Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M503939200 on December 5, 2005

J. Biol. Chem., Vol. 281, Issue 5, 2969-2981, February 3, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/5/2969    most recent
M503939200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Benjanirut, C.
Right arrow Articles by Andrisani, O. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Benjanirut, C.
Right arrow Articles by Andrisani, O. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The cAMP Pathway in Combination with BMP2 Regulates Phox2a Transcription via cAMP Response Element Binding Sites*

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

From the {ddagger}Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907 and the §Molecular Neurobiology Laboratory, MRC215, McLean Hospital, Harvard Medical School, Belmont, Massachusetts 02478

Received for publication, April 12, 2005 , and in revised form, November 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Combined BMP2 and cAMP signaling induces the catechola-minergic lineage in neural crest (NC) cultures by increasing expression of the proneural transcription factor Phox2a, in a cAMP response element (CRE)-binding protein (CREB)-mediated mechanism. To determine whether CREB acts directly on Phox2a transcription induced by BMP2+cAMP-elevating agent IBMX, transient transfections of hPhox2a-reporter constructs were performed in avian NC cultures and murine, catecholaminergic CAD cells. Although BMP2+IBMX increased endogenous Phox2a expression, the 7.5-kb hPhox2a reporters expressing either luciferase or DsRed1-E5 fluorescent protein were unresponsive to BMP2+IBMX, but active in both cell types. Cell sorting of fluorescence-positive NC cells expressing the 7.5-kb hPhox2a fluorescent timer reporter differentiated to equal numbers of catecholaminergic cells as fluorescence-negative cells, suggesting inappropriate transcription from the transfected hPhox2a promoter. NC or CAD cells treated with histone deacetylase inhibitor trichostatin A and BMP2+IBMX display increased endogenous Phox2a transcription and prolonged CREB phosphorylation, indicating Phox2a chromatin remodeling is linked to CREB activation. Chromatin immunoprecipitations employing CREB, CREB-binding protein, and acetylated H4 antibodies identified two CRE half-sites at -5.5 kb in the murine Phox2a promoter, which is also conserved in the human promoter. Proximal to the CRE half-sites, within a 170-bp region, are E-box and CCAAT binding sites, also conserved in mouse and human genes. This 170-bp promoter region confers cAMP, BMP2, and enhanced BMP2+cAMP regulation to Phox2a-luciferase reporters. We conclude these CREs are functional, with CREB directly activating Phox2a transcription. Because the E-box binds bHLH proteins like ASH1 induced in NC cells by BMP2, we propose this novel 170-bp cis-acting element is a composite site, mediating the synergistic regulation by BMP2+cAMP on Phox2a transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pluripotent neural crest (NC)3 cells derived from the neuroepithelium during neural tube closure migrate along defined routes in the developing embryo and differentiate to diverse cell types (1). NC cells from the trunk region of the neural tube differentiate to the catecholaminergic, sympathoadrenal (SA) lineage, including sympathetic neurons and chromaffin cells of the adrenal medulla (2). SA lineage cells synthesize catecholamines (CA) by expressing tyrosine hydroxylase (TH), the rate-limiting enzyme in CA biosynthesis, and dopamine-beta-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 SA lineage by increasing the expression of the homeodomain transcription factor Phox2a (7) in a CREB-mediated mechanism (8). cAMP signaling also regulates the transactivation potential of Phox2a (8), consistent with studies by Lo et al. (6). However, whether cAMP signaling directly regulates transcription of the Phox2a gene remains to be determined. Although the regulation of the human hPhox2a promoter has been studied in human neuroblastoma cell lines expressing the endogenous Phox2a gene (9), the direct transcriptional regulation of the Phox2a promoter 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) and avian/chick (12, 13) homologues, respectively. MASH1 is expressed in both central (CNS) and peripheral (PNS) nervous system (11, 14). Forced MASH1 expression in NC cultures activates Phox2a expression (15), whereas MASH1 -/- mice display defects in sympathetic ganglia, adrenal chromaffin cells (16) and lack Phox2a expression in brain noradrenergic centers and PNS (17). Phox2a regulates transcription of TH and 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-derived catecholaminergic neurons. The Cath.a cell line derived from brain tumors of transgenic mice expressing the SV40 T-antigen under control of the TH promoter (22), like the catecholaminergic SA cells, expresses TH and DBH, synthesizes catecholamines, and also develops neurites. The CAD cell line used herein is a variant of Cath.a cells that undergoes neuronal differentiation by serum withdrawal (23) or by BMP2+cAMP costimulation (24).

Herein, Phox2a promoter activity is examined following costimulation with BMP2+cAMP, employing two differentiation models of catecholaminergic neurons, namely, NC-derived SA cells and CNS-derived CAD cells. We report the identification of a novel, composite cis-acting element, located 5.5-kb upstream from the transcriptional start site of the murine Phox2a gene. This regulatory element is comprised of two functional CRE half-sites in proximity to putative E-box and CCAAT binding sites. The identification of the functional CRE sites conclusively demonstrates the direct transcriptional involvement of the cAMP pathway in Phox2a gene regulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Culture Reagents—Vitrogen 100 was purchased from Collagen Corp.; bovine fibronectin and IBMX from Sigma; trichostatin A (TSA) from A. G. Scientific Inc. Calcium- and magnesium-free phosphate buffer solution (CMF-PBS) and 0.05% trypsin, 0.53 mM EDTA were purchased from Invitrogen. Recombinant human BMP2 (Wyeth Research) was reconstituted to 10 µg/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 Japanese quail (Coturnix coturnix) embryos were prepared as previously described (7). NC cells were isolated from primary outgrowths of neural tube explants plated for 42 h on Vitrogen 100-coated culture dishes. Adherent NC cells from the primary neural tube outgrowths were harvested by trypsin-EDTA, resuspended in growth medium (7), and plated in fibronectin-coated dishes at a density of 320 cells/mm2 resulting in secondary NC cultures. NC cells were allowed to attach for 2 h, and the seeding medium was replaced with 2 ml of growth medium, with or without BMP2 (10 ng/ml) and IBMX (100 µM). NC cultures were fed by exchange of 1 ml of growth medium on day 3 after subculture and every other day thereafter.

