ThecAMPPathwayinCombinationwithBMP2Regulates ...developing embryo and differentiate to diverse cell types (1). NC cells ... chicken embryo extract, and primary NC cultures of 47.5

The cAMP Pathway in Combination with BMP2 RegulatesPhox2a Transcription via cAMP Response ElementBinding Sites*

Received for publication, April 12, 2005, and in revised form, November 21, 2005 Published, JBC Papers in Press, December 5, 2005, DOI 10.1074/jbc.M503939200

Chutamas Benjanirut‡, Maryline Paris‡1, Wen-Horng Wang‡1, Seok Jong Hong§, Kwang Soo Kim§,Ronald L. Hullinger‡, and Ourania M. Andrisani‡2

From the ‡Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907 and the §MolecularNeurobiology Laboratory, MRC215, McLean Hospital, Harvard Medical School, Belmont, Massachusetts 02478

Combined BMP2 and cAMP signaling induces the catechola-minergic lineage in neural crest (NC) cultures by increasing ex-pression of the proneural transcription factor Phox2a, in a cAMPresponse element (CRE)-binding protein (CREB)-mediatedmecha-nism. To determine whether CREB acts directly on Phox2a trans-cription induced by BMP2�cAMP-elevating agent IBMX, transi-ent transfections of hPhox2a-reporter constructs were performedin avian NC cultures and murine, catecholaminergic CAD cells.Although BMP2�IBMX increased endogenous Phox2a expres-sion, the 7.5-kb hPhox2a reporters expressing either lucifer-ase or DsRed1-E5 fluorescent protein were unresponsive toBMP2�IBMX, but active in both cell types. Cell sorting of fluores-cence-positive NC cells expressing the 7.5-kb hPhox2a fluorescenttimer reporter differentiated to equal numbers of catecholaminer-gic cells as fluorescence-negative cells, suggesting inappropriatetranscription from the transfected hPhox2a promoter. NC or CADcells treated with histone deacetylase inhibitor trichostatin A andBMP2�IBMX display increased endogenous Phox2a transcriptionand prolonged CREB phosphorylation, indicating Phox2a chroma-tin remodeling is linked to CREB activation. Chromatin immuno-precipitations employing CREB, CREB-binding protein, and acety-lated H4 antibodies identified two CRE half-sites at �5.5 kb in themurine Phox2a promoter, which is also conserved in the humanpromoter. Proximal to the CRE half-sites, within a 170-bp region,are E-box and CCAAT binding sites, also conserved in mouse andhuman genes. This 170-bp promoter region confers cAMP, BMP2,and enhanced BMP2�cAMP regulation to Phox2a-luciferasereporters. We conclude these CREs are functional, with CREBdirectly activating Phox2a transcription. Because the E-box bindsbHLH proteins like ASH1 induced in NC cells by BMP2, we pro-pose this novel 170-bp cis-acting element is a composite site, medi-ating the synergistic regulation by BMP2�cAMP on Phox2atranscription.

Pluripotent neural crest (NC)3 cells derived from the neuroepithe-lium during neural tube closure migrate along defined routes in the

developing embryo and differentiate to diverse cell types (1). NC cellsfrom the trunk region of the neural tube differentiate to the cat-echolaminergic, sympathoadrenal (SA) lineage, including sympatheticneurons and chromaffin cells of the adrenalmedulla (2). SA lineage cellssynthesize catecholamines (CA) by expressing tyrosine hydroxylase(TH), the rate-limiting enzyme in CA biosynthesis, and dopamine-�-hydroxylase (DBH), which converts dopamine to norepinephrine (2).Bone morphogenetic proteins (BMP2, -4, and -7) (3–5) and cAMP-

elevating agents (6, 7) promote SA lineage development in NC cultures.cAMP signaling in synergy with BMP2 induces development of the SAlineage by increasing the expression of the homeodomain transcriptionfactor Phox2a (7) in a CREB-mediated mechanism (8). cAMP signalingalso regulates the transactivation potential of Phox2a (8), consistentwith studies by Lo et al. (6). However, whether cAMP signaling directlyregulates transcription of the Phox2a gene remains to be determined.Although the regulation of the human hPhox2a promoter has beenstudied in human neuroblastoma cell lines expressing the endogenousPhox2a gene (9), the direct transcriptional regulation of the Phox2apromoter by BMP2�cAMP signaling has not been investigated.BMP2, an inductive signal in SA cell development, induces the bHLH

transcription factor MASH1 (10) or CASH1, the mammalian (11) andavian/chick (12, 13) homologues, respectively. MASH1 is expressed inboth central (CNS) and peripheral (PNS) nervous system (11, 14).Forced MASH1 expression in NC cultures activates Phox2a expression(15), whereasMASH1�/�mice display defects in sympathetic ganglia,adrenal chromaffin cells (16) and lack Phox2a expression in brain nora-drenergic centers and PNS (17). Phox2a regulates transcription of THand DBH genes (18–20), and Phox2a-null mice lack the locus coreulus,a major CNS catecholaminergic (noradrenergic) center (21).The CNS-derived catecholaminergic Cath.a cell line (22) provides a

cellular model for comparative studies of NC-derived and CNS-derivedcatecholaminergic neurons. The Cath.a cell line derived from braintumors of transgenicmice expressing the SV40T-antigen under controlof the TH promoter (22), like the catecholaminergic SA cells, expressesTH and DBH, synthesizes catecholamines, and also develops neurites.The CAD cell line used herein is a variant of Cath.a cells that undergoesneuronal differentiation by serum withdrawal (23) or by BMP2�cAMPcostimulation (24).Herein, Phox2a promoter activity is examined following costimula-

tion with BMP2�cAMP, employing two differentiation models of cat-echolaminergic neurons, namely, NC-derived SA cells and CNS-de-rived CAD cells. We report the identification of a novel, composite

* This work was supported by National Institutes of Health Grant DK59367 (to O. M. A.).The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Dept. of Basic Medical Sciences, Pur-

due University, 625 Harrison St., W. Lafayette, IN 47907-2026. Tel.: 765-494-8131; Fax:765-494-0781; E-mail: [email protected].

3 The abbreviations used are: NC, neural crest; CRE, cAMP response element; CREB, CRE-binding protein; TSA, trichostatin A; ChIP, chromatin immunoprecipitation assay;bHLH, basic helix loop helix; EMSA, electrophoretic mobility shift assay; IBMX,

3-isobutyl-1-methylxanthine; SA, sympathoadrenal; HDAC, histone deacetylases; mt,mutant; PBS, phosphate-buffered saline; UE, upstream element; CA, catecholamines;TH, tyrosine hydroxylase; BMP, bone morphogenetic protein; CNS, central nervoussystem.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 5, pp. 2969 –2981, February 3, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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cis-acting element, located 5.5-kb upstream from the transcriptionalstart site of the murine Phox2a gene. This regulatory element is com-prised of two functional CRE half-sites in proximity to putative E-boxandCCAATbinding sites. The identification of the functional CRE sitesconclusively demonstrates the direct transcriptional involvement of thecAMP pathway in Phox2a gene regulation.

