352-bp
fragment that appeared after forskolin treatment is indeed that derived
from the AluI fragment spanning the CRH gene CRE (Fig. 3A
).
Next, we sought to more precisely localize the region of the CRH gene,
which binds CREB after in vivo forskolin treatment. After
the isolation of HindIII-restricted DNA-CREB complexes by
immunoaffinity magnetic separation and a second round of restriction
enzyme digestion with AluI, the AluI DNA
fragments remaining bound to the CREB antibody (bound fraction) were
separated from all other AluI fragments (AluI
released fraction) (Fig. 2
). After PCR and AluI digestion,
we expected to find only the CRH-CRE 352-bp PCR fragment in the bound
fraction and only the CRH-intronic 163-bp PCR fragment in the unbound
fraction. Indeed, this was the case when gel-separated PCR products
were visualized with ethidium bromide staining (Fig. 3A
). However,
after Southern blot hybridization of these products using a
32P-labeled CRH probe, we observed both fragments in both
the bound and unbound fractions (Fig. 3B
), likely due to the detection
of a small amount of cross-contamination of both fractions using this
much more sensitive procedure. The presence of the CRH intronic
fragment in the bound fraction of forskolin-stimulated cells was likely
due to incomplete AluI restriction enzyme cleavage of the
initial HindIII CRE-containing fragment pulled down by the
anti-CREB antibody. The presence of the CRH promoter fragment in the
unbound fraction was likely due to incomplete cross-linking of this
fragment to the CREB antibody. Nevertheless, the detection of any CRH
promoter sequences was strictly dependent upon cells having been
exposed to forskolin before the UV laser cross-linking procedure.
To ensure that the isolation of CRH promoter fragments exclusively from
forskolin-treated cells was not due to variations in the release of DNA
from agarose-encapsulated nuclei, the total amount of DNA eluted after
HindIII digestion and before the addition of the
biotinylated CREB-specific antibody (Fig. 2
) was measured, and
equivalent amounts of DNA from forskolin- and vehicle-treated cells
were used for the immunoaffinity procedure. Due to the limited
efficiency of the UV cross-linking and immunoaffinity steps (estimated
to be 0.01% overall, S. Wolfl, unpublished data), the vast majority of
all HindIII fragments, whether or not they contain a
cross-linked CREB moiety, will remain in the supernatant after the
addition of streptavidin-coated magnetic particles. To assess the
amount of the specific HindIII fragment containing the CRH
gene in this supernatant, we performed PCR titration experiments with
the CRH-CRE specific primers. Using serial dilutions of 1:5, equivalent
amounts of PCR product were detected after 25 cycles of PCR for both
the vehicle- and the forskolin-treated samples in undiluted supernatant
(Fig. 3C
), indicating that differences in the elution of
HindIII DNA fragments from agarose-encapsulated nuclei
between the two treatment groups did not explain the detection of CRH
promoter-CREB complexes only in forskolin-treated samples.
To determine whether the CREB antiserum we used detected phosphorylated
CREB better than nonphosphorylated CREB, we performed Western blot
analysis of equal amounts of protein extract from forskolin-treated and
vehicle-treated NPLC cells (38). This confirmed that the antibody used
for immunoaffinity separation recognizes CREB from both forskolin- and
vehicle-treated cells, with the 43-kDa species of CREB being detected
in approximately equal amounts after both treatments (Fig. 3D
). This
was expected, as the antiserum was raised against a nonphosphorylated
peptide fragment of CREB and had been previously shown to not
discriminate between CREB and phospho-CREB (39, 40).
As a further control for the specificity of our findings, we were
unable to detect c-myc gene products after
c-myc-specific PCR (28) of DNA collected from the bound and
AluI-released fractions (data not shown). This result was
expected, as the c-myc gene does not contain a cAMP response
element, and indicates that detection of CRH promoter-CREB interaction
solely after stimulation of the PKA pathway was not a nonspecific
consequence of forskolin treatment.
 |
DISCUSSION
|
---|
We have found that in vivo, UV cross-linking of CREB to
a 352-bp region of the hCRH gene promoter containing a canonical,
functional CRE occurs specifically after stimulation of the cAMP
pathway with forskolin, and that this binding is absent in untreated
cells. The structure and function of the consensus CRE of the hCRH
promoter are highly similar to those in other cAMP-regulated genes (5),
suggesting that our observations may apply to other genes regulated by
cAMP. The CRE within the CRH promoter had been previously shown to
confer responsiveness to the CRH gene (21). We have confirmed and
extended these findings by demonstrating that this sequence interacts
with CREB and that this interaction is necessary for PKA-mediated
promoter activation.