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

Western Blot Analysis—Total protein was extracted from NC cultures or CAD cells, grown in 24-well dishes, in radioimmune precipitation assay buffer containing 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 25 µg/ml aprotinin, 1.0% Triton X-100, 50 mM Tris (pH 7.6). Extracts were sonicated on ice for 15 s, and protein concentration was determined by the Bio-Rad protein assay. Total protein (20 µg) was analyzed by 10% SDS-PAGE and transferred to nitrocellulose. The membranes were probed with 1 µg/ml CREB antibody or 1 µg/ml of anti-phospho-CREB (Ser133) (Upstate). Detection was with 1:2,000 dilution of horseradish peroxidase-conjugated anti-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 PBS containing 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) was applied for 40 min followed by three washes in PBT. A 1:400 dilution of Alexa fluor 488 goat anti-rabbit IgG was applied for 45 min followed by three washes in PBS. For immunofluorescence of CAD cells the following modifications were included: nonspecific background was blocked using PBS containing 1% goat serum. Antibody was added in PBS containing 1% goat serum and applied 1 h at room temperature.

Real-time PCR—Total RNA from NC or CAD cells was extracted with TRIzol (Invitrogen). Total RNA (20 µg) was treated with RNase-free DNase I (20 units) in the presence of RNase inhibitor (40 units). cDNA was synthesized using 2.0 µg of total RNA, 0.2 µg of random hexamers, and Superscript II reverse transcriptase as described (Invitrogen). Oligonucleotide primers (Integrated DNA Technologies) were designed using Omega 2.0 software (Accelrys). PCR reactions were performed in identical triplicates using 2 µl of cDNA, 1 µl of 20 µM F-/R- primer, and SYBR Green PCR Master Mix as described by the manufacturer (Applied Biosystems), employing an Applied Biosystems Prism 7000 Real-Time PCR system and the accompanying Sequence Detection Software version 1.0. Data analysis was carried out as described by Lee et al. (25), employing 18 S rRNA as the internal control.

Plasmids and Transfections—hPhox2a promoter-Luc reporter constructs (9) were transiently transfected by Fugene 6 (Roche Applied Sciences) according to the manufacturer's instructions, employing 1 µg of plasmid DNA for a 6-well NC culture or 2 µg for a 6-well of CAD cell culture. 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-1 Vector (BD Biosciences Clontech), encoding the DsRed1-E5 protein, a mutant of Discosoma sp. red fluorescent protein, which changes color over time. Shortly after translation, the protein emits green light; its red fluorophore emerges later, hours after translation. Plasmids were purified by CsCl2 equilibrium gradient centrifugation. Cells were harvested 48 h after transfection, lysed in 0.2 ml of lysis buffer (Promega) and assayed for luciferase activity (Luciferase Assay system, Promega). Luciferase activity was normalized per microgram of protein extract. Cells transfected with the pTimer-1 reporter constructs were assayed by fluorescence microscopy and flow cytometry. Luciferase reporter plasmids were constructed by cloning the mouse Phox2a upstream element (UE and mtUE) spanning nucleotides -5754 to -5340 in the hPhox2a -32Luc and -515-Luc vectors. The resulting vectors were transiently transfected in NC or CAD cells as described earlier.

Electrophoretic mobility shift assays (EMSA) were performed as described (26), employing 250 ng of purified recombinant CREB protein (active motif) per binding reaction and 32P-radiolabeled oligonucleotide probes spanning the CRE half-site 1 or 2. The sequence of the oligonucleotides is as follows: CRE site 1: 5'-GGCTGTAGAATTCGTCATCACTCATTACACTGA-3'; CRE site 2: 5'-AGGAGCACATATGGGGCGTCACTCCTCAGTTAGGAT-3'; mutant CRE: 5'-GGCTGTAGAATTATGGTTCACTCATTACACTGA-3'; Wt CRE: 5'-GGCTGTAGAATTTGACGTCATCACTCATTACACTGA-3'.

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

Chromatin Immunoprecipitation Assays (ChIP)—CAD cells grown to near confluence in 10-cm dishes were treated for 2 h with BMP2(10 ng/ml), IBMX (100 µM), and/or TSA (100 ng/ml). Cellular proteins were cross-linked to chromatinized DNA for 10 min at 37 °C by addition to the medium of 1% formaldehyde. Cells were washed twice using ice-cold PBS containing protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin and 1 µg/ml pepstatin A. Cell pellets were 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 dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) and incubated overnight at 4 °C with 10 µg of CREB antibody or 5 µg of CBP antibody (Upstate) or 5 µg of IgG or 1 µg of acetylated H4 antibody (kindly provided by Dr. S. Briggs, Purdue University). Immune complexes were collected with salmon sperm DNA/protein A agarose slurry (Upstate). Following extensive washing and elution in 1% SDS-0.1 M NaHCO3, DNA-protein cross-links were reversed by incubation at 65 °C overnight. Released DNA was purified by proteinase K digestion, phenol extraction, and ethanol precipitation. Immunoprecipitated DNA was quantified by real-time PCR. The sequences of the forward and reverse primers are as follows: CRE Site 1, 2: F-5'-TGCCTAGCCATTCACATTAG-3'; R-5'-CCTGAGAGGAGAGGCCAAGTG-3'; CRE Site 3: F-5'-TCCTGCCCTGAGAACTCTAT-3'; R-5'-CTCGCAACACTAGCTTTAG-3'; CRE Site 4: F-5'-CCCAGGAGCAGATAGATCCT-3'; R-5'-CATGCTGGGACACTAACAGA-3'; CRE Site 5: F-5'-AATTTGGGAAGATGGGACCC-3'; R-5'-ACCTCTGTCCCACTTCCTCT-3'.


Figure 1
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 1.
A, hPhox2a promoter activity in J. quail primary NC cells with BMP2 (10 ng/ml) +IBMX (100 µM) treatment, as indicated. The hPhox2a-Luc reporter constructs (1 µg) listed were transfected via the Fugene 6 method into 6-well NC cultures at day 0 of replating of primary NC outgrowths. Cells were harvested 48 h after transfection; luciferase activity normalized per microgram of protein extract is expressed relative to luciferase activity of the control pGL3 plasmid, considered as 100%. The result is from three independent experiments, each performed in triplicate. B, real-time PCR for TH mRNA expression, employing total RNA isolated from NC cultures at days 1-5 following replating and treated with BMP2+IBMX, as indicated. Results represent three independent assays, each performed in identical triplicates.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In avian NC cultures, moderate activation of cAMP signaling acts synergistically with BMP2 to promote development of the SA lineage (7), by increasing Phox2a and Phox2b transcription via activation and involvement of the transcription factor CREB (8), the downstream effector of the cAMP pathway (27, 28).