EXPERIMENTAL PROCEDURES

Culture Reagents—Vitrogen 100was purchased fromCollagenCorp.;bovine fibronectin and IBMX from Sigma; trichostatin A (TSA) fromA. G. Scientific Inc. Calcium- and magnesium-free phosphate buffersolution (CMF-PBS) and 0.05% trypsin, 0.53mMEDTAwere purchasedfrom Invitrogen. Recombinant human BMP2 (Wyeth Research) wasreconstituted to 10 �g/ml as recommended and stored at �80 °C.

NC Cultures—Standard NC culture growth medium containing 10%chicken embryo extract, and primary NC cultures of 47.5 h Japanesequail (Coturnix coturnix) embryos were prepared as previouslydescribed (7). NC cells were isolated from primary outgrowths of neuraltube explants plated for 42 h on Vitrogen 100-coated culture dishes.Adherent NC cells from the primary neural tube outgrowths were har-vested by trypsin-EDTA, resuspended in growthmedium (7), and platedin fibronectin-coated dishes at a density of 320 cells/mm2 resulting insecondary NC cultures. NC cells were allowed to attach for 2 h, and theseeding medium was replaced with 2 ml of growth medium, with orwithout BMP2 (10 ng/ml) and IBMX (100�M). NC cultures were fed byexchange of 1 ml of growthmedium on day 3 after subculture and everyother day thereafter.

CAD Cell Culture—CAD cells were grown as described (23) in Dul-becco’s modified Eagle’s medium (low glucose, Invitrogen), supple-mented with 10% fetal bovine serum (HyClone), 5% calf serum (Invitro-gen), and 1% penicillin-streptomycin (100% stocks: 10,000 units/mlpenicillin G sodium and 10,000 mg/ml streptomycin sulfate in 0.85%saline, Invitrogen).

Western Blot Analysis—Total protein was extracted from NC cul-tures or CAD cells, grown in 24-well dishes, in radioimmune precipita-tion assay buffer containing 150 mM NaCl, 2 mM EDTA, 1 mM sodiumorthovanadate, 10 �g/ml leupeptin, 25 �g/ml aprotinin, 1.0% TritonX-100, 50 mMTris (pH 7.6). Extracts were sonicated on ice for 15 s, andprotein concentration was determined by the Bio-Rad protein assay.Total protein (20 �g) was analyzed by 10% SDS-PAGE and transferredto nitrocellulose. The membranes were probed with 1 �g/ml CREBantibody or 1 �g/ml of anti-phospho-CREB (Ser133) (Upstate). Detec-tion was with 1:2,000 dilution of horseradish peroxidase-conjugatedanti-rabbit IgG (Vector) using the enhanced chemiluminescence (ECL)detection system (Amersham Biosciences).

Immunocytochemistry—NC cells were fixed for 20 min with 4%paraformaldehyde. Nonspecific background was blocked using PBScontaining 10% goat serum for 40 min. Antibody for P-CREB (1:200,Upstate) was applied overnight at 4 °C followed by three washes in PBS.Undiluted TH antibody or peripherin antibody (1:200, Chemicon) wasapplied for 40 min followed by three washes in PBT. A 1:400 dilution ofAlexa fluor 488 goat anti-rabbit IgG was applied for 45 min followed bythree washes in PBS. For immunofluorescence of CAD cells the follow-ing modifications were included: nonspecific background was blockedusing PBS containing 1% goat serum. Antibody was added in PBS con-taining 1% goat serum and applied 1 h at room temperature.

Real-time PCR—Total RNA from NC or CAD cells was extractedwith TRIzol (Invitrogen). Total RNA (20 �g) was treated with RNase-free DNase I (20 units) in the presence of RNase inhibitor (40 units).cDNA was synthesized using 2.0 �g of total RNA, 0.2 �g of random

hexamers, and Superscript II reverse transcriptase as described (Invitro-gen). Oligonucleotide primers (Integrated DNA Technologies) weredesigned using Omega 2.0 software (Accelrys). PCR reactions were per-formed in identical triplicates using 2 �l of cDNA, 1 �l of 20 �M F-/R-primer, and SYBR Green PCR Master Mix as described by the manu-facturer (Applied Biosystems), employing an Applied Biosystems Prism7000 Real-Time PCR system and the accompanying Sequence Detec-tion Software version 1.0. Data analysis was carried out as described byLee et al. (25), employing 18 S rRNA as the internal control.

Plasmids and Transfections—hPhox2a promoter-Luc reporter con-structs (9) were transiently transfected by Fugene 6 (Roche AppliedSciences) according to the manufacturer’s instructions, employing 1 �gof plasmid DNA for a 6-well NC culture or 2 �g for a 6-well of CAD cellculture. Luciferase activity was compared with pGL3-control vector(Promega). CMV-nLacZ plasmid was used to optimize transfections.Several hPhox2a promoter constructs were cloned into the pTimer-1Vector (BD Biosciences Clontech), encoding the DsRed1-E5 protein, amutant of Discosoma sp. red fluorescent protein, which changes colorover time. Shortly after translation, the protein emits green light; its redfluorophore emerges later, hours after translation. Plasmids were puri-fied by CsCl2 equilibrium gradient centrifugation. Cells were harvested48 h after transfection, lysed in 0.2 ml of lysis buffer (Promega) andassayed for luciferase activity (Luciferase Assay system, Promega).Luciferase activity was normalized per microgram of protein extract.Cells transfectedwith the pTimer-1 reporter constructswere assayed byfluorescence microscopy and flow cytometry. Luciferase reporter plas-mids were constructed by cloning themouse Phox2a upstream element(UE and mtUE) spanning nucleotides �5754 to �5340 in the hPhox2a�32Luc and �515-Luc vectors. The resulting vectors were transientlytransfected in NC or CAD cells as described earlier.Electrophoretic mobility shift assays (EMSA) were performed as

described (26), employing 250 ng of purified recombinant CREBprotein(activemotif) per binding reaction and 32P-radiolabeled oligonucleotideprobes spanning the CRE half-site 1 or 2. The sequence of the oligonu-cleotides is as follows: CRE site 1: 5�-GGCTGTAGAATTCGTCATC-ACTCATTACACTGA-3�; CRE site 2: 5�-AGGAGCACATATGGGG-CGTCACTCCTCAGTTAGGAT-3�; mutantCRE: 5�-GGCTGTAGA-ATTATGGTTCACTCATTACACTGA-3�; Wt CRE: 5�-GGCTGTA-GAATTTGACGTCATCACTCATTACACTGA-3�.