Because of the short duration (10 nsec) of the laser pulse used for DNA
cross-linking, this technique allows the detection of legitimate
in vivo protein-DNA contacts and avoids secondary cellular
responses that longer periods of UV irradiation or chemical
cross-linking may trigger (41). To detect only cross-linked protein-DNA
complexes, several purification steps were included to disrupt
noncovalent protein-DNA complexes. The stringent purification
procedures used to avoid such artifactual interactions necessarily
reduced the amount of purified fragments. Because the very limited
amount of DNA in the final immunoaffinity step was not sufficient for
direct detection, we combined PCR and Southern blot hybridization to
increase sensitivity and specificity. Although this assay is very
sensitive, it does not allow accurate quantitative evaluation of the
data. However, forskolins effect was unambiguous, as CREB binding to
the CRE-containing hCRH promoter fragment was detected exclusively
after forskolin treatment of cells.
That UV irradiation cross-links CREB to the hCRH promoter only after
cAMP stimulation indicates that this stimulation somehow changes the
interaction between CREB and the promoter. In unstimulated cells, CREB
might be completely dissociated from the CRE of the CRH promoter.
Phosphorylation of ser 133 would then initiate a two-step process,
first leading to CREB-CBP association. The second step, the binding of
this complex to DNA, might be driven by the combined affinities of CREB
for the CRE and CBP for TATA box-associated factors, and/or by the
acetylation of chromatin by the histone aceytltransferase (HAT)
activity of CBP (42) or of an associated molecule such as P/CAF (43),
thereby rendering these DNA regions more accessible (Fig. 4
). Alternatively, nonphosphorylated CREB
might be bound to the CRE, but its affinity or mode of binding might
not allow efficient UV cross-linking to DNA. After CREB phosphorylation
and CREB-CBP binding, the recruitment to the promoter of CBP, perhaps
aided by the HAT activity of CBP or P/CAF, may significantly alter
chromatin organization of the CRH promoter, leading to a change in the
binding mode or affinity of CREB to the DNA that renders it susceptible
to UV cross-linking. Additionally, the HAT activity of CBP might
acetylate CREB or an associated factor, thus enhancing their binding to
DNA (44).

View larger version (31K):
[in this window]
[in a new window]
|
Figure 4. Two-Step Model of CREB/CRE Interaction
In the unstimulated state, CREB is not bound to DNA (left
panel). Phosphorylation of CREB, either by PKA or
calcium/calmodulin-dependent kinase (CaM kinase), leads to association
of CREB and CBP (middle panel). The CREB/CBP complex,
aided by the combined affinities of CREB for CRE and CBP for TATA box
factors, and/or by the HAT activity of CBP or P/CAF causing histone
acetylation and enhanced DNA accessibility, binds to the CRE and TATA
box regions of the promoter, leading to activation of gene
transcription (right panel). DNA is depicted with CRE
(TGACGTCA) and TATA regions boxed. The encircled
P denotes phosphorylation of CREB at ser 133. For clarity, only
monomeric protein and single-stranded DNA are shown.
|
|
The enhanced interaction in vivo between
CREB and the CRE region of the hCRH gene after forskolin treatment is
consistent with in vivo footprinting studies that have shown
that this same treatment enhances protection at the tyrosine
aminotransferase promoter CRE (16). Increased affinity of phospho-CREB
vs. CREB for a CRE in vitro has been demonstrated
by gel mobility shift assay (12, 13), but not by fluorescence
anisotropy (7, 11). Despite these discrepancies under in
vitro conditions, our data suggest that CREB phosphorylation may
enhance CREB-CRE interaction in vivo. In support of this,
phosphorylation-incompetent M1-CREB is completely inactive in F9 cells
(1, 18), whereas in vitro, it activates transcription as
well as does phospho-CREB (18). This implies that the CRE-binding
ability of CREB in vivo requires phosphorylation of ser 133
in the kinase-inducible domain, which is consistent with the fact that
CREB constructs that lack the kinase-inducible domain but contain the
DNA-binding domain of GAL4 are constitutively active in vivo
on a minimal GAL4 promoter (14). Homodimerization is essential for the
proper function of CREB in vivo (45). Heterodimers between
CREB and a CREB DNA-binding mutant interact normally with a CRE
in vitro, but exhibit impaired transcriptional activation of
the same CRE in vivo (45). Our findings suggest that this
could be due to impaired binding of the heterodimer to the CRE under
in vivo, but not in vitro, conditions, rather
than to impaired transcriptional transactivation per se.