To directly demonstrate the mechanism of the combined BMP2 and cAMP signaling on Phox2a gene transcription, we employed the Phox2a promoter and its deletion constructs in transient luciferase reporter assays in avian NC cells. It is well established from studies of developmentally regulated and differentiation-specific genes, that functionally important cis-acting elements are conserved across species (29-33). Accordingly, we employed the human Phox2a promoter to investigate its regulation in the developmentally regulated avian primary NC culture model system. Earlier studies by Hong et al. (9) have reported the cloning of the hPhox2a promoter and its characterization, studied in the human neuroblastoma SK-N-BE (2)M17 and SK-N-BE (2)C Phox2a-expressing cell lines. However, Phox2a regulation in response to BMP2 and cAMP signaling has not been examined.

Employing the Fugene method of transfection in primary NC cultures, we initially established that 40% transfection efficiency was achieved with the CMV-beta-gal reporter, without negative growth effects on the primary culture (data not shown). The hPhox2a-Luc reporter constructs shown in Fig. 1A, spanning sequences from 32 bp to 7.5 kb upstream from the transcriptional start site were transiently transfected in avian NC cells, as a function of cotreatment with BMP2+IBMX (a cAMP-elevating agent). The activity of the hPhox2a promoter tested in the avian NC system is qualitatively identical to that reported by Hong et al. (9). Specifically, the highest activity is observed with the 1.3-kb promoter fragment. These results suggest the Phox2a promoter activity is likely conserved between mammalian and avian species.

The Activity of hPhox2a Promoter Differs from the Endogenous Phox2a Gene in NC Cultures—Interestingly, the transient transfection results do not demonstrate regulation of the hPhox2a promoter constructs by BMP2 and cAMP in NC cells (Fig. 1A). In our previous studies we have shown that cotreatment of NC cells with BMP2 and cAMP synergistically induces expression of the endogenous Phox2a gene (7, 8), as well as of TH mRNA (Fig. 1B). Thus, there appears to be a discrepancy between the in vivo regulation of the Phox2a gene and the transient transfection results of the hPhox2a promoter.

To exclude that the hPhox2a promoter-luciferase reporter constructs might not be suitable for deciphering regulation of the Phox2a promoter (34), the Phox2a promoter was cloned and studied using the fluorescence-timer (pTimer-1) reporter system (35). This reporter monitors both the onset of transcription of a given promoter, by monitoring the appearance of green fluorescence, as well as the time of its transcriptional 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 fluorescence-positive cells were quantified as a function of BMP2+IBMX cotreatment (Fig. 2A). The results demonstrated the promoter is transcriptionally active in the absence of BMP2+IBMX costimulation in NC cultures (Fig. 2A), and that cotreatment with BMP2+IBMX does not mediate any further transcriptional induction (Fig. 2A). Importantly, Phox2a promoter activation starts on day 1 of secondary NC culture (green fluorescence), which is earlier than the known expression pattern of the endogenous Phox2a gene (7), and is turned off by day 2 (red fluorescence), when the endogenous Phox2a gene is known to be transcribed (8, 36).


Figure 2
View larger version (66K):
[in this window]
[in a new window]
  		FIGURE 2.
A, expression of hPhox2a-Timer-1 reporter in primary NC cells. Flow cytometric quantification of fluorescence-positive NC cells, transfected with the 7.5-kb hPhox2a-Timer-1 vector, at days 1 and 2 of secondary NC cultures. The 7.5-kb hPhox2a-Timer-1 vector (1 µg) was transfected via the Fugene 6 method at 24 h of primary NC outgrowths; removal of neural tubes was at 42 h, followed by replating and addition of BMP2+IBMX, as indicated. Green fluorescence indicates the fluorescent reporter protein (DsRed1-E5 protein of Discosoma sp.) shortly after translation; red fluorescence emerges several hours after translation. Data shown represent the average of two independent experiments. B, developmental potential of sorted hPhox2a-fluorescence-positive NC cells, transfected with 7.5-kb hPhox2a-Timer-1 plasmid, as described in A. Fluorescence microscopy of sorted green-red fluorescence-positive and fluorescence-negative NC cells. Following cell sorting, NC cells were re-plated, and treated with BMP2 (10 ng/ml) and IBMX (100 µM) at 2 h after replating. Images are at x10 magnification. C, sorted fluorescence-positive and -negative NC cells from B were cultured for 4 days and immunostained with TH antibody and Hoechst 33402, as indicated.

 
Use of the fluorescent reporter allows cell sorting of the transfected, fluorescence-positive NC cells and thus the study of their phenotype and developmental potential. Following live cell sorting, the Phox2a fluorescence-positive and fluorescence-negative NC cells were placed in culture; 2 h after replating 80% of hPhox2a-fluorescence-positive NC cells remained positive. Likewise, no fluorescence was detected with the sorted fluorescence-negative NC cells (Fig. 2B). The sorted NC cells were then grown either under control conditions or with BMP2+IBMX cotreatment. After 4 days of culture the NC cells were immunostained with TH antibody to monitor TH protein expression, a marker of SA cell development (7, 8). We hypothesized, if the transcriptional activity of the transiently transfected hPhox2a were authentic, these hPhox2a-fluorescence-positive NC cells would be expressing the endogenous Phox2a gene. Because Phox2a expression is required for SA development, we predicted that all of the sorted hPhox2a-fluorescence-positive NC cells would give rise to SA progenitors, i.e. they would be TH immunoreactive. However, following cell sorting and replating, both the hPhox2a fluorescence-positive and -negative NC cells cotreated with BMP2+IBMX, resulted in the appearance of similar numbers of TH-immunoreactive cells (Fig. 2C). Interestingly, the appearance of melanocytes was also observed in the fluorescence-positive NC cultures (data not shown).

These results suggest the hPhox2a promoter constructs were inappropriately activated in NC cells, other than those that became SA cells, supporting that the expression of the transiently transfected hPhox2a reporters does not reflect the expression of the endogenous Phox2a gene. Taken together with previous results (Fig. 1A), these data demonstrate that the hPhox2a-Luc construct, comprised of 7.5kb of upstream sequence, does not respond similarly to the endogenous promoter, suggesting that its expression is either misregulated or unresponsive to signals known to induce the expression of the endogenous Phox2a mRNA.

