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Hans-Knöll-Institut für Naturstoff-Forschung (S.W.)
07745 Jena, Germany
Division of Endocrinology (C.M.,
J.A.M.) Children’s Hospital Harvard Medical School Boston,
Massachusetts 02115
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. 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.
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). 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).
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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).
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, 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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, forskolin’s 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).
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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 CREB’s 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 10–20 fmol of labeled probe] for 20–30 min at
25 C. Reaction mixtures were then separated on a 6%
acrylamide-bisacrylamide 29:1 nondenaturing gel and electrophoresed at
200 V for 1–2 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
manufacturer’s 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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 20, 1999. Accepted for publication February 19, 1999.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 L. F. Michael, H. Asahara, A. I. Shulman, W. L. Kraus, and M. Montminy The Phosphorylation Status of a Cyclic AMP-Responsive Activator Is Modulated via a Chromatin-Dependent Mechanism Mol. Cell. Biol., March 1, 2000; 20(5): 1596 - 1603. [Abstract] [Full Text]
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, 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).