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.) Children’s Hospital Harvard Medical School Boston, Massachusetts 02115


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A schematic diagram of the human CRH (hCRH) gene is shown in Fig. 1AGo. 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. 1AGo 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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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 1086–1885. 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)/{Delta}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. 1BGo). 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. 1BGo). 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. 1CGo). Luciferase activity of this construct was also stimulated 20-fold by treatment with forskolin, 10 µM (Fig. 1DGo). 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/{Delta}224) resulted in a 10-fold decrease in forskolin-stimulated luciferase activity (Fig. 1EGo). 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. 1EGo). 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. 2Go). 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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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. 2Go and 3Go). In both cases, the 352-bp CRH fragment containing the CRE (Fig. 1AGo) was detected only when cells had been initially treated with forskolin (lanes F, Fig. 3Go, A and B), and never in control, untreated cells (lanes C, Fig. 3Go, A and B). This is evident when PCR products were visualized by either ethidium bromide staining (Fig. 3AGo) or after more sensitive Southern blot hybridization of the electrophoresis products to a radioactively labeled hCRH probe (Fig. 3BGo). 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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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. 2Go). 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, {phi}{chi}, 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. 3AGo, 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. 3AGo). 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. 2Go). 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. 3AGo). 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. 3BGo), 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. 2Go) 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. 3CGo), 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. 3DGo). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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, 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. 4Go). 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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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 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {gamma}-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. 2Go. 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. 3CGo), 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. 1AGo). 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. 1AGo 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.


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