Three possibilities account for the observed results. 1) Regulation of the human Phox2a promoter in avian cells differs from regulation in mammalian cells. 2) The chromatin context is an important determinant in the synergistic BMP2+IBMX regulation, and 3) cis-acting elements mediating the BMP2 and cAMP regulation reside outside the 7.5-kb hPhox2a promoter fragment.

Regulation of Phox2a Promoter in Murine, Catecholaminergic CAD Cells—To address whether regulation of the Phox2a promoter in avian cells is different from the regulation in mammalian cells, we employed the murine CAD cell line (23), a variant of the CNS-derived catecholaminergic Cath.a cells (22). The Cath.a cell line was established from a brain tumor that arose in a transgenic mouse carrying the wild-type SV40 T antigen (Tag) under the transcriptional control of the TH promoter (22). Neuronal differentiation in CAD cells is initiated by serum 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 slight increase 1 day after serum starvation, followed by a gradual decrease (Fig. 3, A and B). Importantly, BMP2+IBMX resulted in an obvious increase in Phox2a and TH expression, both in the undifferentiated (day 0 of treatment) and differentiated CAD cells (Fig. 3, A and B) suggesting regulation of the endogenous Phox2a promoter in mammalian cells is similar to avian cells (7, 8). Moreover, BMP2+IBMX cotreatment induced differentiation of CAD cells in the presence of serum.4

Accordingly, employing the CAD cellular model system, the activity of the hPhox2a promoter Luc reporter constructs was investigated following transfection, as a function of BMP2+IBMX cotreatment. The results demonstrate, BMP2+IBMX cotreatment had no effect on the activity of all hPhox2a promoter constructs tested, similar to the results observed in the avian NC cells (Fig. 3C). Thus, we conclude: 1) The hPhox2a promoter responds similarly in the avian NC and murine CAD cells, and 2) transiently transfected hPhox2a-reporter constructs do not respond to the same regulatory mechanism(s) operative on the endogenous Phox2a promoter.

Chromatin Remodeling Regulates Endogenous Phox2a Gene Transcription by BMP2+IBMX—It is well established that reversible histone acetylation by acetyltransferases (HATs) relaxes (opens) chromatin, allowing transcriptional activation (37, 38). Likewise, histone deacetylation by HDACs induces a closed chromatin configuration and transcriptional repression (39-40).

To investigate the second possibility described earlier, namely whether the chromatin context of the Phox2a gene is an important determinant in the synergistic transcription by BMP2+IBMX, we examined the effect of the HDAC inhibitor TSA (41), on endogenous Phox2a transcription. NC cells were treated with various doses of TSA for different periods to determine the TSA effect on NC cell growth (data not shown). Treatment with 100 ng/ml TSA for 3-h intervals at different times during day 1 (24-36 h after re-plating of the primary NC outgrowths) showed no detectable effect on NC cell growth and differentiation. Interestingly, BMP2+IBMX cotreatment in the presence of 100 ng/ml TSA increased endogenous Phox2a mRNA expression compared with treatment with only BMP2+IBMX, assessed by real-time PCR (Fig. 4A). TSA treatment without BMP2+IBMX does not induce Phox2a mRNA expression (Fig. 4A). It is important to note that BMP2+IBMX treatment mediates only minimal Phox2a expression at 24 h of NC cultures, assessed by real-time PCR (8).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3.
Effect of BMP2 and cAMP signaling on endogenous Phox2a transcription in CAD cells. Real-time PCR analyses of Phox2a (A) and TH (B) mRNA expression in total RNA isolated from CAD cells treated with BMP2+IBMX as indicated, starting at day 0 prior to serum withdrawal (day 1). Quantification is from three independent RNA preparations, each PCR reaction analyzed in identical triplicates, and normalized relative to 18 S rRNA used as the internal control, as described in Lee et al. (25). C, hPhox2a promoter activity in murine CAD cells treated with BMP2 (10 ng/ml) +IBMX (100 µM). CAD cells grown in serum-containing medium were transfected with indicated hPhox2a-Luc reporter constructs (2 µg) and treated with BMP2+IBMX, as indicated. Cells were harvested 48 h after transfection; luciferase activity normalized per microgram of protein extract is expressed relative to luciferase activity of pGL3 plasmid, considered as 100%. Results shown are from three independent experiments each performed in triplicates.

 
Similar studies in CAD cells also demonstrated that endogenous Phox2a mRNA expression is further increased by TSA addition in the presence of BMP2+IBMX (Fig. 4B). Likewise, addition of TSA in the presence 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 (Fig. 4B) or CAD cell neuronal differentiation (Fig. 4C). Accordingly, these data support a mechanism whereby the chromatin structure is an important parameter in the transcriptional regulation of the endogenous Phox2a promoter by BMP2+IBMX.


Figure 4
View larger version (34K):
[in this window]
[in a new window]
  		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 with BMP2+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 RNA samples 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. CAD cells 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 cells were 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 three independent RNA preparations, each PCR reaction performed in identical duplicates, and normalized to 18 S rRNA used as the internal control. C, TSA increases neuronal differentiation 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, added at the 6-9-h interval shown in B.

 


Figure 5
View larger version (50K):
[in this window]
[in a new window]
  		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: NC cultures (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, immunofluorescence microscopy employing the phospho-Ser133 CREB antibody, in NC cultures treated with BMP+IBMX as a function of TSA, as indicated.

 
HDAC Inhibition Prolongs CREB Phosphorylation in NC and CAD Cells—Our recent studies (8) have demonstrated that dominant negative CREB suppresses Phox2a mRNA expression by BMP2+IBMX, whereas the constitutively active CREBDIEDML induces Phox2a expression without cAMP signaling. Based on these studies (8) and the effect of TSA on endogenous Phox2a transcription (Fig. 4), a link has been established between Phox2a transcription, CREB activation, and chromatin remodeling.

To further confirm these observations we monitored by Western blot assays the activation of endogenous CREB, following treatment of NC or CAD cells with BMP2+IBMX as a function of TSA addition. Earlier studies (42) demonstrated the phosphorylation of CREB is prolonged following inhibition of HDACs. Following BMP2+IBMX treatment of NC 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 prolonged the phospho-CREB levels in NC cells for 1-1.5 h, in agreement with the observations by Michael et al. (42). Likewise, nuclear phospho-CREB immunostaining was virtually undetectable after 1.5 h of stimulation with BMP2+IBMX treatment only. By comparison, phospho-CREB immunostaining remained elevated in NC cells cotreated with BMP2+IBMX in the presence of TSA (Fig. 5C).