Cell Sorting by Flow Cytometry—NC cells were transfected with 1 �gof hPhox2a-Timer-1 plasmid as described earlier, at 24 h of primaryNCoutgrowths. At 42 h of primary NC outgrowths, neural tubes wereremoved; NC cells were then replated and treated with BMP2�IBMX.After 24 h, NC cells were observed every day for 3 days by fluorescencemicroscopy. Fluorescence-positive cells were quantified by cell sortingusing the Epics Altra flow cytometer (Beckman-Coulter).

Chromatin Immunoprecipitation Assays (ChIP)—CAD cells grownto near confluence in 10-cm dishes were treated for 2 h with BMP2 (10ng/ml), IBMX (100 �M), and/or TSA (100 ng/ml). Cellular proteinswere cross-linked to chromatinized DNA for 10 min at 37 °C by addi-tion to the medium of 1% formaldehyde. Cells were washed twice usingice-cold PBS containing protease inhibitors: 1 mM phenylmethylsulfo-nyl fluoride, 1 �g/ml aprotinin and 1 �g/ml pepstatin A. Cell pelletswere resuspended in 0.2 ml of SDS lysis buffer (1% SDS, 10 mM EDTA,50 mM Tris, pH 8.1) and sonicated to reduce DNA length to �200–1,000 bp. The chromatin mixture was diluted 10-fold in ChIP dilutionbuffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mMNaCl) and incubated overnight at 4 °C with 10 �gof CREB antibody or 5 �g of CBP antibody (Upstate) or 5 �g of IgG or 1�g of acetylated H4 antibody (kindly provided by Dr. S. Briggs, Purdue

Functional CRE Sites in Mammalian Phox2a Promoter

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University). Immune complexes were collected with salmon spermDNA/protein A agarose slurry (Upstate). Following extensive washingand elution in 1% SDS-0.1 M NaHCO3, DNA-protein cross-links werereversed by incubation at 65 °C overnight. Released DNA was purifiedby proteinase K digestion, phenol extraction, and ethanol precipitation.Immunoprecipitated DNA was quantified by real-time PCR. Thesequences of the forward and reverse primers are as follows: CRE Site 1,2: F-5�-TGCCTAGCCATTCACATTAG-3�; R-5�-CCTGAGAG-GAGAGGCCAAGTG-3�; CRE Site 3: F-5�-TCCTGCCCT-GAGAACTCTAT-3�; R-5�-CTCGCAACACTAGCTTTAG-3�; CRESite 4: F-5�-CCCAGGAGCAGATAGATCCT-3�; R-5�-CATGCTGG-GACACTAACAGA-3�; CRE Site 5: F-5�-AATTTGGGAAGATGG-GACCC-3�; R-5�-ACCTCTGTCCCACTTCCTCT-3�.

RESULTS

In avian NC cultures, moderate activation of cAMP signaling actssynergistically with BMP2 to promote development of the SA lineage(7), by increasing Phox2a and Phox2b transcription via activation andinvolvement of the transcription factor CREB (8), the downstreameffector of the cAMP pathway (27, 28).To directly demonstrate the mechanism of the combined BMP2 and

cAMP signaling onPhox2a gene transcription, we employed thePhox2apromoter and its deletion constructs in transient luciferase reporterassays in avian NC cells. It is well established from studies of develop-mentally regulated and differentiation-specific genes, that functionallyimportant cis-acting elements are conserved across species (29–33).Accordingly, we employed the human Phox2a promoter to investigateits regulation in the developmentally regulated avian primary NC cul-ture model system. Earlier studies by Hong et al. (9) have reported thecloning of the hPhox2a promoter and its characterization, studied in thehuman neuroblastoma SK-N-BE (2)M17 and SK-N-BE (2)C Phox2a-expressing cell lines. However, Phox2a regulation in response to BMP2and cAMP signaling has not been examined.Employing the Fugene method of transfection in primary NC cul-

tures, we initially established that 40% transfection efficiency wasachieved with the CMV-�-gal reporter, without negative growth effectson the primary culture (data not shown). The hPhox2a-Luc reporterconstructs shown in Fig. 1A, spanning sequences from 32 bp to 7.5 kbupstream from the transcriptional start site were transiently transfectedin avian NC cells, as a function of cotreatment with BMP2�IBMX (acAMP-elevating agent). The activity of the hPhox2a promoter tested inthe avianNC system is qualitatively identical to that reported byHong etal. (9). Specifically, the highest activity is observed with the 1.3-kb pro-moter fragment. These results suggest the Phox2a promoter activity islikely conserved between mammalian and avian species.

The Activity of hPhox2a Promoter Differs from the EndogenousPhox2a Gene in NC Cultures—Interestingly, the transient transfectionresults do not demonstrate regulation of the hPhox2a promoter con-structs by BMP2 and cAMP inNCcells (Fig. 1A). In our previous studieswe have shown that cotreatment of NC cells with BMP2 and cAMPsynergistically induces expression of the endogenousPhox2a gene (7, 8),aswell as of THmRNA (Fig. 1B). Thus, there appears to be a discrepancybetween the in vivo regulation of the Phox2a gene and the transienttransfection results of the hPhox2a promoter.To exclude that the hPhox2a promoter-luciferase reporter constructs

might not be suitable for deciphering regulation of the Phox2a pro-moter (34), the Phox2a promoter was cloned and studied using thefluorescence-timer (pTimer-1) reporter system (35). This reportermonitors both the onset of transcription of a given promoter, by mon-

itoring the appearance of green fluorescence, as well as the time of itstranscriptional shut off, i.e.when only red fluorescence is observed (35).Employing the hPhox2a-fluorescence-timer reporter constructs

transfected in NC cultures in conjunction with flow cytometry the flu-orescence-positive cells were quantified as a function of BMP2�IBMXcotreatment (Fig. 2A). The results demonstrated the promoter is tran-scriptionally active in the absence of BMP2�IBMX costimulation inNC cultures (Fig. 2A), and that cotreatment with BMP2�IBMX doesnot mediate any further transcriptional induction (Fig. 2A). Impor-tantly, Phox2a promoter activation starts on day 1 of secondary NCculture (green fluorescence), which is earlier than the known expressionpattern of the endogenous Phox2a gene (7), and is turned off by day 2

FIGURE 1. A, hPhox2a promoter activity in J. quail primary NC cells with BMP2 (10 ng/ml)�IBMX (100 �M) treatment, as indicated. The hPhox2a-Luc reporter constructs (1 �g)listed were transfected via the Fugene 6 method into 6-well NC cultures at day 0 ofreplating of primary NC outgrowths. Cells were harvested 48 h after transfection; lucif-erase activity normalized per microgram of protein extract is expressed relative to lucif-erase activity of the control pGL3 plasmid, considered as 100%. The result is from threeindependent experiments, each performed in triplicate. B, real-time PCR for TH mRNAexpression, employing total RNA isolated from NC cultures at days 1–5 following replat-ing and treated with BMP2�IBMX, as indicated. Results represent three independentassays, each performed in identical triplicates.