Likewise, the dominant-negative effect of M1-CREB in transgenic mice
(46) might be evidence for impaired binding of M1-CREB/CREB
heterodimers to CRE motifs, rather than for the binding of
nonphosphorylatable M1-CREB homodimers to these motifs.
Nuclear injection of a CREB-binding peptide fragment of CBP, KIX, into
cells treated with forskolin blocks cAMP-mediated transcription (15).
Although interpreted as evidence that CBP recruitment to the promoter
by phosphorylated CREB is critical for cAMP-induced gene activation
(15), the possibility that phosphorylation stimulates CBP-CREB complex
formation before DNA binding, and that KIX peptide-CREB complexes
cannot so bind, is an alternative explanation for these findings.
Likewise, that addition of exogenous CREB decreases GAL-CAT reporter
activity in cells containing a GAL-CBP fusion protein (6) may be
evidence for binding between GAL-CBP and exogenous CREB remote from
DNA. Consistent with this, in vitro protein-protein
interaction experiments reveal that phosphorylation-dependent CREB-CBP
binding can occur in the absence of CRE-DNA (6, 15).
In summary, our data indicate that, in vivo, CREB, like most
other DNA-binding transcriptional activators, can undergo
signal-dependent promoter interaction. Thus, a normal function of
CREBs kinase-inducible domain, after the recruitment of CBP to CREB
(6), may be to direct this complex to the promoter, thus accounting for
the transactivation properties (5) of this domain.
 |
MATERIALS AND METHODS
|
---|
Mobility-Shift Assay of hCRH CRE-CREB Complexes in
Vitro
Nuclear extracts were prepared from human placental JEG-3 cells
as described previously (47). A double-stranded oligonucleotide
encoding a putative CRE centered 224 bp upstream from the hCRH gene
transcription start site (TCGTTGACGTCACCAA) was end-labeled
with
-32P-ATP. Nuclear extracts (1 µg) were incubated
in 16 µl reaction mixtures [1 µg poly (dI-dC), 1 µg pDN6 random
hexamers, 0.025% BSA, 5 mM dithiothreitol, and 100
mM KCl and 1020 fmol of labeled probe] for 2030 min at
25 C. Reaction mixtures were then separated on a 6%
acrylamide-bisacrylamide 29:1 nondenaturing gel and electrophoresed at
200 V for 12 h. Supershifts were done by adding 1 µl of rabbit
polyclonal anti-CREB antiserum [R1090, kindly provided by J. F.
Habener (39)] to the reaction mixture. Competition experiments were
done by adding indicated amounts of unlabeled oligonucleotide
competitor or an unrelated double-stranded synthetic oligonucleotide
polylinker of the same size. Gels were dried under vacuum and
autoradiographed on Kodak XAR x-ray film.
Regulation of the hCRH Promoter by the PKA Pathway
Fragments of the hCRH promoter containing either 663 bp or 247
bp of DNA upstream from the transcription start site and 127 bp of DNA
downstream of the transcription start site were cloned into the
luciferase expression plasmid, pXP2 [kindly provided by S. Nordeen
(48)] upstream from luciferase. JEG-3 cell culture, transient
transfection, forskolin treatment, and measurement of luciferase
activity were performed as previously described (49), using the calcium
phosphate method followed by glycerol shock. Briefly, JEG-3 cells
(106/plate) were transiently transfected with
CRH-luciferase constructs (1 µg) and 0.5 µg pRSVCAT [a
constitutively driven expression vector for chloramphenicol acetyl
transferase (CAT), used as an internal control for transfection
efficiencies]. In some cases, JEG-3 cells were cotranfected with the
plasmid, RSV-PKAc, containing the catalytic subunit of PKA driven by
the Rous sarcoma virus (RSV) promoter (kindly provided by R. Maurer).