Similar studies investigating the effect of TSA on CREB phosphorylation in CAD cells are shown in Fig. 5B. BMP2+IBMX treatment of CAD cells leads to maximal CREB phosphorylation within 1 h, gradually decreasing to the background level by 4 h (Fig. 5B). Importantly, addition of TSA together with BMP2+IBMX prolonged the phosphorylation of CREB, sustaining the level of phospho-CREB for 4 h, and returning to the background unphosphorylated level 6 h after treatment. Immunofluorescence studies of phospho-CREB performed in CAD cells, as a function of TSA addition in the presence of BMP2+IBMX demonstrate the same effect (data not shown).


Figure 6
View larger version (27K):
[in this window]
[in a new window]
  		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 and human 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 µg) or IgG (negative control). Quantification of Phox2a DNA immunoprecipitated by CREB antibody or IgG was by real-time PCR employing mouse Phox2a gene-specific primers spanning putative CRE half-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 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 µg of CBP antibody and IgG as 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 at Phox2a CRE half-sites. ChIP assays performed with CAD cells treated as indicated, using 1 µg of antibody specific for acetylated H4. The amount of H4 acetylation at each CRE half-site was quantified by real-time PCR. Data are expressed as fold change relative to untreated cells, derived from three independent experiments.

 
Identification and Characterization of cAMP Response Elements in Murine Phox2a Promoter—Because CREB activation is necessary for BMP2+IBMX-induced Phox2a expression (8), and TSA not only enhances Phox2a mRNA expression (Fig. 4), but also prolongs CREB phosphorylation in NC cells (Fig. 5, A and C) and CAD cells (Fig. 5B), we interpret these data to suggest that CREB directly participates in the transcriptional regulation of the Phox2a gene.

To conclusively prove this mechanism, we sought to identify and characterize the cis-acting elements in the Phox2a promoter that respond to cAMP signaling, via CREB. We obtained the Phox2a promoter sequence from the human and mouse genome databanks. Although the chicken genome sequence is also available, the sequence for both the Phox2a gene and promoter are still unavailable. We searched for putative CRE sites in the human and mouse Phox2a promoter sequences. In general, the CRE consists of an 8-bp palindrome (TGACGTCA) typically found within 100-300 nucleotides upstream of the TATA box. The palindromic CRE can be separated into two CGTCA motifs or half-sites, which may be configured on the same or on opposite strands, to function cooperatively in response to cAMP stimulation (28, 43). Computer analyses revealed that completely palindromic CRE sites (TGACGTCA) are not present in the 15 kb of upstream sequence, either in the human or the mouse Phox2a promoters. However, several variant CRE half-sites are found in the Phox2a upstream 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 carried out by real-time PCR, expressed relative to the negative control value obtained in ChIP assays with IgG, performed in parallel. The results demonstrate (Fig. 6B) that CREB binding was detected in all these CRE half-sites in agreement with the studies by Zhang et al. (44). Treatment with BMP2+IBMX mediated a statistically significant (p < 0.05) stimulation of CREB binding at these CRE half-sites, with the exception of CRE site 4. Importantly, the highest level of CREB binding was observed when 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 additional ChIP assays using the CBP antibody, investigating whether activated CREB recruits CBP to these CRE sites. Similar to the ChIP results obtained with the CREB antibody (Fig. 6B), CRE half-sites 1, 2, and 5 also display a statistically significant (p < 0.05) increased association with CBP upon BMP2+IBMX addition. This CBP association with the CRE sites of the Phox2a promoter is more pronounced in the presence of BMP2+IBMX and TSA cotreatment (Fig. 6C), consistent with the enhanced Phox2a expression (Fig. 4) and prolonged CREB phosphorylation (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 is increased with BMP2+IBMX treatment. Furthermore, as expected, with BMP2+IBMX and TSA cotreatment, high H4 acetylation is observed particularly for sites 1, 2, and 3. Because CRE half-sites 1 and 2 demonstrate the most pronounced association with CREB, CBP, and acetylated H4, we conclude that CRE sites 1 and 2 are the major CRE sites participating in Phox2a gene regulation in response to combined BMP2 and cAMP signaling.

To directly demonstrate that CRE half-sites 1 and 2 have the potential to bind CREB, EMSA was performed employing recombinant purified CREB and oligonucleotide probes encompassing the Phox2a CRE half-site 1 or 2, the wild-type palindromic CRE or a mutant CRE oligonucleotide probe (Fig. 7A). Both CRE half-sites 1 or 2 probes display CREB binding, similar to the wild-type CRE probe. The sequence specificity of the observed CREB-DNA complex was determined by competition assays employing 100-fold molar excess of unlabeled wild type versus the mutant CRE oligonucleotides (Fig. 7A).

To further demonstrate the functional significance of the Phox2a UE encompassing CRE half-sites 1 and 2, in conferring cAMP regulation to the 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 in which both CRE half-sites have been mutated (Fig. 7B). The -32-Luc and -515-Luc reporters lacked responsiveness to BMP2+IBMX cotreatment in either NC or CAD cells (Figs. 1A and 3C). Interestingly, transient transfections of these Phox2a reporters containing the upstream Phox2a element (UE) in either NC (Fig. 7C) or CAD (Fig. 7D) cells demonstrate luciferase induction in response to treatment with IBMX or BMP2 alone, and further enhanced expression in response to cotreatment with BMP2+IBMX. Importantly, the Phox2a-luciferase reporters containing the mtUE element with mutated CRE half-sites 1 and 2, did not display luciferase expression in response to BMP2+IBMX treatment in NC (Fig. 7C) or CAD cells (Fig. 7D), confirming the functional significance of the CRE half-sites in Phox2a transcription. These results conclusively demonstrate the functional significance of the Phox2a UE in mediating the transcriptional regulation of the Phox2a promoter by cAMP signaling.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
SA lineage development in cultures of primary NC cells is enhanced by the combined signaling of BMP2 and moderate activation of the cAMP pathway (7). This conclusion is also supported by earlier studies (6, 10) demonstrating, activation of cAMP signaling is required for induction by BMP2 of the SA lineage in NC cultures. Our recent studies (8) have demonstrated the combined BMP2 and cAMP signaling induces transcription of the proneural transcription factor Phox2a. This transcriptional induction of Phox2a requires CREB activation by the cAMP pathway, and CREB-mediated transcription. The mechanism by which cAMP signaling via CREB regulates Phox2a gene expression could be via direct regulation of Phox2a gene transcription, or indirectly, by inducing another transcription factor that in turn, regulates Phox2a transcription.