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(red fluorescence), when the endogenous Phox2a gene is known to betranscribed (8, 36).Use of the fluorescent reporter allows cell sorting of the transfected,

fluorescence-positive NC cells and thus the study of their phenotypeand developmental potential. Following live cell sorting, the Phox2afluorescence-positive and fluorescence-negative NC cells were placedin culture; 2 h after replating 80% of hPhox2a- fluorescence-positiveNCcells remained positive. Likewise, no fluorescence was detected with thesorted fluorescence-negative NC cells (Fig. 2B). The sorted NC cellswere then grown either under control conditions or with BMP2�IBMXcotreatment. After 4 days of culture the NC cells were immunostainedwithTHantibody tomonitorTHprotein expression, amarker of SA celldevelopment (7, 8). We hypothesized, if the transcriptional activity ofthe transiently transfected hPhox2a were authentic, these hPhox2a-flu-orescence-positive NC cells would be expressing the endogenousPhox2a gene. Because Phox2a expression is required for SA develop-ment, we predicted that all of the sorted hPhox2a-fluorescence-positiveNC cells would give rise to SA progenitors, i.e. they would be TH immu-noreactive. However, following cell sorting and replating, both the

hPhox2a fluorescence-positive and -negative NC cells cotreated withBMP2�IBMX, resulted in the appearance of similar numbers of TH-immunoreactive cells (Fig. 2C). Interestingly, the appearance of mela-nocytes was also observed in the fluorescence-positive NC cultures(data not shown).These results suggest the hPhox2a promoter constructs were inap-

propriately activated in NC cells, other than those that became SA cells,supporting that the expression of the transiently transfected hPhox2areporters does not reflect the expression of the endogenous Phox2agene. Taken together with previous results (Fig. 1A), these data demon-strate that the hPhox2a-Luc construct, comprised of 7.5kb of upstreamsequence, does not respond similarly to the endogenous promoter, sug-gesting that its expression is either misregulated or unresponsive tosignals known to induce the expression of the endogenous Phox2amRNA.Three possibilities account for the observed results. 1) Regulation of

the human Phox2a promoter in avian cells differs from regulation inmammalian cells. 2) The chromatin context is an important determi-nant in the synergistic BMP2�IBMX regulation, and 3) cis-acting ele-

FIGURE 2. A, expression of hPhox2a-Timer-1reporter in primary NC cells. Flow cytometricquantification of fluorescence-positive NC cells,transfected with the 7.5-kb hPhox2a-Timer-1 vec-tor, at days 1 and 2 of secondary NC cultures. The7.5-kb hPhox2a-Timer-1 vector (1 �g) was trans-fected via the Fugene 6 method at 24 h of primaryNC outgrowths; removal of neural tubes was at42 h, followed by replating and addition ofBMP2�IBMX, as indicated. Green fluorescenceindicates the fluorescent reporter protein(DsRed1-E5 protein of Discosoma sp.) shortly aftertranslation; red fluorescence emerges severalhours after translation. Data shown represent theaverage of two independent experiments. B,developmental potential of sorted hPhox2a-fluo-rescence-positive NC cells, transfected with 7.5-kbhPhox2a-Timer-1 plasmid, as described in A. Fluo-rescence microscopy of sorted green-red fluores-cence-positive and fluorescence-negative NCcells. Following cell sorting, NC cells werere-plated, and treated with BMP2 (10 ng/ml) andIBMX (100 �M) at 2 h after replating. Images are at�10 magnification. C, sorted fluorescence-posi-tive and -negative NC cells from B were culturedfor 4 days and immunostained with TH antibodyand Hoechst 33402, as indicated.




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ments mediating the BMP2 and cAMP regulation reside outside the7.5-kb hPhox2a promoter fragment.

Regulation of Phox2a Promoter in Murine, Catecholaminergic CADCells—To address whether regulation of the Phox2a promoter in aviancells is different from the regulation in mammalian cells, we employedthe murine CAD cell line (23), a variant of the CNS-derived cat-echolaminergic Cath.a cells (22). The Cath.a cell line was establishedfrom a brain tumor that arose in a transgenic mouse carrying the wild-type SV40 T antigen (Tag) under the transcriptional control of the THpromoter (22). Neuronal differentiation in CAD cells is initiated byserum deprivation (23) or addition of BMP2�IBMX (24).Employing real-time PCR we determined the expression of Phox2a

and TH mRNAs in CAD cells. Phox2a and TH mRNAs display a slightincrease 1 day after serum starvation, followed by a gradual decrease(Fig. 3, A and B). Importantly, BMP2�IBMX resulted in an obviousincrease in Phox2a andTH expression, both in the undifferentiated (day0 of treatment) and differentiated CAD cells (Fig. 3,A and B) suggestingregulation of the endogenous Phox2a promoter in mammalian cells issimilar to avian cells (7, 8). Moreover, BMP2�IBMX cotreatmentinduced differentiation of CAD cells in the presence of serum.4

Accordingly, employing the CAD cellular model system, the activityof the hPhox2a promoter Luc reporter constructs was investigated fol-lowing transfection, as a function of BMP2�IBMX cotreatment. Theresults demonstrate, BMP2�IBMX cotreatment had no effect on theactivity of all hPhox2a promoter constructs tested, similar to the resultsobserved in the avian NC cells (Fig. 3C). Thus, we conclude: 1) ThehPhox2a promoter responds similarly in the avianNC andmurine CADcells, and 2) transiently transfected hPhox2a-reporter constructs do notrespond to the same regulatory mechanism(s) operative on the endog-enous Phox2a promoter.

