HAND2 Synergistically Enhances Transcription of Dopamine-ß

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Developmental Biology 262 (2003) 183–193 www.elsevier.com/locate/ydbio

HAND2 synergistically enhances transcription of dopamine-␤- hydroxylase in the presence of Phox2a

Haiming Xu,a Anthony B. Firulli,b Xiaotong Zhang,a and Marthe J. Howarda,*

a Department of Anatomy and Neurobiology, Medical College of Ohio, 3000 Arlington Ave., Toledo, OH 43614, USA b Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, 702 Barnhill Drive, Room 2666, Indianapolis, IN 46202-5225, USA Received for publication 28 March 2003, revised 5 June 2003, accepted 5 June 2003

Abstract Noradrenergic neuronal identity and differentiation are controlled by cascades of transcription factors acting downstream of BMP4, including the basic helix–loop–helix DNA binding protein HAND2 and the homeodomain factor Phox2a. Dopamine-␤-hydroxylase (DBH) is the penultimate enzyme required for synthesis of norepinephrine and is thus a noradrenergic cell type-specific marker. We have examined the interaction of HAND2 and Phox2a at the DBH promoter. Using transient transfection of P19 or NT-2 cells, HAND2 is shown to synergistically enhance Phox2a-driven transcriptional activity at the DBH promoter, an effect that is enhanced by cAMP. While mutation of the Phox2a homeodomain binding sites HD1, HD2, and HD3 results in the loss of HAND2/Phox2a transactivation of DBH, it is the interaction of HAND2/Phox2a at the CRE/AP1-HD1/2 domains in the DBH enhancer that are required for synergistic activation by HAND2. We find that HAND2 functions as a transcriptional activator without directly binding to E-box sequences in the DBH promoter, suggesting that HAND2-mediated DBH activity occurs by protein–protein interactions with other transcriptional regulators. Although we were unable to detect interaction of HAND2 and Phox2a in IP/Western blots, HAND2 synergistic activation of DBH is blocked by E1A, suggesting that HAND2 interacts with CBP (cAMP response element binding protein) in this transcriptional complex. In the presence of the putative HAND2 dimerization partner, E12, synergistic activation of DBH transcription is titrated away, suggesting that HAND2 does not functionally dimerize with E12 in the DBH transcription complex. Our data suggest that HAND2 regulates cell type-specific expression of norepinephrine in concert with Phox2a by a novel mechanism. © 2003 Elsevier Inc. All rights reserved.

Keywords: HAND2; Phox2a; Noradrenergic differentiation; Neural crest; cAMP; Cell type-speciﬁc gene expression; Sympathetic ganglion neurons; Homeodomain core recognition sites

Introduction pan-neuronal and cell type-speciﬁc patterns of gene expression. The neural crest gives rise to neurons comprising periph- The differentiation of sympathetic ganglion neurons is eral autonomic, sensory, and enteric ganglia, but the under- regulated by a complex of transcriptional regulators, includ- lying mechanisms responsible for generating phenotypic ing the basic helix–loop–helix (bHLH) DNA binding pro- diversity in these neurons remain poorly understood. To teins HAND2 (dHAND) (Howard et al., 1999, 2000) and address this issue, we have focused on the generation of MASH1 (Guillemot et al, 1993; Hirsch et al., 1998; Lo et neural crest-derived peripheral noradrenergic sympathetic al., 1998), as well as the paired homeodomain transcription ganglion neurons. The differentiation of these neurons has factors Phox2a and Phox2b (Morin et al., 1997; Pattyn et al., provided a useful model in which to identify clusters of 1997, 1999; Lo et al., 1999; Stanke et al 1999). In a pathway DNA binding proteins required for the acquisition of both parallel to MASH1, Phox2b, HAND2, and Phox2a function sequentially in the coordinated regulation of cell type-spe- ciﬁc and pan-neuronal genes characteristic of noradrenergic * Corresponding author. Fax: ϩ1-419-383-3008. neurons (Howard et al., 2000). Both the temporal expression E-mail address: [email protected] (M.J. Howard). pattern of transcripts encoding Phox2 proteins and HAND2

0012-1606/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0012-1606(03)00361-0 184 H. Xu et al. / Developmental Biology 262 (2003) 183–193 and ectopic patterns of expression have placed Phox2b in which the effects of cAMP were assessed, 8-Br-cAMP upstream and Phox2a downstream of HAND2 (Howard et was added to a final concentration of 1 mM at 8 h following al., 2000). Bone morphogenetic protein 4 (BMP4), derived transfection. NT-2 cells were maintained in Opti-MEM sup- from the dorsal aorta, is an upstream factor which induces plemented with 10% fetal bovine serum (Invitrogen Life expression of Phox2b and subsequently HAND2 (Reiss- Technologies, Carlsbad, CA). For transfection, 0.3 ␮g DNA mann et al., 1996; Stanke et al., 1999; Howard et al., 2000). was used per well. NT-2 cells were plated at a density of 1 The expression of enzymes required for synthesis of ϫ 105 cells per well, in 12-well tissue culture plates. The norepinephrine is one hallmark of a differentiated noradren- ratio of DBH reporter construct (DBH-luc) to pRL-null ergic neuron. The transcription of tyrosine hydroxylase (Promega Corp., Madison, WI) was maintained constant at (TH) and dopamine-␤-hydroxylase (DBH), the rate limiting 24:1. HAND2 or Phox2a expression plasmids were added at and penultimate enzymes in the norepinephrine biosynthetic 75 ng/well. For NT-2 cells, transfections were done accord- pathway, is regulated by Phox2 proteins (Zellmer et al., ing to manufacturer’s directions using Effectene transfec- 1995; Swanson et al., 1997, 1998; Yang et al., 1998). In tion reagent (Qiagen Inc., Valencia, CA). All cells were rodent neural crest-derived cells, Phox2a alone was insuf- maintained in 5% CO2, 95% air at 37°C. Cells were har- ficient to support expression of noradrenergic characteristics vested and luciferase reporter activity was measured accord- (Lo et al., 1998), but coactivation by cAMP/PKA resulted in ing to manufacturer’s instructions by using a Dual-Lucif- expression of TH (Lo et al., 1999). BMP2, in concert with erase Reporter Assay System (Promega Corp., Madison, Phox2a and PKA, induced TH, DBH, as well as neuronal WI) at 48 h following transfection. As an internal control for differentiation (Lo et al., 1999), suggesting a role for Phox2 transfection efficiency, all transfections contained pRL-null proteins in both expression of pan-neuronal characteristics encoding Renilla luciferase. All luciferase assays are re- as well as regulation of noradrenergic neurotransmitter ported as values normalized to 1 based on the ratio of Firefly identity. Since expression of Phox2a alone was insufficient to Renilla reporter activity in control transfections. Data are to support neuronal differentiation, this raised the possibil- presented as the mean Ϯ S.E.M. of normalized values. ity of additional DNA binding proteins that might function Statistical significance is based on ANOVA and Bonferroni in concert with Phox2a. HAND2 is a likely candidate since post hoc test. overexpression of HAND2 is sufficient to induce transcripts encoding Phox2a and supports synthesis of norepinephrine Mutagenesis as well as expression of pan-neuronal marker genes (Howard et al., 1999, 2000). We directly tested the hypoth- Potential E-box sites in the DBH promoter were mutated esis that HAND2 and Phox2a coordinately regulate the by using the QuickChange Multi Site-Directed Mutagenesis activity of the noradrenergic cell type-specific gene, DBH, Kit (Stratagene, La Jolla, CA), according to manufacturer’s by assessing potential interactions of HAND2 and Phox2a directions. at the DBH promoter and effects of cAMP on DBH promoter activity. Our results demonstrate that HAND2 syner- Biochemical pull-down assay gistically enhances Phox2a activity via a DNA-binding independent mechanism at the DBH promoter most likely in Biochemical pull-down assays were performed as previ- a multiprotein transcriptional complex with cAMP-depen- ously described (Firulli et al., 2000). Briefly, bacterial GST- dent transcriptional coactivators. fusion proteins were induced in BL21 (Novagen) and purified as described (Smith and Johnson 1988). Purified bactrial GST-fusion proteins were bound to glutathione- Materials and methods linked agarose beads and were incubated with 35S-methio- noine labeled in vitro transcribed and translated HAND1, Transfection HAND2, and Phox2a proteins. Incubations were carried out for18hat4°C in binding buffer containing 0.3 M sucrose, P19 cells were maintained in alpha minimal essential 1 mM EDTA, 10 mM Tris, pH 8.0, 0.15 M NaCl, 1% Triton medium (␣-MEM) supplemented with 7.5% bovine calf X-100, 1 ␮g/ml leupeptin, 2 ␮g/ml aprotonin, and 1 mM serum and 2.5% fetal bovine serum (Invitrogen Life Tech- phenylmethylsulfonyl fluoride (Firulli et al., 2000). Samples nologies, Carlsbad, CA). Cells were plated at a density of 1 were washed four times in binding buffer containing 0.6 M ϫ 105 cells per well, in 12-well plates on the day prior to NaCl and fractionated in a 10% SDS-PAGE gel. Recovered transfection. Transfections were performed, using FUGENE proteins were visualized following exposure to Phospho- 6 reagent (Roche Diagnostics Corp., Indianapolis, IN), ac- rImager screens. cording to manufacturer’s directions. In each well, 0.5 mg DNA was used for transfection. The ratio of DBH reporter Reporter and plasmid constructs construct (DBH-luc) to pRL-null (Promega Corp., Madison, WI) was maintained constant at 24:1. HAND2 or Phox2a The DBH-luc construct and mutants were kindly pro- expression plasmids were added at 150 ng/well. For studies vided by Elaine Lewis. The DBH-luc reporter construct H. Xu et al. / Developmental Biology 262 (2003) 183–193 185

(Swanson et al., 2000) contains the DB1 enhancer and the proximal DBH promoter (Ϫ232/ϩ12) inserted into pGL3- Basic (Promega Corp., Madison, WI). The homeodomain binding mutants (Swanson et al., 2000) were made by site- directed mutagenesis (QuickChange mutagenesis kit; Pro- mega, Corp., Madison, WI) of the DBH-luc reporter construct. To construct the HAND2 expression plasmid, the 1100-bp BssHII–DraIII fragment of mouse HAND2 was cloned into the RsaI site in pcDNA1 (Invitrogen Life Tech- nologies, Carlsbad, CA). The construction of Arix-pcDNA3 was previously described (Swanson et al., 2000). dHAND⌬basic was generously provided by D. McFadden and E. Olson and has been described (McFadden et al., 2002). Potential E-box sites in the DBH promoter were mutated at Ϫ68 to Ϫ73bp (CAAATG mutated to AG- GCCT), Ϫ88 to Ϫ93bp (CAATTG mutated to AAATTT), and Ϫ336 to Ϫ341bp (CAGTTG mutated to AAGCTT). A new restriction site was created in each mutated sequence to allow for conﬁrmation of the mutation.

Statistical analysis

The data are presented as the mean Ϯ S.E.M. Treatment groups were compared by ANOVA and Bonferroni’s multiple comparison posthoc test used to determine statistical signiﬁcance.

Results

HAND2 synergistically enhances Phox2a activity at the DBH promoter

The differential requirement for cAMP in the cell type- specific expression of DBH in noradrenergic precursor cells in rodent and avian suggested the presence of cell type- specific coactivators of DBH in addition to Phox2a. HAND2 was a likely candidate because ectopic expression of HAND2 is sufficient for the expression of both pan- neuronal and cell type-specific noradrenergic marker genes (Howard et al., 2000). As an initial step in elucidating the function of HAND2 as a coactivator in cell type-specific transcription of DBH, we employed transient transfection assays to assess DBH activity. To determine whether HAND2 functions with Phox2a in regulating transcriptional activity at the DBH promoter, P19 cells and NT2 cells were transiently transfected with a DBH-luciferase (DBH-luc) Fig. 1. HAND2 synergistically augments Phox2a transcriptional activity at reporter construct (Swanson et al., 2000) and basal promoter the DBH promoter. P19 cells (A) and NT-2 cells (B) were transiently transfected with HAND2, Phox2a, or both expression constructs and wild type DBH promoter using FuGENE6 (Roche Diagnostics Corp., Indianap- olis, IN) or Effectene transfection reagent (Qiagen, GMBH, Germany), on cell lysates. In control studies (C), basal reporter activity of pGL3-basic respectively. For each transfection, pRL-null was included as an internal and pGL3-promoter (192 ng/well) and reporter activity in the presence of control for transfection efficiency. A total of 0.5 or 0.3 ␮g/well DNA was HAND2 was tested in transient transnfections of P19 cells. Each well used for P19 cells and NT2 cells, respectively. Then 75 ng/well of each included pRL-null (8 ng/well) as an internal control for transfection effi- expression plasmid was included for P19 cells or NT2 cells, respectively, ciency. The data are presented as the mean Ϯ S.E.M. (three transfections) as indicated. Cells were harvested after 48 and luciferase assays performed of normalized Firefly/Renilla luciferase units. 186 H. Xu et al. / Developmental Biology 262 (2003) 183–193 activity was determined (Fig. 1). DBH promoter activity was then assessed following cotransfection in P19 (Fig. 1A) and NT-2 (Fig. 1B) cells of the DBH-luc construct and pcDNA1-HAND2, pcDNA-3-Phox2a, or both expression constructs. HAND2 stimulates DBH promoter activity only minimally when expressed alone; on average, DBH reporter activity is increased 0.3 Ϯ 0.11-fold over control values. When transfected alone, Phox2a activates the DBH enhancer driving transcription of the reporter in P19 and NT2 cells. In response to Phox2a, DBH reporter activity was significantly increased 8.9 Ϯ 0.7-fold (P Ͻ 0.001) in P19 cells and 6.4 Ϯ 0.7-fold (P Ͻ 0.001) in NT2 cells. In the Fig. 2. cAMP augments HAND2 activation with Phox2a of the DBH presence of HAND2, activation of the DBH promoter by promoter. P19 cells were transiently transfected with pCDNA1-HAND2 Phox2a is significantly increased (P Ͻ 0.001) compared (75 ng/well), and/or pCDNA3-Phox2a (75 ng/well) expression constructs with the stimulated activity of Phox2a alone. In P19 cells and wild type DBH promoter reporter construct using FuGENE6 transfec- and NT-2 cells, coexpression of HAND2 and Phox2a sig- tion reagent (Roache Diagnostics Corp., Indianapolis, IN). Cells were Ϯ grown in the presence or absence of 1 mM 8-Br-cAMP for 48 h and nificantly increased reporter activity by 6.5-fold (57.4 Ϯ Ϯ luciferase assays performed on cell lysates. Data are presented as mean 3.7-fold over control) and 3-fold (19.8 2.5-fold over S.E.M. normalized luciferase activity from three independent transfections. control) compared with Phox2a, respectively. To confirm that HAND2 does not interact with pGL3-basic or pGL3- promoter in our system (Hong et al., 2002), P19 cells were that could bind HAND2, we sought to define the critical transiently transfected with pGL3-basic, pGL3-promoter, cis-elements, which mediate the effects of HAND2/Phox2a and pCDNA1 (Fig. 1C) and Luciferase activity was mea- and cAMP within the DBH promoter. sured in response to HAND2. HAND2 failed to increase To determine whether HAND2 influences transcriptional Luciferase reporter activity, suggesting that activation of activity of Phox2a by affecting some change in Phox2a DBH with HAND2 and Phox2a is specific to DBH. The binding, we tested the ability of HAND2 to synergistically increased activity of Phox2a in the presence of HAND2 is augment DBH promoter activity in response to Phox2a more than an additive effect and thus shows synergistic binding in Phox2a homeodomain core recognition site mu- activation of DBH. This transcriptional synergy between tants. There are three homeodomain binding sites in the HAND2 and Phox2a suggests that these transcription fac- DBH proximal promoter (Swanson et al., 2000) which act in tors do not function independently of each other at the DBH a coordinated manner in the cell type-specific regulation of promoter, implying a physical interaction between Phox2a DBH expression. Homeodomain core recognition sites 1 and HAND2 or association through an intermediate pro- and 2 (HD1/2) impart cAMP responsiveness (Swanson et tein(s). al., 2000). In both P19 and NT-2 cells, stimulated transcription is reduced in the HD1/2 mutant (Fig. 3). Neither cAMP augments HAND2 and Phox2a transcriptional HAND2 (Fig. 3) nor Phox2a in the presence or absence of activity cAMP (Fig. 4) stimulated transcription of the HD1/2 mutant reporter, suggesting that the homeodomain sites in the DB1 Previous promoter analysis of DBH has identified a func- enhancer region of the DBH promoter are necessary for tional cAMP-response element/activator protein 1 (CRE/ transcriptional activation and HAND2-Phox2a synergy. AP1) domain (Swanson et al., 1998). This CRE/AP1 site in This conclusion was further tested by repeating this assay the DBH enhancer mediates responsiveness to cAMP and using a reporter construct lacking the more proximal home- the synergistic activation of DBH by Phox2a and PKA odomain core recognition site 3 (HD3) (Swanson et al, (Swanson et al., 1998). As HAND2 can synergistically 2000). Wild-type promoter reporter activity was increased augment activity of Phox2a on DBH transcription, we next 8-fold by Phox2a and 35-fold in the presence of HAND2 examined effects of cAMP on HAND2/Phox2a modulation plus Phox2a (Fig. 3). Wild-type activity was stimulated an of DBH expression in P19 cells. Results show basal DBH additional 2.5-fold by treatment with 1 mM 8-Br-cAMP promoter activity in the presence of HAND2, Phox2a or enhancing basal promoter activity to 87-fold (Fig. 4). In the HAND2 plus Phox2a is significantly (P Ͻ 0.001) enhanced HD3 mutant, HAND2 and Phox2a show no significant (2.0 to 2.4-fold) by the addition of 1 mM 8-Br-cAMP (Fig. change in luciferase activity, while HAND2 in the presence 2). That cAMP could enhance the synergistic activation of of 1 mM 8-Br-cAMP increased reporter activity 2.8-fold. DBH reporter activity by HAND2 and Phox2a suggests that HAND2 and Phox2a, in the presence of 1mM 8-Br-cAMP, HAND2 is a cell type-specific coactivator involved in the significantly (P Ͻ 0.01) increased basal reporter activity stimulated transcription of the DBH promoter. Since the 9-fold (Fig. 4), suggesting that HD1/2 mediates a portion of DBH promoter contains both Phox2a homeodomain core HAND2-Phox2a DBH activation. Taken together, the data recognition (HD) sites as well as three putative E-box sites suggest that, in P19 cells, HD1/2 and HD3 are required for H. Xu et al. / Developmental Biology 262 (2003) 183–193 187

and DBH-reporter activity was measured after 48 h. Basal reporter activity was increased 10-fold by Phox2a at the native DBH promoter or in the E-box mutants. In the presence of HAND2, Phox2a increased DBH reporter activity 38- to 48-fold (Fig. 5A). There was no signiﬁcant difference between transactivation of the native versus any of the three E-box mutants. This suggested that direct DNA binding was not required for HAND2 to function as a coactivator with Phox2a at the DBH promoter. We further tested the conclusion that HAND2 does not bind DNA directly (Fig. 5B) by determining the activity of a basic domain deletion mutant (dHAND⌬basic, kindly provided by E. Olson) on Phox2a stimulation of DBH reporter activity. Basal DBH promoter activity was increased 13-fold in the presence of Phox2a and either native HAND2 or HAND2⌬basic. Deletion of the HAND2 DNA binding domain had no effect on reporter activity in response to Phox2a, suggesting that HAND2 functions by protein–protein interaction and not by binding to the DBH promoter.

HAND2 does not physically interact with Phox2a

HAND2 can function as a coactivator of DBH transcription independent of DNA binding. This result raises the possibility that HAND2 physically interacts with Phox2a. To test the possibility that HAND2 physically interacts with Phox2a, we used a biochemical pull-down assay. A bacterial HAND2-GST fusion protein was immobilized onto glutathione-linked agarose beads and incubated with 35S-me- thionine-labeled in vitro transcribed and translated protein Fig. 3. HAND2 transcriptional activity requires Phox2a binding at HD (Firulli et al., 2000). Protein retained on glutathione-linked sites. P19 cells (A) and NT-2 cells (B) were transiently transfected, as beads was fractionated in a SDS-PAGE gel and detected described above in Fig. 1, with HAND2, Phox2a, or both expression constructs with either the wild type DBH-luc or HD mutant reporter constructs (described under Materials and methods). For each transfection, pRL-null was included as an internal control for transfection efﬁciency. Luciferase assays were performed on cell lysates following 48 h of cell growth. Data are presented as the normalized luciferase activity based on the ratio of Fireﬂy/Renilla luciferase activity. Each point represents the mean Ϯ S.E.M. of three independent transfections.

Phox2a activity but that HD1/2 maintains signiﬁcant Phox2a activity in the absence of HD3. In addition, HD1/2 is required for the cAMP effect in the presence of HAND2.

HAND2 function at the DBH promoter does not require direct DNA binding Fig. 4. cAMP augmentation of DBH transcriptional activity requires Phox2a HD binding domains. P19 cells were transiently transfected with In initial studies to investigate the mechanism of either the wild type DBH-luc or HD mutant reporter constructs, pCDNA1- HAND2 activity at the DBH promoter, we took two ap- HAND2 and pCDNA3-Phox2a, in the presence or absence of 1 mM proaches. In the first, we used site-directed mutagenesis to 8-Br-cAMP. Following 48 h of cell growth, luciferase assays were per- abolish each of three potential E-box binding domains in the formed on cell lysates. pRL-null was included in each transfection as an internal control for transfection efficiency. Data are presented as the nor- DBH promoter and assessed the effect on DBH-luc reporter malized luciferase activity based on the ratio of Firefly/Renilla luciferase activity (Fig. 5A). For each assay, P19 cells were transiently activity. Each point represents the mean Ϯ S.E.M. of three independent transfected with HAND2, Phox2a, or HAND2 plus Phox2a transfections. 188 H. Xu et al. / Developmental Biology 262 (2003) 183–193

Fig. 6. HAND2 and Phox2a do not physically interact. Either GST or a GST fusion for HAND2 or HAND1, were bound to Glutathione beads and incubated with 35S-labeled, in vitro transcribed, and translated HAND2 or Phox 2a. The bead-bound proteins were fractionated in a 10% SDS-PAGE gel and then visualized via phosphorimager.

Fig. 5. HAND2 synergistically augments Phox2a stimulated DBH pro- Phox2a stimulated transcription (Fig. 7). In agreement with moter activity in the absence of DNA binding. (A) P19 cells were tran- Ͻ siently transfected with wild type DBH-luc (192 ng/well) or E-box mutant Dai et al. (2002b). E12 signiﬁcantly reduced (P 0.001) DBH-luc (192 ng/well) in the presence of pCDNA1-HAND2, pCDNA3- synergistic activation of DBH by HAND2 without affecting Phox2a, or pCDNA1-HAND2 plus pCDNA3-Phox2a (75 ng/well). Cell a change in the activity of Phox2a. This result suggests that lysates were made after 48 h growth and luciferase reporter activity was measured. (B) P19 cells were transiently transfected with wild type pCDNA1-HAND2 or pCDNA1-HAND2⌬basic (75 ng/well), lacking the DNA binding domain, and the wild type DBH-luc reporter construct (192 ng/well). pRL-null was included in each transfection as an internal control for transfection efﬁciency. After 48, cells were harvested and luciferase assays on cell lysates were performed. The data are presented as the mean Ϯ S.E.M. of normalized luciferase activity from three independent transfections. following exposure on a PhosphorImager (Fig. 6). GST- HAND2 failed to pull-down Phox2a, suggesting that HAND2 and Phox2a do not physically interact in the transcription complex formed at the DBH promoter. As a control, GSTHAND2 beads were incubated with HAND2 and HAND1. The formation of HAND2 homodimers and HAND1–HAND2 heterodimers has been previously re- Fig. 7. E12 titrates HAND2 synergy with Phox2a. P19 cells were tran- ported (Firulli et al., 2000) and suggested the possibility that siently transfected with a mixture of DNA containing the reporter plasmid HAND2 homodimers might be functional in the combina- DBH-luc (192 ng/well) and pRL-null (8 ng/well) using FuGENE6 Trans- torial synergy observed at the DBH promoter. This was fection Reagent (Roche, Indianapolis, IN) according to manufacturer’s tested indirectly by determining whether HAND2 function- directions. Then 75 ng/well of pCDA1-HAND2, pCDNA3-Phox2a, or ally dimerizes with E12 to enhance Phox2a transcriptional CS-MT2E12 were included as indicated. The total amount of DNA was ϩ adjusted to 0.5 ␮g/well using pCDNA1. Cells were harvested at 48 h and activity. Cotransfection of pCS2 E12, pcDNA1-HAND2, luciferase activity was measured in cell lysates. The data are presented as and pcDNA3-Phox2a in P19 cells demonstrates that E12 the mean Ϯ S.E.M. of normalized luciferase activity from three indepen- has no effect on either basal DBH promoter activity or dent transfections. H. Xu et al. / Developmental Biology 262 (2003) 183–193 189

transfection of P19 cells with wild-type DBH-luc or with a CRE mutant (CRE⌬DBH) resulted in a complete loss of HAND2 synergy with Phox2a (Fig. 9B). Mutation of the CRE domain also resulted in a signiﬁcant reduction in HAND2 basal activity, suggesting that, in the basal state, CBP/P300 may constitutively reside at this site as is suggested by the loss of basal promoter activity in the presence of EIA (Fig. 8). Within HAND2, the CBP/P300 binding domain lies within the bHLH domain (Dai et al., 2002). We deleted the bHLH (HAND2⌬bHLH) domain from HAND2 (Fig. 9C) and tested synergistic activation of DHB with Phox2a. All synergistic activity of HAND2 is lost if the bHLH domain is deleted. Together, the data suggest that Fig. 8. HAND2 synergy with Phox2a is dependent on transcriptional synergistic activation of DBH occurs with Phox2a binding coactivators. P19 cells were transiently transfected (FuGENE 6 Transfec- at HD binding sites and HAND2 interacting in a higher tion Reagent) with DBH-luc (192 ng/well) and pCDNA1-HAND2 (75 order complex with CBP/P300 which occurs independent of ng/well), pCDNA3-Phox2a (75 ng/well), or E1A 12S (75 ng/well), as DNA binding but requiring the bHLH domain of HAND2. indicated. pCDNA1 vector DNA was added to adjust the ﬁnal DNA to 0.5 ␮g/well. Cells were harvested at 48 h and Luciferase activity was measured in cell lystes. The data are presented as the mean Ϯ S.E.M. of normalized luciferase activity from three independent transfections. Discussion

One hallmark of the differentiation of neurons is the HAND2 dimerizes with another partner, independent of coordinated expression of pan-neuronal and cell type-spe- E-proteins, which may be HAND2 in this context. These cific phenotypic characteristics. For neural crest-derived results suggest that synergy between HAND2 and Phox2a is noradrenergic neurons, many of the critical regulatory genes the result of protein–protein interaction in a transcriptional have been identified (for review, see Goridis and Rohrer, complex at the DBH promoter. 2002), but the mechanisms governing their interactions have not been well established. HAND2 has been impli- Synergistic activation of DBH by HAND2 and Phox2a cated as a pivotal player in the differentiation of neural requires CBP/P300 crest-derived noradrenergic sympathetic ganglion neurons. Unlike other transcription factors required for the differen- The functional synergy between HAND2 and Phox2a tiation of noradrenergic neurons, such as Phox2a, Phox2b, could result from the recruitment of other regulatory mole- and MASH-1, which are also expressed in a diverse array of cules. Both HAND2 and Phox2a interact with CBP/P300 nonnoradrenergic neurons, the neural expression of (Dai et al., 2002; Swanson et al., 2000), and this interaction HAND2 is confined to sympathetic and enteric neurons has been shown to increase transcriptional activity. We used (Howard et al., 1999; Wu and Howard, 2002). In the present the CBP/P300 inhibitory protein E1A to determine whether studies, we undertook to examine the function of HAND2 in CBP/P300 is a cofactor that might mediate the functional regulating the cell type-specific gene, dopamine-␤-hydrox- interaction of HAND2 and Phox2a. Cotransfection of ylase (DBH). Our data suggest that HAND2 is a cotrans- pBJ9E1A significantly inhibited DBH reporter activity (Fig. criptional regulator of DBH. HAND2 transcriptional regu- 8). In the presence of E1A, promoter activity was potently lation of DBH is independent of direct DNA binding and inhibited (ϳ100%) in response to HAND2, Phox2a, or dependent on the HLH domain. The HAND2 synergy with HAND2 plus Phox2a. This result suggests that the syner- Phox2a is mediated not by direct protein–protein interaction gistic activation of DBH reporter activity by HAND2- but through an intermediate factor that is likely CBP/P300. Phox2a requires CBP/P300. This conclusion was further Collectively, our data suggest HAND2 is a cell type-specific tested by deleting the majority of the DBH enhancer, con- regulatory molecule contributing to the expression of nor- taining the CRE/AP1, HD1, and HD2 domains, from Ϫ232 epinephrine. to Ϫ125 of the DBH promoter (DBH-125-luc) leaving only Previous results suggested the possibility that HAND2 the HD3 domain and the TATA box (Fig. 9A) or by mu- and Phox2a act together in the cell type-specific expression tating the CRE to render it inactive (Fig. 9B). In P19 cells of the noradrenergic marker gene DBH. The ectopic expres- cotransfected with HAND2, Phox2a, and DBH-125-luc, sion of HAND2, both in vitro (Howard et al., 1999) and in synergistic activation of DBH by HAND2 is reduced 100%. vivo (Howard et al., 2000), is sufficient for development of This result suggests that synergy requires sites within the cells expressing both pan-neuronal and noradrenergic cell DB1 enhancer and that Phox2a alone is inadequate for type-specific markers. The induction of Phox2a by HAND2 HAND2 function. The site within the DB1 enhancer re- most likely accounts, in part, for the subsequent expression quired for HAND2 synergy is the CRE domain. Transient of TH, DBH, and NE (Howard et al., 2000). Thus, HAND2 190 H. Xu et al. / Developmental Biology 262 (2003) 183–193

Fig. 9. The cis-acting domain for HAND2 synergistic activation of Phox2a resides in the DB1 enhancer. (A) The DB1 enhancer was deleted from DBH-luc by removing bases from Ϫ225 to Ϫ126 with EcoRI and HindII. The resulting mutant (DBH-125-luc) was used (192 ng/well) in transient transfection assays in P19 cells. Cells were transfected with 75 ng/well of pCDNA1-HAND2, and pCDNA3-Phox2a as indicated. The total DNA was adjusted with pCDNA1 to 0.5 ␮g/well. At 48 h, cells were harvested, and lysates were made by using the Dual-luciferase Reporter Assay System from Promega (Promega Corp., Madison, WI). The data are presented as the mean Ϯ S.E.M. of normalized luciferase activity from three independent transfectons. (B) The CRE motif of the DBH promoter was mutated by using the primer DBH-CREM (CTTGAATGATGTCCATGaatATTAGTGTCAATTAGGG), from Ϫ188 to Ϫ150 bp by site-directed mutagenesis (DBH⌬CRE). Wild-type and DBH⌬CRE were used in transient transfection assays, as in (A), to assess the effect of the CRE on H. Xu et al. / Developmental Biology 262 (2003) 183–193 191 can be added to the growing list of bHLH DNA binding to mediate the response to cAMP, at the DBH promoter, via proteins that link expression of pan-neuronal and cell type- a higher order complex containing HAND2, CBP/P300, and specific genes. Evidence is mounting implicating bHLH Phox2a. In the presence of EIA, which blocks a broad array factors as transcriptional coactivators, regulating function of of transcriptional coactivators, including CBP/P300 (Moran phenotype-specific marker genes, in a variety of cell types. 1993; Arany et al., 1995; Lundblad et al., 1995), transcrip- In addition to the interaction we demonstrate for HAND2, tion of DBH is refractory to Phox2a, HAND2, and cAMP. Phox2a, and DBH, synergistic interactions have been dem- Basal DBH reporter activity is also reduced, suggesting that onstrated for HAND2, GATA4, and Nkx2.5 in the regula- CBP/P300, or a similar molecule, is constitutively present tion of the cardiac-specific gene, atrial naturetic peptide regulating basal promoter activity. This idea is supported by (Thattallyath et al., 2002; Dai et al., 2002). Both GATA4 the finding that DBH basal promoter activity can be in- and Phox2a bind the transcriptional coactivator CBP/P300 creased up to three times by incubating cells with 8-bromo- (Dai et al., 2002; Dai and Markham, 2001; Swanson et al., cAMP (M.J.H., unpublished data; Swanson et al., 1997) and 2000), suggesting a model by which cAMP stimulated that mutation of the CRE domain within the DB1 enhancer downstream effector molecules might interact in a higher results in a loss of HAND2 synergistic activation of DBH order transcriptional complex to regulate DBH. with Phox2a. HAND2 physically interacts with the CH3 Cyclic-AMP regulates transcription of the DBH gene domain of P300 via its bHLH domain (Dai et al., 2002) and (Shaskus et al., 1992; Ishiguro et al., 1993 Kim et al., 1994; Phox2a interacts with the CH3 domain of CBP (Swanson et Swanson et al., 1997). Although cAMP/PKA had a modest al., 2000), suggesting that transcription of DBH is regulated effect on HAND2 transactivation of DBH in the absence of by a scaffold of transcription factors and coactivators. In the Phox2a, the interaction of HAND2 with Phox2a is enhanced context of the DBH promoter, the bHLH domain of HAND2 greater than two-fold in the presence of cAMP/PKA. The is required for synergistic activation of DBH transcription. function of cAMP/PKA in norepinephrine synthesis most All synergistic activity is lost if the bHLH domain is re- likely occurs by regulating Phox2a activity and by recruit- moved from HAND2. Similar results have been reported in ment of an AP1 complex to the CRE site adjacent to the tissue-specific regulation of ANP (Dai et al., 2002) as well Phox2a binding site HD1 (Swanson et al., 2000; Adachi and as proper patterning of the limb by HAND2 (McFadden et Lewis, 2002; Kim et al., 1998). Regions within the DBH al., 2002). Taken together, similar results in three different enhancer which regulate basal activity, cell type-specific systems suggest that, for some aspects of tissue-specific expression, and cAMP responsiveness are located between gene regulation, the functional activity of HAND2 resides Ϫ180 and Ϫ150 in the rat gene (Shaskus et al., 1992; within the bHLH domain, although interactions with addi- Ishiguro et al., 1993; Afar et al., 1996). The close proximity tional coactivators cannot be ruled out. of these regulatory domains suggested interactions between Classically, tissue-specific bHLH DNA binding proteins, HAND2, Phox2a, and transcriptional coactivators to regu- such as HAND2, are thought to form heterodimers with late tissue specific DBH expression. Synergistic activation ubiquitous bHLH DNA binding proteins, and this complex of DBH by HAND2 is mediated by interaction with se- then binds to E-box consensus (CANNTG) motifs to regu- quences included within the DB1 enhancer and independent late transcription of target genes (Massari and Murre, 2000). of HD3. In our domain mapping studies, we have shown Our results could be explained by HAND2 heterodimers that deletion of CRE/AP1, HD1, and HD2 sites from the binding to putative E-box sites within the DBH promoter or DBH promoter results in a complete loss of HAND2-me- by HAND2 interacting with Phox2a in the transcriptional diated synergistic activation of Phox2a supported transcrip- complex. If the molecular mechanism underlying HAND2 tion at HD3. This result suggests that Phox2a binding at synergistic activation of DBH were mediated through bind- HD3 is inadequate for transactivation of DBH (Swanson et ing to E-box sequences, we would have expected to dimin- al., 2000) and that Phox2a alone is insufficient for syner- ish synergistic activation by mutation of potential E-box gistic activation by HAND2. Synergistic activation of sites within the DBH promoter. Our finding that mutation of Phox2a by both cAMP and HAND2 raised the possibility each of three potential E-box sites within the DBH promoter that HAND2 interacts in a combinatorial fashion in a tran- did not abrogate the synergistic enhancement of transcrip- scription factor scaffold at the DBH promoter. tion suggests that HAND2 can regulate the activity of target In the heart, HAND2 participates in transcriptional com- genes in a DNA-independent manner. As we were unable to plexes comprised of CBP/P300, GATA4, and Nkx2.5, that construct the triple E-box mutant, we used a basic domain regulate tissue-specific gene expression (Thattallyath et al., mutant lacking the DNA binding motif (McFadden et al., 2002; Dai et al., 2002). It is possible that HAND2 functions 2002) to confirm that HAND2 function is independent of

HAND2 synergy. (C) To determine whether HAND2 functional activity resides within the bHLH domain, this region was deleted by digesting the dHAND⌬basic construct with HindIII and BamHI and the resulting fragment blunt ended and religated to remove a KpnI site within the vector backbone. This plasmid was digested with AvrII and KpnI to yield a 4.4-kb fragment. The KpnI terminus was blunt ended and religated with a 1.5 kb StuI/AvrII fragment to obtain HAND2⌬bHLH. These constructs were identified by restriction digestion, PCR, and functional assay. Transient transfections were performed as described in Materials and methods. 192 H. Xu et al. / Developmental Biology 262 (2003) 183–193 direct DNA binding. Removal of the basic DNA binding demonstrate such interactions using both IP/Westerns domain from HAND2 did not negatively influence HAND2 (A.B.F., unpublished observation) and GST-fusion protein synergistic enhancement of Phox2a transcriptional activity, pull down assays. Based on our results and those of Dai et indicating that HAND2 regulates Phox2a directed DBH al. (2002) and McFadden et al. (2002), HAND2 can func- transcription independent of DNA binding. A similar mech- tion in tissue-specific gene expression by recruiting proteins anism has been reported for HAND2 in limb bud and heart. to a transcriptional complex and synergistically altering In the heart, HAND2 regulates transcription of the cardiac- transcription in the absence of direct DNA binding. We specific gene, ANP, independent of DNA binding (Thattal- propose that HAND2 acts as a bridge between Phox2a and iyath et al., 2002; Dai et al., 2002). HAND2 is required for CBP/P300 and modulates transcription of DBH, thus influ- proper limb morphogenesis (Charite et al., 2000; Fernan- encing tissue-specific gene expression in noradrenergic neu- dez-Teran et al., 2000; te Welscher et al., 2002) but func- rons. At the present time, we are unable to rule out the tions in limb patterning independent of direct DNA binding possibility that HAND2 recruits additional coactivators to (McFadden et al., 2002). In transgenic mice, wild-type the DBH promoter, carrying out its function at a site discrete HAND2 and dHAND⌬baic have equivalent activity in gen- from those we have identified in the DB1 enhancer. eration of preaxial polydactly, similar to the equivalence we report for synergistic activation of DBH. This provides support for the notion that the basic region of HAND2 is not Acknowledgments necessary for mediating all of the biological functions as- cribed to HAND2. This would suggest that the ability of We thank, with appreciation, Drs. Hermann Rohrer, HAND2 to interact with other proteins within a transcrip- Donald Newgreen, and Jane Johnson for helpful discussions tional complex is mediated by targeting independent of and comments. Dr. Elaine Lewis (Oregon health Sciences DNA binding. University) generously provided the DBH-luciferase re- It remained possible that we had not identified all poten- porter construct, pcDNA3.1-Phox2a, and homeodomain tial E-box sites within the DBH promoter. A recent report core recognition site mutant constructs. Drs. D. McFadden by Hong et al. (2002) suggested that HAND proteins may and E. Olson (University of Texas, Southwestern) gener- ⌬ directly activate Luciferase reporter activity independent of ously provided the dHAND basic construct. Dr. Tony promoter activity. However, in our system, interaction of Kouzarides (Wellcome/CRC Institute and Department of HAND2 with sequence motifs in pGL3 does not occur. Pathology, Cambridge University, Cambridge, UK) kindly Deletion of the DBI enhancer abrogated HAND2 synergis- provided E1A.12S. Dr. David L. Turner (Mental Health tic activation of Phox2a-mediated DBH transcription, sug- Research Institute, University of Michigan) generously pro- ϩ gesting specificity of HAND2-Phox2a transcriptional con- vided mouse E12 (pCS2 E12). The authors acknowledge trol. Our studies further suggest that HAND2 does not the excellent technical assistance of Beth Firulli and YanLi function as a heterodimer with the E-protein E12, in the Zhao. This work was supported by grants from the NIH context of the DBH promoter. Since E12 can titrate HAND2 (NS40644, DK57237) to M.J.H., and (HLA61677, and a transcriptional activity of DBH, HAND2-E12 dimers must March of Dimes Award) to A.B.F. not be active at this complex, although HAND2 has been shown in a mammalian two-hybrid assay to dimerize with References E12 (Dai and Cserjesi, 2002). The formation of HAND2 homodimers has been demonstrated in vitro and in vivo Adachi, M., Lewis, E.J., 2002. The paired-like homeodomain protein, Arix, (Dai and Cserjesi, 2002; Firulli et al., 2000), although there mediates protein kinase A-stimulated dopamine-␤-hydroxylase gene is not general agreement as to the transcriptional activity of transcription through its phosphorylation states. J. Biol. Chem. 277, HAND2 homodimers. In the developing limb, HAND2 does 22915–22924. not appear to dimerize with E-proteins in the DNA binding- Afar, R., Silverman, R., Aguanno, A., Albert, V.R., 1996. Positive and negative elements contribute to the cell-specific expression of the rat independent regulation of limb patterning (McFadden et al., dopamine beta-hydroxylase gene. Mol. Brain Res. 36, 79–92. 2002). We suggest that HAND2 functions in tissue-specific Arany, Z., Newsome, D., Oldread, E., Livingston, D.M., Eckner, R., 1995. gene regulation in a novel manner encompassing both the A family of transcriptional adaptor proteins targeted by the E1A on- choice of dimerization partner as well as DNA binding coprotein. Nature 374, 81–84. activity. HAND2 is expressed and has been shown to have Charite´, J., McFadden, D.G., Olson, E.N., 2000. The bHLH transcription factor dHAND controls sonic hedgehog expression and establishment a role in a variety of tissues with very different develop- of the zone of polarizing activity during limb development. Develop- mental history and function. The diverse array of HAND2 ment 127, 2461–2470. mediated responses may be due in part to the novel ability Dai, Y.-S., Cserjesi, P., 2002. The basic helix-loop-helix factor, HAND2, of HAND2 to function independent of DNA binding, to functions as a transcriptional activator by binding to E-boxes as a bind to a large array of dimerization partners, and partici- heterodimer. J. Biol. Chem. 277, 12604–12612. Dai, Y.-S., Cserjesi, P., Markhami, B.E., Molkentin, J.D., 2002. The pate in scaffolds of transcription factors and coactivators. transcription factors GATA4 and dHAND physically interact to syn- We tested the possibility that the proteins encoded by ergistically activate cardiac gene expression through a p300-dependent HAND2 and Phox2a bind each other directly but could not mechanism. J. Biol. Chem. 277, 24390–24398. H. Xu et al. / Developmental Biology 262 (2003) 183–193 193

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