To determine whether cAMP directly regulates Phox2a gene transcription, we investigated Phox2a promoter activity in the developmentally responsive avian NC cell culture system, and the murine, CNS-derived, catecholaminergic CAD cell line (23). Herein, we report the identification within the Phox2a promoter of functional CRE sites that are evolutionarily conserved in the human and mouse promoters. We have identified two functional CRE half-sites located ~100 or 250 bp from each other in the 5' upstream region of the mouse and the human promoters 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 Phox2a promoter mediates the combined synergistic effect of BMP2 and cAMP signaling (Fig. 8B).


Figure 7
View larger version (39K):
[in this window]
[in a new window]
  		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 the Phox2a 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. These reporter 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.

 
Transfected 7.5-kb hPhox2a Reporter Responds Differently to Endogenous Phox2a Promoter—Earlier studies by Hong et al. (9) reported the cloning and characterization of the human Phox2a promoter employing human neuroblastoma cell lines expressing the endogenous Phox2a gene. We employed the same hPhox2a constructs and studied them in the developmentally regulated avian NC cellular model system, as a function of BMP2+IBMX costimulation. Although employing the avian promoter would have been an ideal approach, we were unable to clone the avian Phox2a promoter. However, as shown by other studies (29-33) and confirmed in our studies herein (Fig. 8A) functionally important, 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 activity of the hPhox2a promoter studied in the context of the two reporters, and in the context of the avian NC cells versus the murine 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 activity in all cellular models tested, i.e. human cell lines (9), avian NC cells, and murine CAD cells. These results support that similar regulatory mechanisms 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 upstream sequence, were responsive to the costimulation by BMP2 and IBMX. We conclude that the transfected hPhox2a promoter does not parallel the regulation of the endogenous Phox2a gene in response to the combined BMP2+cAMP signaling. These conclusions are further supported by the developmental potential of the NC cells that were sorted based on the hPhox2a-driven expression of the fluorescent reporter (pTimer-1 vector). Both the sorted hPox2a-fluorescence-positive as well as the fluorescence-negative NC cells gave rise to similar numbers of TH-immunoreactive cells, in response to BMP2+IBMX. If the activity of the transfected hPhox2a had been authentic, the expression of the endogenous Phox2a gene would have resulted in all hPhox2a-fluorescence-positive cells also being TH immunoreactive, following treatment with BMP2+IBMX.

Involvement of Chromatin Remodeling in Phox2a Gene Expression—The importance of chromatin structure in gene regulation is well established (37, 38, 40, 45). However, transiently transfected plasmid templates are thought to assemble incompletely into chromatin, unless it is experimentally demonstrated that they do. Accordingly, we investigated whether the transcription of the endogenous Phox2a gene in NC and CAD cells is modulated by treatment with TSA, an inhibitor of HDACs. The rationale for these studies is based on the work of Michael et al. (42) that demonstrated treatment with the HDAC inhibitor TSA, prolonged the phosphorylation of CREB following activation of the cAMP pathway. In primary NC cultures and the CAD cell line, we demonstrate treatment with the HDAC inhibitor TSA enhanced the BMP2+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 activation. Because CREB is required for Phox2a transcription (8) these observations support a model linking directly CREB activation to a chromatin-dependent mechanism in Phox2a transcription by BMP2+cAMP.

Functional CRE Half-sites in the 5'-Upstream Region and Also in the Intron of the Phox2a Gene—We employed computer analyses to address the validity of this proposed model by identifying putative CRE sites located in the 5'-flanking region of the human and mouse Phox2a genes as well as in the Phox2a intron 1. We analyzed ~15 kb of the upstream sequence. Interestingly, perfectly palindromic CRE sites (TGACGTCA) have not been found in these sequences, and only five imperfect CRE half-sites have been located within the examined 15 kb of the upstream Phox2a 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 in CREB-responsive genes. Three of these CRE 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, control conditions in agreement with recent findings (44); 2) display higher CREB binding with BMP2+IBMX cotreatment, which is further enhanced by TSA addition, supporting the demonstrated synergy of the BMP2 and cAMP signaling pathways (7, 8); 3) display enhanced CBP binding, especially evident upon costimulation with BMP2+IBMX+TSA; CBP is the known, functionally associated CREB co-activator (46); and 4) display enhanced association with acetylated histone 4, a marker of transcriptionally active genes (37, 45). These observations derived from the in vivo association of CREB, CBP, and acetylated histone 4 with specific regions of the Phox2a promoter demonstrate the functional significance of the CRE half-sites in Phox2a transcription. Importantly, this analysis is consistent with the conclusions by Cha-Molstad et al. (47) namely, that CREB binding and function is regulated in a cell-specific manner by epigenetic mechanisms, determining active chromatin conformation.

CRE Half-sites 1 and 2 Are the Major Sites Mediating cAMP Regulation of Phox2a Promoter—Because CBP interacts only with activated CREB (46, 48), the ChIP assays employing the CBP antibody conclusively demonstrate the functional involvement of the CRE half-sites in the 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, identify these sites as the major cis-acting elements mediating the combined BMP2+cAMP transcriptional regulation of the Phox2a gene. In support of these conclusions we demonstrate that CRE half-sites 1 or 2 bind purified CREB in in vitro DNA binding assays and importantly the upstream Phox2a element (UE) encompassing these CRE half-sites confers 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 approach for characterizing transcription factor regulatory regions in vivo (43). Thus, these independent observations by Impey et al. (43) further validate our conclusions that cAMP signaling via CREB regulates Phox2a gene transcription.