Chromatin Remodeling Regulates Endogenous Phox2a Gene Tran-scription by BMP2�IBMX—It is well established that reversible histoneacetylation by acetyltransferases (HATs) relaxes (opens) chromatin,allowing transcriptional activation (37, 38). Likewise, histone deacety-lation by HDACs induces a closed chromatin configuration and tran-scriptional repression (39–40).To investigate the second possibility described earlier, namely

whether the chromatin context of the Phox2a gene is an importantdeterminant in the synergistic transcription by BMP2�IBMX, weexamined the effect of the HDAC inhibitor TSA (41), on endogenousPhox2a transcription. NC cells were treated with various doses of TSAfor different periods to determine the TSA effect on NC cell growth(data not shown). Treatment with 100 ng/ml TSA for 3-h intervals atdifferent times during day 1 (24–36 h after re-plating of the primaryNCoutgrowths) showed no detectable effect on NC cell growth and differ-entiation. Interestingly, BMP2�IBMX cotreatment in the presence of100 ng/ml TSA increased endogenous Phox2a mRNA expression com-pared with treatment with only BMP2�IBMX, assessed by real-timePCR (Fig. 4A). TSA treatment without BMP2�IBMX does not inducePhox2a mRNA expression (Fig. 4A). It is important to note thatBMP2�IBMX treatment mediates only minimal Phox2a expression at24 h of NC cultures, assessed by real-time PCR (8).Similar studies in CAD cells also demonstrated that endogenous

Phox2a mRNA expression is further increased by TSA addition in thepresence of BMP2�IBMX (Fig. 4B). Likewise, addition of TSA in thepresence of BMP2�IBMX increases CAD cell neuronal differentiation,assessed by peripherin immunostaining and TH immunoreactivity (Fig.4C). By contrast, TSA alone does not induce Phox2a mRNA expression

4 M. Paris and O. M. Andrisani, unpublished results.

FIGURE 3. Effect of BMP2 and cAMP signaling on endogenous Phox2a transcriptionin CAD cells. Real-time PCR analyses of Phox2a (A) and TH (B) mRNA expression in totalRNA isolated from CAD cells treated with BMP2�IBMX as indicated, starting at day 0prior to serum withdrawal (day 1). Quantification is from three independent RNA prep-arations, each PCR reaction analyzed in identical triplicates, and normalized relative to 18S rRNA used as the internal control, as described in Lee et al. (25). C, hPhox2a promoteractivity in murine CAD cells treated with BMP2 (10 ng/ml) �IBMX (100 �M). CAD cellsgrown in serum-containing medium were transfected with indicated hPhox2a-Lucreporter constructs (2 �g) and treated with BMP2�IBMX, as indicated. Cells were har-vested 48 h after transfection; luciferase activity normalized per microgram of proteinextract is expressed relative to luciferase activity of pGL3 plasmid, considered as 100%.Results shown are from three independent experiments each performed in triplicates.




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FIGURE 4. The histone deacetylase inhibitor TSA increases BMP2�IBMX-induced Phox2a expression in NC cultures. A, NC cultures at day 0 of secondary culture treated withBMP2�IBMX for 24 h. TSA (100 ng/ml) was added for 3 h after 24, 27, 30, and 33 h of treatment with BMP2�IBMX, as indicated. After each 3-h treatment TSA was removed. The RNAsamples were collected on day 2 for real-time PCR quantification of endogenous Phox2a mRNA. B, TSA increases BMP2�IBMX-induced Phox2a mRNA expression in CAD cells. CADcells grown in serum-containing medium with BMP2�IBMX added at time 0, followed by TSA addition for 3 h, as indicated. After each 3-h treatment, TSA was removed, and CAD cellswere incubated with BMP2�IBMX for a total of 24 h, when RNA was collected for real-time PCR quantification of endogenous Phox2a mRNA. Data shown (A and B) are from threeindependent RNA preparations, each PCR reaction performed in identical duplicates, and normalized to 18 S rRNA used as the internal control. C, TSA increases neuronal differen-tiation in CAD cells treated with BMP2�IBMX. Immunofluorescence microscopy of TH and peripherin expression in CAD cells treated with BMP2�IBMX in the presence of TSA, addedat the 6 –9-h interval shown in B.




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(Fig. 4B) or CAD cell neuronal differentiation (Fig. 4C). Accordingly,these data support a mechanism whereby the chromatin structure is animportant parameter in the transcriptional regulation of the endoge-nous Phox2a promoter by BMP2�IBMX.

HDAC Inhibition Prolongs CREB Phosphorylation in NC and CADCells—Our recent studies (8) have demonstrated that dominant nega-tive CREB suppresses Phox2a mRNA expression by BMP2�IBMX,whereas the constitutively active CREBDIEDML induces Phox2a expres-sionwithout cAMP signaling. Based on these studies (8) and the effect ofTSA on endogenous Phox2a transcription (Fig. 4), a link has been estab-lished between Phox2a transcription, CREB activation, and chromatinremodeling.To further confirm these observationswemonitored byWestern blot

assays the activation of endogenous CREB, following treatment of NC

or CAD cells with BMP2�IBMX as a function of TSA addition. Earlierstudies (42) demonstrated the phosphorylation of CREB is prolongedfollowing inhibition of HDACs. Following BMP2�IBMX treatment ofNC cells, CREB phosphorylation was detected after 15 min (Fig. 5A).Whereas TSA addition alone had no effect on CREB phosphorylation(data not shown), TSA in combination with BMP2�IBMX prolongedthe phospho-CREB levels in NC cells for 1–1.5 h, in agreement with theobservations by Michael et al. (42). Likewise, nuclear phospho-CREBimmunostaining was virtually undetectable after 1.5 h of stimulationwith BMP2�IBMX treatment only. By comparison, phospho-CREBimmunostaining remained elevated in NC cells cotreated withBMP2�IBMX in the presence of TSA (Fig. 5C).Similar studies investigating the effect of TSA on CREB phosphoryl-

ation in CAD cells are shown in Fig. 5B. BMP2�IBMX treatment of

FIGURE 5. TSA treatment prolongs CREB phosphorylation in NC and CAD cell cultures. Western blot assays of phospho-Ser133 CREB in whole cell extracts isolated from: NCcultures (A) or CAD cells (B), following stimulation with BMP2� IBMX in the presence (�) or absence (�) of 100 ng/ml TSA treatment for the indicated times. C, immunofluorescencemicroscopy employing the phospho-Ser133 CREB antibody, in NC cultures treated with BMP�IBMX as a function of TSA, as indicated.




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FIGURE 6. A, diagram illustrates the putative CRE half-sites and their nucleotide position relative to the �1 transcriptional start site, identified by computer analyses of the mouse andhuman Phox2a genes. B, ChIP assays, employing murine CAD cells treated with BMP2�IBMX as a function of TSA addition as indicated, with the CREB antibody (10 �g) or IgG (negativecontrol). Quantification of Phox2a DNA immunoprecipitated by CREB antibody or IgG was by real-time PCR employing mouse Phox2a gene-specific primers spanning putative CREhalf-sites 1 and 2, site 3, site 4, and site 5. Data are expressed as fold change of CREB binding quantified relative to IgG, and represent the average of at least three independent




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CADcells leads tomaximal CREBphosphorylationwithin 1 h, graduallydecreasing to the background level by 4 h (Fig. 5B). Importantly, addi-tion of TSA together with BMP2�IBMX prolonged the phosphoryla-tion of CREB, sustaining the level of phospho-CREB for 4 h, and return-ing to the background unphosphorylated level 6 h after treatment.Immunofluorescence studies of phospho-CREB performed in CADcells, as a function of TSA addition in the presence of BMP2�IBMXdemonstrate the same effect (data not shown).

Identification and Characterization of cAMP Response Elements inMurine Phox2a Promoter—Because CREB activation is necessary forBMP2�IBMX-induced Phox2a expression (8), and TSA not onlyenhances Phox2a mRNA expression (Fig. 4), but also prolongs CREBphosphorylation inNC cells (Fig. 5,A andC) andCADcells (Fig. 5B), weinterpret these data to suggest that CREB directly participates in thetranscriptional regulation of the Phox2a gene.

To conclusively prove this mechanism, we sought to identify andcharacterize the cis-acting elements in the Phox2a promoter thatrespond to cAMP signaling, via CREB. We obtained the Phox2a pro-moter sequence from the human and mouse genome databanks.Although the chicken genome sequence is also available, the sequencefor both the Phox2a gene and promoter are still unavailable. Wesearched for putative CRE sites in the human and mouse Phox2a pro-moter sequences. In general, the CRE consists of an 8-bp palindrome(TGACGTCA) typically found within 100–300 nucleotides upstreamof the TATA box. The palindromic CRE can be separated into twoCGTCA motifs or half-sites, which may be configured on the same oron opposite strands, to function cooperatively in response to cAMPstimulation (28, 43). Computer analyses revealed that completely palin-dromic CRE sites (TGACGTCA) are not present in the 15 kb ofupstream sequence, either in the human or the mouse Phox2a promot-ers. However, several variant CRE half-sites are found in the Phox2aupstream promoter region as well as within intron 1 (Fig. 6A).To determine whether CREB interacts with these CRE half-sites,

ChIP assays were performed in CAD cells using the CREB antibody;quantification of the associated Phox2a promoter sequence was carriedout by real-time PCR, expressed relative to the negative control valueobtained in ChIP assays with IgG, performed in parallel. The resultsdemonstrate (Fig. 6B) that CREB binding was detected in all these CREhalf-sites in agreement with the studies by Zhang et al. (44). Treatmentwith BMP2�IBMX mediated a statistically significant (p � 0.05) stim-ulation of CREB binding at these CRE half-sites, with the exception ofCRE site 4. Importantly, the highest level of CREB binding was observedwhen CAD cells were cotreated with BMP2�IBMX and TSA.To establish that these CRE half-sites mediate Phox2a transcription

in response to BMP2 and cAMP signaling, we performed additionalChIP assays using the CBP antibody, investigating whether activatedCREB recruits CBP to these CRE sites. Similar to the ChIP resultsobtainedwith theCREB antibody (Fig. 6B), CREhalf-sites 1, 2, and 5 alsodisplay a statistically significant (p � 0.05) increased association withCBP upon BMP2�IBMX addition. This CBP association with the CREsites of the Phox2a promoter is more pronounced in the presence ofBMP2�IBMX and TSA cotreatment (Fig. 6C), consistent with theenhanced Phox2a expression (Fig. 4) and prolonged CREB phosphoryl-ation (Fig. 5) detected under these treatment conditions.To investigate whether the CRE sites of the Phox2a promoter are

associated with nucleosomal modifications that reflect the activation

state of the promoter, we examined the acetylation status of histone H4(Fig. 6D). H4 acetylation in regions encompassing the CRE half-sites isincreased with BMP2�IBMX treatment. Furthermore, as expected,with BMP2�IBMX and TSA cotreatment, high H4 acetylation isobserved particularly for sites 1, 2, and 3. Because CRE half-sites 1 and 2demonstrate the most pronounced association with CREB, CBP, andacetylated H4, we conclude that CRE sites 1 and 2 are the major CREsites participating in Phox2a gene regulation in response to combinedBMP2 and cAMP signaling.To directly demonstrate that CRE half-sites 1 and 2 have the potential

to bind CREB, EMSA was performed employing recombinant purifiedCREB and oligonucleotide probes encompassing the Phox2a CRE half-site 1 or 2, the wild-type palindromic CRE or a mutant CRE oligonu-cleotide probe (Fig. 7A). Both CRE half-sites 1 or 2 probes display CREBbinding, similar to the wild-type CRE probe. The sequence specificity ofthe observed CREB-DNA complex was determined by competitionassays employing 100-fold molar excess of unlabeled wild type versusthe mutant CRE oligonucleotides (Fig. 7A).To further demonstrate the functional significance of the Phox2aUE

encompassing CRE half-sites 1 and 2, in conferring cAMP regulation tothe Phox2a promoter, we cloned the fragment spanning nucleotides�5754 to �5340 (UE) in the �32-Luc and �515-Luc reporters (Fig.7B). In addition, we cloned in these reporters the mtUE element inwhich both CRE half-sites have been mutated (Fig. 7B). The �32-Lucand �515-Luc reporters lacked responsiveness to BMP2�IBMXcotreatment in either NC or CAD cells (Figs. 1A and 3C). Interestingly,transient transfections of these Phox2a reporters containing theupstream Phox2a element (UE) in either NC (Fig. 7C) or CAD (Fig. 7D)cells demonstrate luciferase induction in response to treatment withIBMX or BMP2 alone, and further enhanced expression in response tocotreatment with BMP2�IBMX. Importantly, the Phox2a-luciferasereporters containing the mtUE element with mutated CRE half-sites 1and 2, did not display luciferase expression in response toBMP2�IBMXtreatment in NC (Fig. 7C) or CAD cells (Fig. 7D), confirming the func-tional significance of the CRE half-sites in Phox2a transcription. Theseresults conclusively demonstrate the functional significance of thePhox2a UE in mediating the transcriptional regulation of the Phox2apromoter by cAMP signaling.

DISCUSSION

SA lineage development in cultures of primary NC cells is enhancedby the combined signaling of BMP2 and moderate activation of thecAMP pathway (7). This conclusion is also supported by earlier studies(6, 10) demonstrating, activation of cAMP signaling is required forinduction by BMP2 of the SA lineage in NC cultures. Our recent studies(8) have demonstrated the combined BMP2 and cAMP signalinginduces transcription of the proneural transcription factor Phox2a. Thistranscriptional induction of Phox2a requires CREB activation by thecAMP pathway, and CREB-mediated transcription. The mechanism bywhich cAMP signaling via CREB regulates Phox2a gene expressioncould be via direct regulation of Phox2a gene transcription, or indi-rectly, by inducing another transcription factor that in turn, regulatesPhox2a transcription.To determine whether cAMP directly regulates Phox2a gene tran-

scription, we investigated Phox2a promoter activity in the developmen-tally responsive avian NC cell culture system, and the murine, CNS-

experiments (p � 0.05 where shown). C, CREB recruits CBP at CRE half-sites in mouse Phox2a gene. ChIP assays in CAD cells treated as indicated, using 5 �g of CBP antibody and IgGas the negative control. Quantification is by real-time PCR, as described in B. Data are from at least three independent assays. The p value is indicated. D, histone H4 acetylation atPhox2a CRE half-sites. ChIP assays performed with CAD cells treated as indicated, using 1 �g of antibody specific for acetylated H4. The amount of H4 acetylation at each CRE half-sitewas quantified by real-time PCR. Data are expressed as fold change relative to untreated cells, derived from three independent experiments.




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FIGURE 7. A, CRE half-sites 1 or 2 bind CREB in vitro. EMSA of purified recombinant CREB, 250 ng per binding reaction, and the indicated 32P-radiolabeled CRE oligonucleotide probes.Competition assays performed with 100-fold excess of the indicated unlabeled oligonucleotides. B, diagram illustrates Phox2a promoter constructs �32-Luc and �515-Luc containing thePhox2a UE. The nucleotide positions of the UE and the CRE half sites 1 and 2 are indicated. mtUE contains both CRE half-sites 1 and 2 mutated to the mt CRE sequence shown in A. Thesereporter plasmids were transfected in NC (C) and CAD (D) cells, as a function of BMP2, IBMX, or BMP2�IBMX cotreatment, as indicated. Results are from three independent experiments.




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derived, catecholaminergic CAD cell line (23). Herein, we report theidentification within the Phox2a promoter of functional CRE sites thatare evolutionarily conserved in the human and mouse promoters. Wehave identified two functional CRE half-sites located �100 or 250 bpfrom each other in the 5� upstream region of the mouse and the humanpromoters at 5.5 kb and 10.3 kb, respectively (Fig. 8A). Interestingly,these CRE sites are in proximity to several E-box cis-acting elements,putative binding sites for bHLH transcription factors such as MASH1(Fig. 8A), suggesting that a composite response element in the Phox2apromoter mediates the combined synergistic effect of BMP2 and cAMPsignaling (Fig. 8B).

Transfected 7.5-kb hPhox2a Reporter Responds Differently to Endog-enous Phox2a Promoter—Earlier studies by Hong et al. (9) reported thecloning and characterization of the humanPhox2a promoter employinghuman neuroblastoma cell lines expressing the endogenous Phox2agene. We employed the same hPhox2a constructs and studied them inthe developmentally regulated avian NC cellular model system, as afunction of BMP2�IBMX costimulation. Although employing theavian promoter would have been an ideal approach, we were unable toclone the avian Phox2a promoter. However, as shown by other studies(29–33) and confirmed in our studies herein (Fig. 8A) functionallyimportant, regulatory cis-acting elements are evolutionarily conserved.The hPhox2a promoter was studied in NC and CAD cell cultures

using either the luciferase or fluorescence-timer reporters. The activityof the hPhox2a promoter studied in the context of the two reporters,and in the context of the avianNC cells versus themurine CAD cell line,is qualitatively similar to results reported by Hong et al. (9). Specifically,the 1.3-kb hPhox2a promoter fragment demonstrates the highest activ-ity in all cellular models tested, i.e. human cell lines (9), avian NC cells,and murine CAD cells. These results support that similar regulatorymechanisms are operative between the avian and mammalian cells.Furthermore, in contrast to the endogenous Phox2a gene (7, 8), none

of the hPhox2a constructs, even that comprised of 7.5 kb of upstreamsequence, were responsive to the costimulation by BMP2 and IBMX.We conclude that the transfected hPhox2a promoter does not parallelthe regulation of the endogenous Phox2a gene in response to the com-bined BMP2�cAMP signaling. These conclusions are further sup-ported by the developmental potential of the NC cells that were sortedbased on the hPhox2a-driven expression of the fluorescent reporter(pTimer-1 vector). Both the sorted hPox2a-fluorescence-positive aswell as the fluorescence-negative NC cells gave rise to similar numbersof TH-immunoreactive cells, in response to BMP2�IBMX. If the activ-ity of the transfected hPhox2a had been authentic, the expression of theendogenous Phox2a gene would have resulted in all hPhox2a-fluores-cence-positive cells also being TH immunoreactive, following treat-ment with BMP2�IBMX.

Involvement of Chromatin Remodeling in Phox2a Gene Expression—The importance of chromatin structure in gene regulation is well estab-lished (37, 38, 40, 45). However, transiently transfected plasmid tem-plates are thought to assemble incompletely into chromatin, unless it isexperimentally demonstrated that they do. Accordingly, we investi-gated whether the transcription of the endogenous Phox2a gene in NCand CAD cells is modulated by treatment with TSA, an inhibitor ofHDACs. The rationale for these studies is based on the work ofMichaelet al. (42) that demonstrated treatment with the HDAC inhibitor TSA,prolonged the phosphorylation of CREB following activation of thecAMP pathway. In primary NC cultures and the CAD cell line, wedemonstrate treatment with the HDAC inhibitor TSA enhanced theBMP2�cAMP-mediated expression of the endogenous Phox2a gene,and also prolonged the phosphorylation of CREB. We conclude that

inhibition of histone deacetylation increases the BMP2�IBMX-induced Phox2a transcription by extending the duration of CREB ac-tivation. Because CREB is required for Phox2a transcription (8) theseobservations support a model linking directly CREB activation to achromatin-dependent mechanism in Phox2a transcription byBMP2�cAMP.

Functional CRE Half-sites in the 5�-Upstream Region and Also in theIntron of the Phox2aGene—Weemployed computer analyses to addressthe validity of this proposed model by identifying putative CRE siteslocated in the 5�-flanking region of the human andmouse Phox2a genesas well as in the Phox2a intron 1. We analyzed �15 kb of the upstreamsequence. Interestingly, perfectly palindromicCRE sites (TGACGTCA)have not been found in these sequences, and only five imperfect CREhalf-sites have been located within the examined 15 kb of the upstreamPhox2a sequence and intron 1. Furthermore, none of these CRE half-sites reside within 100–300 nucleotides from the TATA box where,CRE sites are typically located inCREB-responsive genes. Three of theseCRE half-sites are in the upstream 5�-flanking region (Fig. 8A).In murine CAD cells, employing ChIP assays, we demonstrate that

these CRE sites: 1) are occupied by CREB in the unstimulated, controlconditions in agreement with recent findings (44); 2) display higherCREB binding with BMP2�IBMX cotreatment, which is furtherenhanced by TSA addition, supporting the demonstrated synergy ofthe BMP2 and cAMP signaling pathways (7, 8); 3) display enhancedCBP binding, especially evident upon costimulation withBMP2�IBMX�TSA; CBP is the known, functionally associated CREBco-activator (46); and 4) display enhanced association with acetylatedhistone 4, a marker of transcriptionally active genes (37, 45). Theseobservations derived from the in vivo association of CREB, CBP, andacetylated histone 4 with specific regions of the Phox2a promoter dem-onstrate the functional significance of theCREhalf-sites inPhox2a tran-scription. Importantly, this analysis is consistent with the conclusionsby Cha-Molstad et al. (47) namely, that CREB binding and function isregulated in a cell-specific manner by epigenetic mechanisms, deter-mining active chromatin conformation.

CRE Half-sites 1 and 2 Are the Major Sites Mediating cAMP Regula-tion of Phox2a Promoter—Because CBP interacts only with activatedCREB (46, 48), the ChIP assays employing the CBP antibody conclu-sively demonstrate the functional involvement of the CRE half-sites inthe BMP2�cAMP-mediated regulation of Phox2a gene transcription.The pronounced association of CBP and acetylated H4 with CRE half-sites 1 and 2, following BMP2�IBMX�TSA costimulation, identifythese sites as the major cis-acting elements mediating the combinedBMP2�cAMP transcriptional regulation of the Phox2a gene. In sup-port of these conclusionswe demonstrate that CRE half-sites 1 or 2 bindpurified CREB in in vitro DNA binding assays and importantly theupstreamPhox2a element (UE) encompassing theseCREhalf-sites con-fers cAMP regulation to unresponsive Phox2a promoter constructs.Significantly, the Phox2a gene has been identified as a CREB/cAMP-regulated gene in PC12 cells using the SACO method, a new approachfor characterizing transcription factor regulatory regions in vivo (43).Thus, these independent observations by Impey et al. (43) further vali-date our conclusions that cAMP signaling via CREB regulates Phox2agene transcription.Concerning sites 3, 4, and 5, theChIP assays do not identify these sites

as major regulators of Phox2a gene expression, although we cannotexclude the possibility that all sites may work together in the overalltranscriptional regulation of the Phox2a gene.Interestingly, further computer sequence analysis of the nucleotide

sequence near the CRE half-sites 1 and 2 revealed that they are in prox-




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imity with other transcription factor binding sites. Of special interestare the E-box (CANNTG) and CCAAT binding sites (Fig. 8A). Intrigu-ingly, each CRE half-site 1 or 2 is located 50 bp from an E-box, andlikewise the CCAAT site is 50 bp away from an E-box. The E-box is theputative binding site for bHLH transcription factors such as ASH1. Inagreement with this observation the upstream Phox2a element confersenhanced BMP2�cAMP responsiveness to previously unresponsivePhox2a promoter constructs. Whether concurrent ASH1 and CREBinteractions with CBP, mediate Phox2a gene expression in NC culturesremains to be determined.We have assessed by in vitro protein-protein

interaction assays, the occurrence of interactions between recombinantASH1with the E1A-binding region of CBP.5 Studies by others have alsoshown the interaction of ASH1 and CREB with CBP in mediating VGFtranscription (49). The CCAAT binding site, located 50 bp from a puta-tive E-box binds the CCAAT enhancer-binding protein (C/EBP), whichis proposed to have intrinsic cAMP-inducible activity, capable of medi-ating cAMP responsiveness (50).Moreover, C/EBP� is also able to stim-

5 S. Chen and O. M. Andrisani, unpublished results.

FIGURE 8. A, nucleotide sequence of the mouse and human Phox2a promoter sequences. The CRE half-sites 1 and 2 identified by ChIP assays as the major functional CRE sites are inproximity to putative bHLH E-box and C/EBP (CCAAT) binding sites, conserved in mouse and human Phox2a promoters. B, model depicting the transcriptional regulation of thePhox2a gene by the combined action of the BMP2 and cAMP signaling pathways. cAMP induces CREB Ser133 phosphorylation, which recruits the co-activator CBP. ASH1, thedownstream, indirect effector of BMP2, binds to the E-box and interacts with the CBP/phospho-CREB complex bound to each CRE half-sites 1 and 2 to initiate Phox2a transcription.




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ulate CBP HAT activity (51). Further studies are required to define thefunctional significance of these sites in Phox2a gene regulation.In summary, we propose amodel (Fig. 8B) depicting the regulation of

Phox2a gene transcription by the combined BMP2�cAMP signaling.Phox2a transcription in response to the combined BMP2�cAMP sig-naling involves a composite regulatory, cis-acting element comprised ofCRE-half-sites 1 and 2 in proximity (50 bp) to E-box sites and theCCAAT binding site. This composite cis-acting element results in theformation of a protein complex containing CREB, CBP, and ASH1induced by BMP2 signaling. This model offers an explanation for thesynergistic effect of BMP2 and cAMP signaling on Phox2a gene tran-scription (7, 8).

Acknowledgments—We thank Dr. S. Briggs for providing acetylated H4 anti-body and Wyeth Research for providing BMP2.

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Kim, Ronald L. Hullinger and Ourania M. AndrisaniChutamas Benjanirut, Maryline Paris, Wen-Horng Wang, Seok Jong Hong, Kwang Soo

via cAMP Response Element Binding SitesThe cAMP Pathway in Combination with BMP2 Regulates Phox2a Transcription

doi: 10.1074/jbc.M503939200 originally published online December 5, 20052006, 281:2969-2981.J. Biol. Chem.

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http://www.jbc.org/lookup/doi/10.1074/jbc.M503939200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;281/5/2969&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/281/5/2969

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Documents

ThecAMPPathwayinCombinationwithBMP2Regulates ...developing embryo and differentiate to diverse cell types (1). NC cells ... chicken embryo extract, and primary NC cultures of 47.5