Total transfected DNA was held constant by the addition of the plasmid,
pBluescript (Stratagene, La Jolla, CA). After
transfection, cells were treated with either forskolin, 10
µM (Sigma Chemical Co., St. Louis, MO),
dissolved in ethanol, or ethanol (vehicle) alone, for 6 h, after
which cell lysates were prepared. Luciferase activity (mean ±
SEM) was measured using a luminometer (EG&G Berthold, Bad
Wildbad, Germany), CAT activity was measured by phase extraction
followed by liquid scintillation counting, and luciferase activity was
corrected for differences in CAT expression.
Growth and Forskolin Treatment of NPLC Cells
NPLC/PRF/5, a human hepatocellular carcinoma-derived cell line,
was grown in DMEM supplemented with 10% FBS under 10% CO2
(33). Cells were plated 48 h before the experiments and were
subconfluent when collected. Forskolin, 10 µM,
(Sigma Chemical Co.) dissolved in ethanol was added to
108 cells. The same amount of ethanol vehicle was added to
the otherwise untreated control cells. After 1 h of incubation,
cells were collected and washed twice in PBS (Gibco BRL,
Gaithersburg, MD).
In Vivo Cross-linking and Isolation of DNA-CREB
Complexes
Overview
To directly analyze the interaction of CREB with the CRE of the hCRH
promoter, we used a short pulse of high-energy UV laser light (266 nm,
12 mJoules 10 nsec) to cross-link bound proteins to DNA in
situ (50), as we have previously described (28, 34). An outline of
the experimental protocol is given in Fig. 2
. NPLC cells are treated
for 1 h with forskolin, an inducer of the cAMP/PKA pathway. After
incubation, cells are trypsinized and embedded in agarose microbeads.
In protocol 1, intact, agarose-encapsulated cells are irradiated with
UV laser light and then lysed with Triton X-100. In protocol 2, UV
cross-linking is carried out after the encapsulated cells were lysed
with Triton X-100. The subsequent steps are identical in both
experimental protocols. The permeabilized nuclei are treated with high
salt to remove unbound proteins. After equilibration in
HindIII restriction digestion buffer, the DNA in the
encapsulated nuclei is digested with HindIII. This step
generates genomic DNA fragments of a median length of 6 kb. The
HindIII-cut DNA fragments diffuse out of the nuclei and are
recovered in the supernatant after pelleting of the agarose beads. The
supernatant contains the cross-linked protein-DNA complexes as well as
free DNA. To disrupt potential artifactual protein-DNA complexes formed
after the cross-linking step, the supernatant is treated with phenol.
After extraction of the aqueous phase and interphase with chloroform,
the solvent is removed under vacuum. The buffer volume is readjusted,
and the samples are incubated with biotinylated CREB-specific antibody.
Streptavidin-coated magnetic particles are added to isolate DNA-CREB
antibody complexes. For higher resolution at the DNA level, the
HindIII-digested DNA fragments bound to the
streptavidin-coated magnetic particles are digested with
AluI. The specific AluI DNA fragments
cross-linked to CREB, which should remain bound to the magnetic
particle (bound fraction), are separated from all other AluI
fragments, which should be released (AluI-released
fraction). Finally, the bound fragments are released from the magnetic
particles by proteinase K digest and analyzed by PCR and Southern blot
hybridization.
Preparation of Agarose-Encapsulated Cells/Nuclei
Aliquots of 108 cells were encapsulated as described (34, 50). To prepare metabolically active, permeabilized
agarose-encapsulated nuclei, encapsulated cells were treated with
buffer (130 mM KCl, 1 mM MgCl2, 1
mM Na2HPO4, pH 7.4) containing
0.25% Triton (Sigma Chemical Co.) on ice for 20 min. The
detergent was removed by washing the beads five times in buffer without
Triton.
Cross-linking
Either agarose-encapsulated cells or nuclei (from protocol 1 or 2,
respectively) were exposed in a quartz cuvette with a 5-mm path length
to a single 10-nsec pulse of the fourth harmonic wavelength at 266 nm
of a Nd-YAG laser. The pulse energy was about 12 mJoules with the beam
focused on an area of 100 mm2.
Isolation of CREB-Bound DNA Fragments
After cross-linking, all samples were incubated with high salt (final
concentration, 2 M NaCl) for at least 4 h to remove
unbound protein and washed several times with HindIII
digestion buffer. DNA was digested with HindIII for at least
6 h at 37 C. The supernatant of the HindIII digest was
treated with phenol to disrupt non-cross-linked protein-DNA complexes.
Chloroform was added to facilitate separation of the aqueous phase,
remnants of solvent were removed under vacuum, and sample volumes were
readjusted with water. A rabbit antihuman CREB polyclonal antiserum
that does not discriminate between nonphosphorylated and phosphorylated
CREB [R1090, kindly provided by J. F. Habener (39, 40)] was
biotinylated using Enzotin (Enzo, Inc., New York, NY) according to the
manufacturers instructions. For immunoprecipitation, equal amounts of
the samples, based on their DNA content, were incubated with
biotinylated CREB antiserum for 30 min, added to streptavidin-coated
magnetic particles (DynAl, Great Neck, NY), and incubated
for another 1.5 h. The unbound supernatant was kept for control
reactions (see Fig. 3C
), and the beads were washed three times with
AluI restriction enzyme buffer. Bound DNA was digested with
AluI for 2 h at 37 C. The supernatant
(AluI-released fraction) and the bound fraction were washed
three times. Bound DNA fragments were then released from the magnetic
beads by proteinase K digestion. Before further PCR analysis, samples
were phenol extracted, ethanol precipitated, and resuspended in 50 µl
of water.
Detection of CREB-Bound DNA Fragments
PCR primers used to amplify specific AluI restriction
fragments of the CRH gene were 1) CRE-containing promoter fragment:
CRH352F (nt 368), AAGATGGTGGGACTC; CRH352R (nt 719).
CAACAGATATTTATCGCC; 2) intron fragment: CRH163F (nt 1142),
ATGTGCGCCGCGGAG; CRH163R (nt 1304), TCTTAAGGAATAGTCCGCGAAC. Each primer
is named by the length of the corresponding PCR product followed by
either F (for forward or sense strand) or R (for reverse or antisense
strand). The numbers in parentheses correspond to the nucleotide
position of the 5'-end of the primer in the hCRH gene sequence (Fig. 1A
). To assess the relative recovery of CRH genomic fragments from
untreated and forskolin-treated cells up to the point after the
addition of streptavidin-coated magnetic particles, DNA from the
supernatants after this step were used as templates for PCR with
CRH352F and CRH163R. A 937-bp fragment is expected from this PCR
reaction.
As a further control, PCR of c-myc genomic fragments was
also performed using primers previously described (34) and PCR
conditions described below. Under these conditions, c-myc
genomic fragments are readily detected after PCR (28).
PCR was carried out in 50 µl of 1.5 mM MgCl2, 0.1%
Triton, 70 mM Tris HCl, pH 8.8, containing 1 µl of the
immunoprecipitated fraction and 30 pmol of each primer. After a 5-min
denaturation at 98 C, samples were kept at 85 C, and 2 µl of start
mixture containing deoxynucleoside triphosphates (5
mM) and 2.5 U of Taq DNA polymerase
(Ampli-Taq, Perkin Elmer Corp., Norwalk, CT)
were added to the hot sample. A total of 35 cycles (94 C for 1 min and
65 C for 45 sec) were carried out.
To visualize PCR products, agarose gel electrophoresis and Southern
blotting were performed using standard methods (51). DNA was hybridized
with a 32P-labeled hCRH cRNA probe spanning nucleotides
315-1316 (Fig. 1A
and Ref. 52). Filters were washed and exposed to
x-ray film (Kodak XAR5, Eastman Kodak Co., Rochester,
NY).
Western Blot Analysis of CREB in NPLC Cells
Western blots of protein extracts of NPLC cells cultured in the
presence or absence of forskolin were performed following the protocol
of Schreiber et al. (38). Extracts derived from equal
numbers of cells were loaded onto an SDS-polyacrylamide gel and
transferred to a nylon membrane. Visualization of the fragments
recognized by the CREB antiserum (R1090) was carried out following
standard procedures using a secondary antibody conjugate, and size
estimates were made using a protein molecular mass marker mixture
(Amersham, Arlington Heights, IL; sizes 95, 67, 44, and 30
kDa).
 |
ACKNOWLEDGMENTS
|
---|
We thank R. Maurer and S. Nordeen for plasmids, J. Habener for
the anti-CREB antiserum, and K. Seth, S. Orkin, and M. Greenberg for
helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Stefan Wölfl, Hans-Knöll Institut für Naturstoff-Forschung, 07745 Jena, Germany. E-mail: stefan{at}hki-jena.de, or to Dr. Joseph Majzoub,
Pediatrics and Medicine, Harvard Medical School, Childrens Hospital,
300 Longwood Avenue, Boston, Massachusetts 02115. E-mail:
majzoub{at}a1.tch.harvard.edu
Received for publication January 20, 1999.
Accepted for publication February 19, 1999.
 |
REFERENCES
|
---|
-
Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates
somatostatin gene transcription by phosphorylation of CREB at serine
133. Cell 59:675680[Medline]
-
Hagiwara M, Brindle P, Harootunian A, Armstrong R, Rivier J,
Vale W, Tsien R, Montminy MR 1993 Coupling of hormonal stimulation and
transcription via the cyclic AMP-responsive factor CREB is rate limited
by nuclear entry of protein kinase A. Mol Cell Biol 13:48524859[Abstract]
-
Sheng M, Thompson MA, Greenberg ME 1991 CREB: a
Ca(2+)-regulated transcription factor phosphorylated by
calmodulin-dependent kinases. Science 252:14271430[Medline]
-
Deisseroth K, Heist EK, Tsien RW 1998 Translocation of
calmodulin to the nucleus supports CREB phosphorylation in hippocampal
neurons. Nature 392:198202[CrossRef][Medline]
-
Montminy M 1997 Transcriptional regulation by cyclic AMP.
Annu Rev Biochem 66:807822[CrossRef][Medline]
-
Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman
RH 1993 Phosphorylated CREB binds specifically to the nuclear protein
CBP. Nature 365:855859[CrossRef][Medline]
-
Kwok RP, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP,
Brennan RG, Roberts SG, Green MR, Goodman RH 1994 Nuclear protein CBP
is a coactivator for the transcription factor CREB. Nature 370:223226[CrossRef][Medline]
-
Ferreri K, Gill G, Montminy M 1994 The cAMP-regulated
transcription factor CREB interacts with a component of the TFIID
complex. Proc Natl Acad Sci USA 91:12101213[Abstract]
-
Yamamoto KK, Gonzalez GA, Biggs WH, Montminy MR 1988 Phosphorylation-induced binding and transcriptional efficacy of nuclear
factor CREB. Nature 334:494498[CrossRef][Medline]
-
Montminy MR, Bilezikjian LM 1987 Binding of a nuclear protein
to the cyclic-AMP response element of the somatostatin gene. Nature 328:175178[CrossRef][Medline]
-
Richards JP, Bachinger HP, Goodman RH, Brennan RG 1996 Analysis of the structural properties of cAMP-responsive
element-binding protein (CREB) and phosphorylated CREB. J Biol
Chem 271:1371613723[Abstract/Free Full Text]
-
Nichols M, Weih F, Schmid W, DeVack C, Kowenz-Leutz E, Luckow
B, Boshart M, Schutz G 1992 Phosphorylation of CREB affects its binding
to high and low affinity sites: implications for cAMP induced gene
transcription. EMBO J 11:33373346[Abstract]
-
Bullock BP, Habener JF 1998 Phosphorylation of the cAMP
response element binding protein CREB by cAMP-dependent protein kinase
A and glycogen synthase kinase-3 alters DNA-binding affinity,
conformation, and increases net charge. Biochemistry 37:37953809[CrossRef][Medline]
-
Brindle P, Linke S, Montminy M 1993 Protein-kinase-A-dependent
activator in transcription factor CREB reveals new role for CREM
repressors. Nature 364:821824[CrossRef][Medline]
-
Parker D, Ferreri K, Nakajima T, LaMorte VJ, Evans R, Koerber
SC, Hoeger C, Montminy MR 1996 Phosphorylation of CREB at Ser-133
induces complex formation with CREB-binding protein via a direct
mechanism. Mol Cell Biol 16:694703[Abstract]
-
Weih F, Stewart AF, Boshart M, Nitsch D, Schutz G 1990 In vivo monitoring of a cAMP-stimulated DNA-binding
activity. Genes Dev 4:14371449[Abstract]
-
Boshart M, Weih F, Nichols M, Schutz G 1991 The
tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of
cAMP-dependent protein kinase. Cell 66:849859[Medline]
-
Alberts AS, Arias J, Hagiwara M, Montminy MR, Feramisco JR 1994 Recombinant cyclic AMP response element binding protein (CREB)
phosphorylated on Ser-133 is transcriptionally active upon its
introduction into fibroblast nuclei. J Biol Chem 269:76237630[Abstract/Free Full Text]
-
Pogo BG, Allfrey VG, Mirsky AE 1966 RNA synthesis and histone
acetylation during the course of gene activation in lymphocytes. Proc
Natl Acad Sci USA 55:805812[Medline]
-
Muglia L, Jacobson L, Dikkes P, Majzoub JA 1995 Corticotropin-releasing hormone deficiency reveals major fetal but not
adult glucocorticoid need. Nature 373:427432[CrossRef][Medline]
-
Seasholtz AF, Thompson RC, Douglass JO 1988 Identification of
a cyclic adenosine monophosphate-responsive element in the rat
corticotropin-releasing hormone gene. Mol Endocrinol 2:13111319[Abstract]
-
Guardiola-Diaz HM, Boswell C, Seasholtz AF 1994 The
cAMP-responsive element in the corticotropin-releasing hormone gene
mediates transcriptional regulation by depolarization. J Biol Chem 269:1478414791[Abstract/Free Full Text]
-
Adler GK, Smas CM, Fiandaca M, Frim DM, Majzoub JA 1990 Regulated expression of the human corticotropin releasing hormone gene
by cyclic AMP. Mol Cell Endocrinol 70:165174[CrossRef][Medline]
-
Dorin RI, Zlock DW, Kilpatrick K 1993 Transcriptional
regulation of human corticotropin releasing factor gene expression by
cyclic adenosine 3',5'-monophosphate: differential effects at proximal
and distal promoter elements. Mol Cell Endocrinol 96:99111[CrossRef][Medline]
-
Van LP, Spengler DH, Holsboer F 1990 Glucocorticoid repression
of 3',5'-cyclic-adenosine monophosphate-dependent human
corticotropin-releasing-hormone gene promoter activity in a transfected
mouse anterior pituitary cell line. Endocrinology 127:14121418[Abstract]
-
Majzoub JA, Emanuel R, Adler G, Martinez C, Robinson B,
Wittert G 1993 Second messenger regulation of mRNA for
corticotropin-releasing factor. Ciba Found Symp 172:3043[Medline]
-
Spengler D, Rupprecht R, Van LP, Holsboer F 1992 Identification and characterization of a 3',5'-cyclic adenosine
monophosphate-responsive element in the human corticotropin-releasing
hormone gene promoter. Mol Endocrinol 6:19311941[Abstract]
-
Wolfl S, Martinez C, Rich A, Majzoub JA 1996 Transcription of
the human corticotropin-releasing hormone gene in NPLC cells is
correlated with Z-DNA formation. Proc Natl Acad Sci USA 93:36643668[Abstract/Free Full Text]
-
Scatena CD, Adler S 1998 Characterization of a human-specific
regulator of placental corticotropin-releasing hormone. Mol Endocrinol 12:12281240[Abstract/Free Full Text]
-
Kovacs KJ, Sawchenko PE 1996 Sequence of stress-induced
alterations in indices of synaptic and transcriptional activation in
parvocellular neurosecretory neurons. J Neurosci 16:262273[Abstract]
-
Takahashi H, Teranishi Y, Nakanishi S, Numa S 1981 Isolation
and structural organization of the human corticotropin-beta-lipotropin
precursor gene. FEBS Lett 135:97102[CrossRef][Medline]
-
Deutsch PJ, Hoeffler JP, Jameson JL, Lin JC, Habener JF 1988 Structural determinants for transcriptional activation by
cAMP-responsive DNA elements. J Biol Chem 263:1846618472[Abstract/Free Full Text]
-
Carlin CR, Simon D, Mattison J, Knowles BB 1988 Expression and
biosynthetic variation of the epidermal growth factor receptor in human
hepatocellular carcinoma-derived cell lines. Mol Cell Biol 8:2534[Medline]
-
Wittig B, Wolfl S, Dorbic T, Vahrson W, Rich A 1992 Transcription of human c-myc in permeabilized nuclei is associated with
formation of Z-DNA in three discrete regions of the gene. EMBO J 11:46534663[Abstract]
-
Hecht A, Laroche T, Strahl-Bolsinger S, Gasser SM, Grunstein M 1995 Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins:
a molecular model for the formation of heterochromatin in yeast. Cell 80:583592[Medline]
-
Kuo MH, Zhou J, Jambeck P, Churchill ME, Allis CD 1998 Histone
acetyltransferase activity of yeast Gcn5p is required for the
activation of target genes in vivo. Genes Dev 12:627639[Abstract/Free Full Text]
-
Kadosh D, Struhl K 1998 Targeted recruitment of the Sin3-Rpd3
histone deacetylase complex generates a highly localized domain of
repressed chromatin in vivo. Mol Cell Biol 18:51215127[Abstract/Free Full Text]
-
Schreiber E, Matthias P, Muller MM, Schnaffner W 1989 Rapid
detection of octamer binding proteins with mini-extracts, prepared
from a small number of cells. Nucleic Acids Res 17:6419[Medline]
-
Waeber G, Meyer TE, LeSieur M, Herman HL, Gerard N, Habener JF 1991 Developmental stage-specific expression of cyclic adenosine
3',5'-monophosphate response element-binding protein CREB during
spermatogenesis involves alternative exon splicing [published erratum
appears in Mol Endocrinol 1993 Nov;7(11):1501]. Mol Endocrinol 5:14181430[Abstract]
-
Walker WH, Fucci L, Habener JF 1995 Expression of the gene
encoding transcription factor cyclic adenosine 3',5'-monophosphate
(cAMP) response element-binding protein (CREB): regulation by
follicle-stimulating hormone-induced cAMP signaling in primary rat
Sertoli cells. Endocrinology 136:35343545[Abstract]
-
Wolfl S, Wittig B, Dorbic T, Rich A 1997 Identification of
processes that influence negative supercoiling in the human c-myc gene.
Biochim Biophys Acta 1352:213221[Medline]
-
Bannister AJ, Kouzarides T 1996 The CBP co-activator is a
histone acetyltransferase. Nature 384:641643[CrossRef][Medline]
-
Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y 1996 A
p300/CBP-associated factor that competes with the adenoviral
oncoprotein E1A. Nature 382:319324[CrossRef][Medline]
-
Gu W, Roeder RG 1997 Activation of p53 sequence-specific DNA
binding by acetylation of the p53 C-terminal domain. Cell 90:595606[Medline]
-
Dwarki VJ, Montminy M, Verma IM 1990 Both the basic region and
the leucine zipper domain of the cyclic AMP response element
binding (CREB) protein are essential for transcriptional activation.
EMBO J 9:225232[Abstract]
-
Struthers RS, Vale WW, Arias C, Sawchenko PE, Montminy MR 1991 Somatotroph hypoplasia and dwarfism in transgenic mice expressing a
non-phosphorylatable CREB mutant. Nature 350:622624[CrossRef][Medline]
-
Shapiro DJ, Sharp PA, Wahli WW, Keller MJ 1988 A
high-efficiency HeLa cell nuclear transcription extract. DNA 7:4755[Medline]
-
Nordeen SK 1988 Luciferase reporter gene vectors for analysis
of promoters and enhancers. Biotechniques 6:454458[Medline]
-
Iwasaki Y, Oiso Y, Saito H, Majzoub JA 1997 Positive and
negative regulation of the rat vasopressin gene promoter. Endocrinology 138:52665274[Abstract/Free Full Text]
-
Jackson DA, Cook PR 1985 A general method for preparing
chromatin containing intact DNA. EMBO J 4:913918[Abstract]
-
Southern EM 1975 Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J Mol Biol 98:503517[Medline]
-
Adler GK, Smas CM, Majzoub JA 1988 Expression and
dexamethasone regulation of the human corticotropin-releasing hormone
gene in a mouse anterior pituitary cell line. J Biol Chem 263:58465852[Abstract/Free Full Text]