Concerning sites 3, 4, and 5, the ChIP assays do not identify these sites as major regulators of Phox2a gene expression, although we cannot exclude the possibility that all sites may work together in the overall transcriptional 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 proximity with other transcription factor binding sites. Of special interest are the E-box (CANNTG) and CCAAT binding sites (Fig. 8A). Intriguingly, each CRE half-site 1 or 2 is located 50 bp from an E-box, and likewise the CCAAT site is 50 bp away from an E-box. The E-box is the putative binding site for bHLH transcription factors such as ASH1. In agreement with this observation the upstream Phox2a element confers enhanced BMP2+cAMP responsiveness to previously unresponsive Phox2a promoter constructs. Whether concurrent ASH1 and CREB interactions with CBP, mediate Phox2a gene expression in NC cultures remains to be determined. We have assessed by in vitro protein-protein interaction assays, the occurrence of interactions between recombinant ASH1 with the E1A-binding region of CBP.5 Studies by others have also shown the interaction of ASH1 and CREB with CBP in mediating VGF transcription (49). The CCAAT binding site, located 50 bp from a putative E-box binds the CCAAT enhancer-binding protein (C/EBP), which is proposed to have intrinsic cAMP-inducible activity, capable of mediating cAMP responsiveness (50). Moreover, C/EBP{alpha} is also able to stimulate CBP HAT activity (51). Further studies are required to define the functional significance of these sites in Phox2a gene regulation.


Figure 8
View larger version (30K):
[in this window]
[in a new window]
  		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 in proximity 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 the Phox2a gene by the combined action of the BMP2 and cAMP signaling pathways. cAMP induces CREB Ser133 phosphorylation, which recruits the co-activator CBP. ASH1, the downstream, 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.

 
In summary, we propose a model (Fig. 8B) depicting the regulation of Phox2a gene transcription by the combined BMP2+cAMP signaling. Phox2a transcription in response to the combined BMP2+cAMP signaling involves a composite regulatory, cis-acting element comprised of CRE-half-sites 1 and 2 in proximity (50 bp) to E-box sites and the CCAAT binding site. This composite cis-acting element results in the formation of a protein complex containing CREB, CBP, and ASH1 induced by BMP2 signaling. This model offers an explanation for the synergistic effect of BMP2 and cAMP signaling on Phox2a gene transcription (7, 8).


   FOOTNOTES
 
* 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 page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Basic Medical Sciences, Purdue University, 625 Harrison St., W. Lafayette, IN 47907-2026. Tel.: 765-494-8131; Fax: 765-494-0781; E-mail: andrisao{at}purdue.edu.

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 nervous system. Back

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

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


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



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Le Douarin, N. M., Creuzet, S., Couly, G., and Dupin, E. (2004) Development 131, 4637-4650[Abstract/Free Full Text]
  2. Anderson, D. J., Carnahan, J. F., Michelsohn, A., and Patterson, P. H. (1991) J. Neurosci. 11, 3507-3519[Abstract]
  3. Varley, J. E., and Maxwell, G. D. (1996) Exp. Neurol. 140, 84-94[CrossRef][Medline] [Order article via Infotrieve]
  4. Varley. J. E., Wehby, R. G., Rueger, D. C., and Maxwell, G. D. (1995) Dev. Dyn. 203, 434-447[Medline] [Order article via Infotrieve]
  5. Reissmann, E., Ernsberger, U., Francis-West, P. H., Rueger, D., Brickell, P. M., and Rohrer, H. (1996) Development 122, 2079-2088[Abstract]
  6. Lo, L., Morin, X., Brunet, J. F., and Anderson, D. J. (1999) Neuron 22, 693-705[CrossRef][Medline] [Order article via Infotrieve]
  7. Bilodeau, M. L., Boulineau, T., Hullinger, R. L., and Andrisani, O. M. (2000) Mol. Cell Biol. 20, 3004-3014[Abstract/Free Full Text]
  8. Chen, S., Ji, M., Paris, M., Hullinger, R. L., and Andrisani, O. M. (2005) J. Biol. Chem. 280, 41025-41036[Abstract/Free Full Text]
  9. Hong, S. J., Kim, C. H., and Kim, K. S. (2001) J. Neurochem. 79, 1225-1236[CrossRef][Medline] [Order article via Infotrieve]
  10. Shah, N. M., Groves, A. K., and Anderson, D. J. (1996) Cell 85, 331-343[CrossRef][Medline] [Order article via Infotrieve]
  11. Lo, L. C., Johnson, J. E., Wuenschell, C. W., Saito, T., and Anderson, D. J. (1991) Genes Dev. 5, 1524-1537[Abstract/Free Full Text]
  12. Ernsberger, U., Patzke, H., Tissier-Seta, J. P., Reh, T., Goridis, C., and Rohrer, H. (1995) Mech. Dev. 52, 125-136[CrossRef][Medline] [Order article via Infotrieve]
  13. Groves, A. K., George, K. M., Tissier-Seta, J. P., Engel, J. D., Brunet, J. F., and Anderson, D. J. (1995) Development 121, 887-901[Abstract]
  14. Guillemot, F., and Joyner, A. L. (1993) Mech. Dev. 42, 171-185[CrossRef][Medline] [Order article via Infotrieve]
  15. Lo, L., Tiveron, M. C., and Anderson, D. J. (1998) Development 125, 609-620[Abstract]
  16. Huber, K., Bruhl, B., Guillemot, F., Olson, E. N., Ernsberger, U., and Unsicker, K. (2002) Development 129, 4729-4738[Medline] [Order article via Infotrieve]
  17. Hirsch, M. R., Tiveron, M. C., Guillemot, F., Brunet, J. F., and Goridis, C. (1998) Development 125, 599-608[Abstract]
  18. Kim, H. S., Seo, H., Yang, C., Brunet, J. F., and Kim, K. S. (1998) J. Neurosci. 18, 8247-8260[Abstract/Free Full Text]
  19. Yang, C., Kim, H. S., Seo, H., Kim, C. H., Brunet, J. F., and Kim, K. S. (1998) J. Neurochem. 71, 1813-1826[Medline] [Order article via Infotrieve]
  20. Zellmer, E., Zhang, Z., Greco, D., Rhodes, J., Cassel, S., and Lewis, E. J. (1995) J. Neurosci. 15, 8109-8120[Abstract]
  21. Morin, X., Cremer, H., Hirsch, M. R., Kapur, R. P., Goridis, C., and Brunet, J. F. (1997) Neuron 18, 411-423[CrossRef][Medline] [Order article via Infotrieve]
  22. Suri, C., Fung, B. P., Tischler, A. S., and Chikaraishi, D. M. (1993) J. Neurosci. 13, 1280-1291[Abstract]
  23. Qi, Y., Wang, J. K., McMillian, M., and Chikaraishi, D. M. (1997) J. Neurosci. 17, 1217-1225[Abstract/Free Full Text]
  24. Bilodeau, M. L., Ji, M., Paris, M., and Andrisani, O. M. (2005) Mol. Cell. Neurosci. 29, 394-404[CrossRef][Medline] [Order article via Infotrieve]
  25. Lee, S., Tarn, C., Wang, W-H., Chen, S., Hullinger, R. L., and Andrisani, O. M. (2002) J. Biol. Chem. 277, 8730-8740[Abstract/Free Full Text]
  26. Andrisani, O. M., Pot, D. A., Zhu, Z., and Dixon, J. E. (1988) Mol. Cell. Biol. 8, 1947-1956[Abstract/Free Full Text]
  27. Andrisani, O. M. (1999) Crit. Rev. Eukaryot. Gene Expr. 9, 19-32[Medline] [Order article via Infotrieve]
  28. Montminy, M. (1997) Annu. Rev. Biochem. 66, 807-822[CrossRef][Medline] [Order article via Infotrieve]
  29. Creaser, P. C., D'Argenio, D. A., and Williams, T. (1996) Nucleic Acids Res. 24, 2597-2605[Abstract/Free Full Text]
  30. Ghislain, J., Marquet-Trin-Dinh, C., Gilardi-Hebenstreit, P., Charnay, P., and Frain, M. (2003) Development 130, 941-953[Abstract/Free Full Text]
  31. Kilav, R., Silver, J., and Naveh-Many, T. (2001) J. Biol. Chem. 276, 8727-8733[Abstract/Free Full Text]
  32. Liu, T., Wu, J., and He, F. (2000) J. Mol. Evol. 50, 22-30[Medline] [Order article via Infotrieve]
  33. Rowitch, D. H., Echelard, Y., Danielian, P. S., Gellner, K., Brenner, S., and McMahon, A. P. (1998) Development 125, 2735-2746[Abstract]
  34. Hong, S. J., Chae, H., and Kim, K. S. (2002) BioTechniques 33, 1236-1238, 1240[Medline] [Order article via Infotrieve]
  35. Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X., Lukyanov, S., Matz, M., Kim, S., Weissman, I., and Siebert, P. (2000) Science 290, 1585-1588[Abstract/Free Full Text]
  36. Bilodeau, M. L., Boulineau, T., Greulich, J. D., Hullinger, R. L., and Andrisani, O. M. (2001) In Vitro Cell Dev. Biol. Anim. 37, 185-192[Medline] [Order article via Infotrieve]
  37. Roth, S. Y., Denu, J. M., and Allis, C. D. (2001) Annu. Rev. Biochem. 70, 81-120[CrossRef][Medline] [Order article via Infotrieve]
  38. Smale, S. T., and Fisher, A. G. (2002) Annu. Rev. Immunol. 20, 427-462[CrossRef][Medline] [Order article via Infotrieve]
  39. Johnstone, R. W. (2002) Nat. Rev. Drug Discov. 1, 287-299[CrossRef][Medline] [Order article via Infotrieve]
  40. Workman, J. L., and Kingston, R. E. (1998) Annu. Rev. Biochem. 67, 545-579[CrossRef][Medline] [Order article via Infotrieve]
  41. Finnin, M. S., Donigian, J. R., Cohen, A., Richon, V. M., Rifkind, R. A., Marks, P. A., Breslow, R., and Pavletich, N. P. (1999) Nature 401, 188-193[CrossRef][Medline] [Order article via Infotrieve]
  42. Michael, L. F., Asahara, H., Shulman, A. I., Kraus, W. L., and Montminy, M. (2000) Mol. Cell. Biol. 20, 1596-1603[Abstract/Free Full Text]
  43. Impey, S., McCorkle, S. R., Cha-Molstad, H., Dwyer, J. M., Yochum, G. S., Boss, J. M., McWeeney, S., Dunn, J. J., Mandel, G., and Goodman, R. H. (2004) Cell 119, 1041-1054[Medline] [Order article via Infotrieve]
  44. Zhang, X., Odom, D. T., Koo, S. H., Conkright, M. D., Canettieri, G., Best, J., Chen, H., Jenner, R., Herbolsheimer, E., Jacobsen, E., Kadam, S., Ecker, J. R., Emerson, B., Hogenesch, J. B., Unterman, T., Young, R. A., and Montminy, M. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4459-4464[Abstract/Free Full Text]
  45. Spector, D. L. (2003) Annu. Rev. Biochem. 72, 573-608[CrossRef][Medline] [Order article via Infotrieve]
  46. Goodman, R. H., and Smolik, S. (2000) Genes Dev. 14, 1553-1577[Free Full Text]
  47. Cha-Molstad, H., Keller, D. M., Yochum, G. S., Impey, S., and Goodman, R. H. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13572-13577[Abstract/Free Full Text]
  48. Kwok, R. P., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan, R. G., Robers, S. G., Green, M. R., and Goodman, R. H. (1994) Nature 370, 177-178[CrossRef][Medline] [Order article via Infotrieve]
  49. Mondolesi, G., Gargano, S., Pennuto, M., Illi, B., Molfetta, R., Soucek, L., Mosca, L., Levi, A., Jucker, R., and Nasi, S. (2002) FEBS Lett. 10, 50-56
  50. Wilson, H. L., and Roesler, W. J. (2002) Mol. Cell. Endocrinol. 188, 15-20[CrossRef][Medline] [Order article via Infotrieve]
  51. Chen, C. J., Deng, Z., Kim, A. Y., Blobel, G. A., and Lieberman, P. M. (2001) Mol. Cell. Biol. 21, 476-487[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
W.-H. Wang, R. L. Hullinger, and O. M. Andrisani
Hepatitis B Virus X Protein via the p38MAPK Pathway Induces E2F1 Release and ATR Kinase Activation Mediating p53 Apoptosis
J. Biol. Chem., September 12, 2008; 283(37): 25455 - 25467.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Paris, W.-H. Wang, M.-H. Shin, D. S. Franklin, and O. M. Andrisani
Homeodomain Transcription Factor Phox2a, via Cyclic AMP-Mediated Activation, Induces p27Kip1 Transcription, Coordinating Neural Progenitor Cell Cycle Exit and Differentiation
Mol. Cell. Biol., December 1, 2006; 26(23): 8826 - 8839.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/5/2969    most recent
M503939200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Benjanirut, C.
Right arrow Articles by Andrisani, O. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Benjanirut, C.
Right arrow Articles by Andrisani, O. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement