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Mol Cell Biol. Nov 2010; 30(22): 5348–5363.
Published online Sep 20, 2010. doi:  10.1128/MCB.00353-09
PMCID: PMC2976382

Cooperative Action of Multiple cis-Acting Elements Is Required for N-myc Expression in Branchial Arches: Specific Contribution of GATA3 [down-pointing small open triangle]

Abstract

The precise expression of the N-myc proto-oncogene is essential for normal mammalian development, whereas altered N-myc gene regulation is known to be a determinant factor in tumor formation. Using transgenic mouse embryos, we show that N-myc sequences from kb −8.7 to kb +7.2 are sufficient to reproduce the N-myc embryonic expression profile in developing branchial arches and limb buds. These sequences encompass several regulatory elements dispersed throughout the N-myc locus, including an upstream limb bud enhancer, a downstream somite enhancer, a branchial arch enhancer in the second intron, and a negative regulatory element in the first intron. N-myc expression in the limb buds is under the dominant control of the limb bud enhancer. The expression in the branchial arches necessitates the interplay of three regulatory domains. The branchial arch enhancer cooperates with the somite enhancer region to prevent an inhibitory activity contained in the first intron. The characterization of the branchial arch enhancer has revealed a specific role of the transcription factor GATA3 in the regulation of N-myc expression. Together, these data demonstrate that correct N-myc developmental expression is achieved via cooperation of multiple positive and negative regulatory elements.

The N-myc gene belongs to a family of proto-oncogenes, including the highly related c-myc, N-myc, and L-myc genes, that encode transcription factors sharing many structural and biochemical functions (1, 18, 38). The structural motifs conserved between the MYC proteins include a basic helix-loop-helix domain and a leucine zipper domain involved in DNA-protein and protein-protein interactions, respectively. These motifs are also found in numerous transcription factors that control cell determination during diverse cell processes such as myogenesis, neurogenesis, and sex determination (8, 49, 63, 65).

Deregulation of myc gene expression is known to initiate and enhance the progression of naturally occurring neoplasms, and the etiology of the resulting tumors appears to be linked to the normal expression pattern of myc genes during development (29, 37, 53, 73). For example, neuroblastomas are embryonic tumors of the peripheral sympathetic nervous system. They originate from the neural crest-derived precursor cells (neuroblasts) that migrate ventrally to give rise to the sympathetic nervous system. Little is known about the precise embryonic precursor cell types from which neuroblastomas are derived (29). Neural crest-derived structures, such as the somites, the branchial arches (BA), and the limb buds (LB), show N-myc expression. The tumorigenic potential of the MYC proteins has been demonstrated by transformation assays both in vitro and in vivo (44, 47). The tumorigenic capacities of c-MYC and N-MYC most likely reside in their ability to drive cells into the cell cycle (2, 20).

During early mouse embryogenesis, the expression patterns of the c-myc and N-myc genes are distinct, albeit overlapping. Both genes are expressed at preimplantation stages, but their profiles diverge with advancing development. The c-myc gene is widely expressed in tissues and organs in proliferation. In contrast, N-myc expression is restricted to a limited number of tissues and organs and is not necessarily associated with proliferating cells (Fig. (Fig.1)1) (10, 12, 15, 22, 23, 26, 34, 43, 46, 55, 58). At embryonic day 9.5 (E9.5), N-myc is highly expressed in the central nervous system (CNS), including the mesencephalon, the proencephalon, and the neural tube (26). At this stage, N-myc expression is also detected in neural crest-derived tissues such as the facial primordia, the BA, and the dorsal root ganglia (DRG), as well as in mesodermal derivatives, including the caudal half of the somites and their sclerotome derivatives, the mesonephric tubules, and the LB. In the BA, N-myc is expressed in the mesenchyme as well as in the ectoderm (26). In some tissues, N-myc is expressed in a complementary fashion to c-myc, for example, in the early gut, brain, lung, and kidney, in which c-myc expression is often associated with rapidly proliferating cells, while N-myc and even L-myc expression persists through cell differentiation (22, 42, 45). These distinct patterns suggest specific roles for myc genes, with expression of c-MYC being associated with cell proliferation while N- and L-MYC expression is often detected in postmitotic cells (11, 38). However, c- and N-myc genes have been shown to be involved in cell proliferation and differentiation both in vitro and in vivo (2, 4, 28, 41, 48).

FIG. 1.
Expression profile of N-myc at E10.5. Whole-mount in situ hybridization was performed on E10.5 wild-type embryos (A to C, E, and F) and on E12.5 lung tissue (D). N-myc expression was detected in the central nervous system (A), in tissues derived from ...

Studies on the control of the N-myc gene have revealed that transcriptional and posttranscriptional mechanisms are involved in its regulation. The N-myc promoter contains a functional TATA box, a CT box, and multiple binding sites for transcriptional factors, such as E2F, Sp1, Oct, WT1, and a TIE site, all of which are involved in the basal, the autoregulatory, and the tissue-specific expression of the N-myc gene (reviewed in reference 60). Other regions of the N-myc gene have also been associated with its regulation. For instance, the first intron contains DNA elements that act as an attenuator or silencer to regulate the levels of N-myc transcripts in a tissue-specific manner (56, 66, 68, 70). A neural-specific enhancer has also been identified in the mouse N-myc upstream sequences (24, 25). N-myc gene expression is further regulated at the posttranscriptional level by the control of pre-mRNA processing and mRNA stability. The 3′ untranslated region (UTR) of the N-myc gene contains DNA elements that can interact with ELAV-like RNA binding proteins involved in regulating mRNA turnover (3, 5, 9, 33, 40). MicroRNAs can also modulate gene expression by mediating the degradation of mRNA. A recent study showed that N-myc is a direct target of miR-34a, which maps within a chromosomal region frequently deleted in neuroblastomas (67). Finally, protein stability and activation by phosphorylation also impinge on N-MYC function (27, 39, 57).

Transgenic mouse analyses have demonstrated that human and mouse genomic fragments containing all the regulatory elements described above are able to direct N-myc expression in newborn and adult tissues in a manner which closely parallels that of the endogenous N-myc gene (21, 62, 72). However, these sequences can only in part direct the correct N-myc expression profile of a transgene during embryonic development, suggesting that other cis-acting sequences are required to recapitulate the entire developmental regulation of N-myc (10). In the present study, we sought to identify the spatial regulatory elements necessary for the N-myc developmental expression by using transgenic mouse embryos that carry chimeric constructs containing N-myc genomic sequences fused to the lacZ reporter gene. Our findings establish that N-myc gene regulation in BA and LB requires several upstream, downstream, and intragenic DNA regions not previously identified that cooperate in an intricate manner to establish the N-myc developmental expression profile. Moreover, the BA enhancer has been reduced to a 230-bp fragment containing a GATA binding site that is required for its activity.

MATERIALS AND METHODS

Construction of N-myc-lacZ transgenes.

Constructs 1 to 30 were derived from construct pJC41, previously described by Charron et al. (10), and from a 16.2-kb N-myc mouse genomic fragment encompassing sequences from kb −8.7 to kb +7.5 relative to the major transcription initiation site (14). In construct pJC41, the bacterial lacZ gene was inserted into the second exon of the N-myc gene between the PvuII and BssHII sites in the context of a 16-kb N-myc genomic fragment extending from kb −4.4 to kb +11.6. Construct 2 was generated by cloning the XbaI fragment of pJC41 into the vector pUC18 (Fig. (Fig.22 A). Construct 1 was obtained by cloning a 7.8-kb SalI-RsrII genomic fragment containing the 5′-flanking sequences extending from kb −8.7 to kb −0.9 kb into the SalI-RsrII sites of construct 2 to replace and extend the 5′ N-myc genomic sequences (Fig. (Fig.2A).2A). The XbaI-EcoRI fragment (from kb −2.0 to kb −1.6) of construct 2 was deleted to generate construct 3 (Fig. (Fig.2A).2A). Constructs 5 and 6 were generated by first cloning the 2.4-kb BamHI-XbaI genomic fragment, extending from kb −4.4 to kb −2.0 in the BamHI-XbaI site of pBluescript II SK(+) and then by inserting a 4.3-kb BamHI fragment from the phspPTlacZpA plasmid vector into the BamHI site of the resulting construct in both orientations (Fig. (Fig.2A).2A). The BamHI fragment of the phspPTlacZpA plasmid provides a reporter gene containing the hsp68 minimal promoter fused to the lacZ gene with simian virus 40 (SV40) sequences that supply intron/exon and poly(A) signals (31). Transgenes 4, 7, and 8 were obtained directly from construct 2 after RsrII-XbaI, RsrII-BspHI, and StuI digestions (Fig. (Fig.2A2A and and33 A). Construct 10 was derived from construct 2 by deleting the 1.3-kb EcoRI-XbaI fragment at the 3′ end (Fig. (Fig.3A).3A). Transgene 9 was obtained directly from construct 10 after RsrII-SalI digestion (Fig. (Fig.3A).3A). For construct 11, a 1.3-kb EcoRI-XbaI genomic fragment containing the 3′-flanking sequences extending from kb +5.9 to kb +7.2 was cloned in front of the hspPTlacZpA cassette as described above (Fig. (Fig.3A).3A). Constructs 12 and 13 were generated similarly by cloning the 2.3-kb HincII-XbaI N-myc genomic fragment, extending from kb +4.9 to kb +7.2 upstream or downstream of the hspPTlacZpA cassette, respectively (Fig. (Fig.3A).3A). Construct 14 was produced by introducing the HindIII-BamHI lacZ cassette from pCH110 between the BamHI and HincII sites of construct 1 (Fig. (Fig.44 A). Construct 15 was obtained from construct 1 by deleting most of the intron 2 sequences located between the NruI and the BclI restriction sites from kb +2.4 to kb +4.1 (Fig. (Fig.4A).4A). Construct 16 was obtained by digesting constructs 1 and 14 with RsrII and EcoRV, which are unique sites in both constructs, allowing the replacement of the RsrII-EcoRV fragment of construct 1 by the one of construct 14 and leading to the deletion of most of the first and second exons and the first intron sequences (Fig. (Fig.4A).4A). A 2.65-kb BstBI-ClaI genomic fragment containing intron 2 sequences extending from kb +1.65 to kb +4.3 was cloned in both orientations in front of the hspPTlacZpA cassette as described above to generate constructs 17 and 18 (Fig. (Fig.4A).4A). The RsrII-NruI fragment of construct 16 containing the lacZ sequences was introduced into the RsrII-NruI sites of construct 10 to generate construct 19 (Fig. (Fig.4A).4A). Transgenes 20 to 22 were obtained directly from construct 17 after SphI-NotI, ScaI-NotI, and XmnI-NotI digestions, respectively (Fig. (Fig.55 A), whereas transgenes 23 and 24 were obtained from construct 18 after XmnI-NotI and ScaI-NotI digestion, respectively. Construct 25 was made by cloning the N-myc promoter region (RsrII-BamHI; kb −0.9 to kb +0.2) in front of the HindIII-BamHI lacZ cassette from pCH110 (Fig. (Fig.5B).5B). Construct 26 was generated by cloning the HindIII-BglII fragment, extending from kb +3.0 to kb +3.6 in front and in the reverse orientation of the N-myc promoter region of construct 25 (Fig. (Fig.5B).5B). Transgene 27 was obtained directly from construct 26 after MfeI-BamHI digestion (Fig. (Fig.5B).5B). Construct 28 was generated by amplifying the sequences located between positions kb +3.17 and kb +3.4 (forward primer, 5′-TTAAGCTTTCCTCCTGGGCTGTGGAG-3′; reverse primer, 5′-TAATGCGGCCGCGAGGTTTCTTCTTC-3′) and by cloning it in front of the N-myc promoter in construct 25.

FIG. 2.
Identification of a limb bud enhancer in upstream sequences of the mouse N-myc gene. (A) Structure of the mouse N-myc gene from which the various constructs were derived and diagram of the N-myc-lacZ transgenes used to generate F0 transgenic embryos. ...
FIG. 3.
Downstream sequences of the N-myc gene are required for branchial arch expression and contain a somite enhancer. (A) Schematic representation of the N-myc-lacZ 3′ deletion transgenes used to generate F0 transgenic embryos. B, BamHI; Bc, BclI; ...
FIG. 4.
The second exon-intron of N-myc acts as a branchial arch enhancer but is not sufficient to direct branchial arch expression in the N-myc context. (A) Schematic representation of N-myc-lacZ intragenic deletion constructs used to generate F0 transgenic ...
FIG. 5.
Localization of the BA enhancer in the N-myc second intron. (A) Schematic representation of N-myc exon 2-intron 2 deletions tested with the hsp68-lacZ reporter in F0 transgenic embryos and summary of the transgenic expression analysis. B, BamHI; Bc, BclI; ...

Deleted and mutant BA enhancers in constructs 29 and 30, respectively, were generated by PCR using overlapping fragments (see Fig. 9A, below). The Vent DNA polymerase (New England BioLabs) was used to avoid unwanted mutations. The two first fragments for the deleted amplicon were generated using primer DELFWDNRU (5′-ATCCCCTTTTTCGCGAATCC-3′) and PFUDPNR primer (5′-GCCAAAGAATAAAAAGTCTAAAACAATTGTTCCGCTTTCCGGTC-3′) and PFUDPNF (5′-GACCGGAAAGCGGAACAATTGTTTTAGACTTTTTATTCTTTGGC-3′) and 609RXHO (5-AGATCTCAATTATAAAGCTC-3′). The two other fragments that contained the mutated GATA site were amplified with the DELFWDNRU primer and the S1.1B* reverse and the S1.1B* forward primers (see “Electrophoretic mobility shift assays” below) and the 609RXHO primer. The final products were generated by a third amplification that included the initially obtained fragments as template and the two end primers DELFWDNRU and 609RXHO. These products were digested with NruI and MfeI for subcloning. Finally, the plasmids obtained after transformation were analyzed by restriction digest and sequenced prior to transgenesis.

Production and characterization of transgenic mice.

Transgenic mouse embryos were generated as described previously (10). Transgenic embryos were recovered from foster mothers at E10.5, the morning of vaginal plug detection being considered E0.5. Embryos were stained for β-galactosidase activity as previously described (10). Transgenic specimens were identified by Southern blot analysis of DNA extracted from yolk sacs with an N-myc or a lacZ probe. All animal experiments were performed according to the guidelines of the Canadian Council on Animal Care and approved by the institutional animal care committee.

Electrophoretic mobility shift assays.

Gel electrophoretic mobility shift assays (EMSA) were performed using PCR DNA fragments or annealed oligonucleotides. The full-length S1 DNA probe was amplified by PCR using Oligo 1 (5′-TTAAGCTTTCCTCCTGGGCTGTGGAG-3′) and Oligo 2 (5′-TAATGCGGCCGCGAGGTTTCTTCTTC-3′). Internal subprobes were also amplified by PCR with Oligos 1 and 3 (5′-TACCCGGGACAGACATTAAAGGGACAG-3′) for the S1.1 fragment, Oligo 4 (5′-TACCCGGGCTGTCCCTTTAATGTCTGT-3′) and Oligo 5 (5′-TACCCGGGCAGTGTTTTGGTTTTCAGCC-3′) for the S1.2 fragment, and Oligos 2 and 6 (5′-TACCCGGGGGCTGAAAACCAAAACACTG-3′) for the S1.3 fragment. Probe labeling was performed by restriction-fill-in reactions with restriction sites incorporated into the PCR products: HindIII/NotI (fragment S1), HindIII/XmaI (S1.1), XmaI (S1.2), and XmaI/NotI (S1.3). The fill-in reaction was performed with the large Klenow fragment of polymerase I. For the S1.1B and S1.1C fragments, complementary oligonucleotides were annealed and end labeled using [γ-32P]ATP and T4 polynucleotide kinase (see the sequences in Fig. 7B, below). The sequence of the S1.1B* fragment containing a mutation in the GATA binding site is 5′-GGAAACTTGGTGTCGCAGCCTGCACTTTGAAAGGGC-3′ (the underlined sequence portion corresponds to the mutated GATA site).

E10.5 whole-cell extract (WCE) was prepared as described in reference 61. Briefly, E10.5 B6CBAF2 embryos were collected in 3 volumes of WCE buffer (20 mM HEPES [pH 7.9], 400 mM KCl, 1 mM EDTA, 10% glycerol, 0.5 mM dithiothreitol [DTT], 1 μg/ml leupeptin, 2 μM pepstatin, 0.1 mM phenylmethylsulfonyl fluoride). Cells were disrupted by two freeze-thaw cycles, and debris was removed by centrifugation.

EMSA reactions were performed by incubating 1 ng of labeled DNA probe with 2 μl of whole-cell protein extract (5 μg) or recombinant GATA proteins (1 μg; TNT Quick Coupled transcription/translation kit; Promega), 1 μg of poly(dI-dC), and 100 ng yeast tRNA in 12 μl of 15 mM HEPES (pH 7.9), 50 mM NaCl, 0.08 mM ZnCl2, 2 mM MgCl2, 0.8 mM DTT, 0.5% NP-40, and 3% Ficoll. The reactions were equilibrated for 30 min at room temperature and separated on a 6% nondenaturating polyacrylamide (29:1) gel containing 0.25× Tris-borate-EDTA. Radioactive bands were visualized by autoradiography.

Cotransfection experiments.

Mouse GATA1, -3, and -5 and rat GATA4 and -6 cDNAs encompassing the full-length coding sequences were cloned in the pcDNA3 expression vector. The 600-bp BA enhancer was cloned in the XhoI site of a pGL3 luciferase reporter expression vector (Promega). HEK293 cells were transiently cotransfected in six-well plates with 0.5 μg/well of luciferase reporter construct and 2 μg/well of GATA expression vector and using the CaPO4 precipitation technique. The pRL-SV Renilla reniformis luciferase expression vector was used as a control for transfection efficiency (0.25 μg/well; Promega, Madison, WI). Forty-eight hours after transfection, cell lysates were assayed for luciferase activity using the Dual-Glo luciferase system (Promega). Data are presented as the fold induction ± the standard error of the mean (SEM) of normalized relative luciferase units consisting of the ratio of luciferase activity produced by the studied luciferase vectors divided by the R. reniformis luciferase activity.

Chromatin immunoprecipitation.

Subconfluent cultures of murine neuroblastoma NBA2 cells were used for formaldehyde chromatin cross-linking. NBA2 nuclear extracts were then resuspended in 1 ml of chromatin immunoprecipitation (ChIP) lysis buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-laurylsarcosine). Sonication was performed using a Bioruptor (Diagenode, Belgium) for 10 cycles of a 30-s pulse followed by a 30-s pause at the highest setting. Two hundred micrograms of chromatin fragments was used for immunoprecipitation with Dynabeads linked to protein G (Invitrogen, Burlington, ON, Canada) and 2 μg of either anti-GATA3 (HG3-31; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-H3 (ab1791; Abcam Inc., Cambridge, MA), or control mouse IgG (M5284; Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). The immunocomplexes were washed five times with 0.5 ml radioimmunoprecipitation assay (RIPA) wash buffer (50 mM HEPES-KOH [pH 7.6], 1 mM EDTA, 500 mM LiCl, 1% NP-40 [IGEPAL], 0.7% sodium deoxycholate), with a final wash in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl. Protein-DNA complexes were eluted from Dynabeads by addition of elution buffer (1% SDS, 50 mM Tris [pH 8.0], 10 mM EDTA) at 65°C for 15 min. Cross-links were reversed by addition of 200 mM NaCl at 65°C overnight. DNA fragments were purified using a QIAquick gel extraction kit (Qiagen, Mississauga, Canada) after RNase and proteinase K treatments.

Quantitative PCR (qPCR) amplifications were performed with specific primers: BA enhancer forward (5′-CCCTTGTCCTTCGAGCTGTA-3′) and reverse (5′-TTAGAGCGATCCCTCCACAG-3′); second N-myc exon forward (5′-CCTCACTCCTAATCCGGTCA-3′) and reverse (5′-GCCGTGCTGTAGTTTTTCGT-3′); third N-myc exon forward (5′-AACTATGCTGCACCCTCACC-3′) and reverse (5′-CTCTTCGCTTTTGCTGGAAC-3′); 160 kb downstream of the N-myc locus forward (5′-GCCTCCACCTGAGGGCTCCA-3′) and reverse (5′-GCCCTCCTGCACCTCCTCT-3′). Amplification of each fragment was monitored by using a 7000 real-time PCR system (Applied Biosystems, Foster City, CA). The values for samples immunoprecipitated by anti-GATA3, anti-H3, or control IgG were recorded as the percentage relative to input. ChIP results were confirmed by three independent experiments, and qPCR was performed in triplicate for each sample.

Whole-mount in situ hybridization analysis.

The whole-mount in situ hybridization protocol was based on that described in reference 32. The 817-bp PvuII-ClaI fragment of the murine N-myc cDNA contains sequences from the second exon and was used as template for synthesizing the digoxigenin UTP-labeled riboprobe.

RESULTS

Identification of limb bud enhancer activity upstream of the N-myc gene.

Our previous transgenic studies showed that N-myc 41X and N-myc 41S constructs, which contained N-myc genomic sequences from kb −2.0 to kb +7.2 and from kb −4.4 to kb +11.8, respectively, relative to the major transcription initiation site (14), can direct expression of the lacZ reporter gene in the neural crest-derived branchial arches and the limb buds in established transgene mouse lines (10). We undertook a deletion analysis to further define the DNA domains involved in this regulation (Fig. (Fig.2).2). First, construct 1, including N-myc genomic sequences from kb −8.7 to kb +7.2, and the N-myc 41X construct (construct 2) were tested in F0 transgenic embryos. Expression levels in BA and LB were revealed by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) staining for construct 1 as previously observed for established transgenic mouse lines with the N-myc 41S construct, indicating that the addition of 4.3 kb more of upstream sequence does not impact on transgene expression (Fig. (Fig.2)2) (10). In contrast, none of the eight transgenic embryos obtained with construct 2 showed lacZ expression in LB. These findings indicated that the sequences located between kb −4.4 and kb +7.2 were sufficient for LB and BA expression and suggested that the LB regulatory sequences were located in a 2.4-kb fragment between position kb −4.4 and kb −2.0.

The importance of the 2.4-kb DNA fragment for N-myc LB expression was directly assessed by putting the sequence either in front or downstream (constructs 5 and 6; arrows indicate the 5′-to-3′ orientation of the DNA fragment) of the mouse heat shock gene hsp68 promoter fused to the lacZ reporter gene (hsp68-lacZ), which does not direct any tissue-specific expression by itself during development (31). All transgenic embryos obtained with constructs 5 and 6 showed a strong LB expression (Fig. (Fig.2).2). Thus, the 2.4-kb regulatory region (identified as the R1 domain) (see Fig. 10, below) possesses the characteristics of an LB enhancer.

Transcriptional regulatory sequences in the N-myc 3′-flanking region.

To localize the regulatory sequences involved in BA expression, we performed 5′ and 3′ deletions. First, 5′ deletions of construct 2 were designed to generate constructs 3 and 4, which contained 1.6 kb and 0.9 kb of sequence upstream of the transcriptional initiation site, respectively. Both constructs were able to specifically direct lacZ expression in BA with no additional site of expression being observed (Fig. (Fig.2).2). Thus, DNA elements involved in BA regulation are included in the sequences localized between kb −0.9 and kb +7.2.

Removal of the 3′-flanking sequences of construct 4, the smallest version directing BA expression, gave rise to constructs 7 and 9, which included N-myc genomic sequences from position kb −0.9 to kb +6.5 or kb +5.9 kb, respectively (Fig. (Fig.3).3). Like construct 4, construct 7 directed lacZ expression in BA. However, none of the F0 transgenic embryos obtained with construct 9 showed X-Gal staining. Addition of 5′-flanking sequences at up to kb −2.0 to construct 9 did not reestablish BA expression (construct 10), indicating that the sequences located between positions kb +5.9 and kb +6.5 were essential to direct expression in the BA. To precisely define the control region, a transgene was obtained by digesting construct 2 with StuI, deleting the 3′ sequences from kb +6.5 to kb +6.1 and the 5′ sequences from kb −0.9 to kb −0.5 (construct 8) (Fig. (Fig.3).3). Transgenic embryos obtained with the transgene showed X-Gal staining in BA, indicating that critical regulatory sequences driving BA expression are restricted to a 200-bp fragment located between positions kb +5.9 and kb +6.1.

To further assess the presence of a BA regulatory element in the 3′-flanking sequences, a DNA fragment from kb +5.9 to kb +7.2 was placed in front of the hsp68-lacZ reporter gene (construct 11) (Fig. (Fig.3).3). Three out of five transgenic embryos showed weak and ectopic X-Gal staining without any BA expression (Fig. 3B and C). A larger DNA region was therefore tested in order to rule out the possibility that the BA control sequences extend upstream of the kb +5.9 limit. A 2.3-kb genomic fragment from kb +4.9 to kb +7.2 was inserted in front or downstream of the hsp68-lacZ reporter gene (constructs 12 and 13) (Fig. (Fig.3A).3A). None of the transgenic embryos generated with these constructs showed expression in the BA (Fig. 3B and C), whereas a majority displayed X-Gal staining in the somites, a normal site of N-myc expression (Fig. (Fig.1).1). These results suggested the presence of a somite-specific enhancer in the DNA fragment located between kb +4.9 and kb +7.2 (identified as the R4 domain in Fig. 10, below). Even though the DNA fragments extending from kb +5.9 to kb +6.1 or from kb +5.9 to kb +7.2 are required to direct BA expression when present in an N-myc genomic context, they do not possess the properties of an independent BA enhancer, as they cannot confer BA expression when linked to a heterologous promoter.

Intragenic sequences are involved in the regulation of N-myc branchial arch expression.

Data obtained with transgene 8 indicated that the N-myc sequences located between kb −0.5 and kb +6.1 were sufficient to correctly direct expression in the BA. Previous studies had established the presence of regulatory elements in the first intron of the mouse and human N-myc genes (68, 71). To determine the importance of the exon and intron sequences in N-myc branchial arch expression, we designed constructs in which both N-myc introns (construct 14), intron 1 (construct 16), or intron 2 (construct 15) were deleted in the context of construct 1 (Fig. (Fig.4).4). In these constructs, the R1 domain, which directs expression in limb buds, was retained and used as a marker of the activity of the transgenes. Construct 14 carried a deletion from kb +0.2 to kb +4.6 that included exons 1, 2, and 3 and intron 1 and 2 sequences. This transgene directed expression in LB but not in BA (Fig. (Fig.4).4). This result suggested that the BA regulatory sequences associated with the R4 domain were not sufficient to allow expression in BA and required additional N-myc exon or intron sequences. If the deletion was restricted to the first intron and the beginning of exon 2 (kb +0.2 to +1.8), BA expression was observed (construct 16) (Fig. (Fig.4),4), whereas the deletion of the second intron (kb +2.4 to kb +4.1; construct 15) led to a lack of BA staining. Together, these results suggested the presence of BA positive regulatory sequences in the second intron.

To confirm that intron 2 sequences can target expression to the BA, a DNA region encompassing sequences from kb +1.65 to kb +4.3 (named the R3 domain) was tested in front of the hsp68-lacZ reporter gene in either orientation (constructs 17 and 18) (Fig. (Fig.4A).4A). In both cases, transgenic embryos displayed X-Gal staining in the BA, supporting the notion that the R3 domain can act as a BA enhancer. However, in the context of the N-myc gene, the R3 region was not sufficient to drive expression in BA, as shown by constructs 9 and 10, which both contain the R3 sequences (Fig. (Fig.3).3). Comparison of constructs 2 and 4 with constructs 10 and 9, respectively, thus suggested that both R3 and R4 sequences are required for branchial arch expression (Fig. (Fig.22 and and33).

Results obtained with construct 16 demonstrated that the first intron and part of the second exon sequences (named the R2 domain [see Fig. 10, below]) were not required for BA expression. Previous work had shown that this N-myc region is involved in gene attenuation and possesses a silencer activity in the human gene (68, 71). We thus verified if the R2 sequences from kb +0.2 to kb +1.8 can act negatively on BA expression. The R2 domain was deleted in construct 10, generating construct 19, to test if the R3 BA enhancer triggers BA expression in the absence of the R4 domain in the N-myc context (Fig. (Fig.4).4). All embryos expressing the transgene presented BA expression, revealing the inhibitory action of R2 sequences on the R3 enhancer.

The N-myc branchial arch enhancer is located in intron 2 sequences.

To determine which region of the R3 domain (kb 1.65 to kb +4.3), the second exon or intron, contains the BA enhancer, 5′ and 3′ deletions of the R3 domain were generated using constructs 17 and 18. As shown in Fig. Fig.5,5, the deletion of the sequences located between kb +1.65 and kb +2.8 (constructs 20 and 21) did not affect BA expression, whereas further deletion to kb +3.3 led to the loss of BA lacZ staining (construct 22), whereas all the 3′ deletions tested were unable to direct expression in the BA with the exception of one F0 transgenic construct for construct 23 (constructs 23 and 24) (Fig. (Fig.5A).5A). Altogether these data indicated that the BA enhancer is located in the second intron of N-myc between positions kb +2.8 and kb +4.3. An interspecies sequences comparison showed a highly conserved region in the N-myc second intron (see Fig. 7A, below). Moreover, computational predicted transcriptional regulatory modules within the human and the mouse genomes colocalized with this conserved sequence (16). To test if this conserved sequence contains the BA enhancer, this sequence as well as 5′ and 3′ deletions were tested in front of an N-myc-lacZ minimal promoter (kb −0.9 to kb +0.2) that on its own cannot direct transgene expression (construct 25) (Fig. (Fig.5B).5B). As shown in Fig. 5B and C, constructs 26, 27, and 28, carrying a 600-, 400-, and 230-bp fragment located between positions kb +3.0 and kb +3.6, kb +3.0 and kb +3.4, and kb +3.17 and kb +3.4, respectively, directed expression in BA.

To confirm that the staining pattern obtained with the different N-myc constructs agreed with in situ hybridization data (26), embryos with a BA lacZ-positive signal were sectioned. All embryos observed mimicked the native profile of N-myc expression in the mesenchymal and ectodermal tissues of the BA, as shown by in situ hybridization at E10.5 (Fig. (Fig.1F)1F) as well as by the data obtained at E9.5 as published by Kato et al. (see Fig. Fig.22 of reference 26). However, the N-MYC protein was not detected in the ectodermal tissue (26). This difference may be explained by a change in mRNA translatability or in protein stability between mesenchymal and ectodermal tissues. Detection of β-galactosidase activity in the BA ectoderm of N-myc-lacZ transgenic embryos supports variation in N-MYC protein stability between the two tissues (Fig. (Fig.66).

FIG. 6.
Targeted expression of the lacZ reporter constructs in the developing BA. Transgenic embryos produced by the different N-myc-lacZ constructs were embedded in paraffin and sectioned to show in more detail the staining patterns. (A and B) Embryos derived ...

Altogether, these data show that the 230-bp conserved region contains DNA elements with enhancer properties that can induce N-myc expression in the BA.

N-myc branchial arch expression involves a GATA binding site.

To gain insight into the critical sequences involved in BA expression, we investigated whether the conserved region of intron 2, included in the 600-bp BA enhancer fragment located between position kb +3.0 and kb +3.6, contains functional binding sites for known trans-acting factors that may be involved in BA gene expression. To do so, we performed EMSA with protein extracts from E10.5 embryos. The 600-bp fragment was subdivided in three fragments, and only the central fragments (S1) corresponding to the 230-bp BA enhancer fragment showed specific binding activity in the EMSA (Fig. (Fig.77 A to C and data not shown). As shown in the left panel of Fig. Fig.7C,7C, E10.5 proteins can form binding complexes with the S1 fragment. The specificity of protein binding to the probe was confirmed by competition studies with a 100-fold excess of unlabeled S1 probe, which resulted in a total loss of protein binding (Fig. (Fig.7C,7C, lane 3 of left panel), whereas unrelated competitor did not interfere with binding (lane 4). Further characterization of specific protein interactions with different S1 subfragments (S1.1, S1.2, and S1.3 [Fig. [Fig.7B])7B]) revealed that protein binding to S1 could specifically be competed with the S1.1 fragment (Fig. (Fig.7C,7C, middle panel). No competition was observed with fragments S1.2, S1.3, or an unrelated competitor of similar size. Moreover, binding was observed only with fragment S1.1, indicating that putative binding sites are located in the 78 bp unique to the S1.1 fragment and may be involved in BA expression (Fig. (Fig.7C,7C, right panel).

FIG. 7.
The 230-pb BA enhancer contains a consensus GATA binding motif. (A) Nucleotide sequence conservation in the N-myc second intron. A genomic sequence comparison of the mouse N-myc second intron with the genome of several species was performed using the ...

Since the S1.1 fragment enclosed highly conserved sequences containing predicted transcriptional factor binding sites (Fig. (Fig.7B),7B), we performed additional EMSA on the S1.1 subfragment to determine the exact sequence recognized by proteins in the E10.5 embryo extract. Only the fragments S1.1B and S1.1C permitted the formation of specific binding complexes (Fig. (Fig.77 and data not shown). Fragment S1.1B contains a potential GATA binding site conserved between mammalian species, whereas a potential LEF/TCF binding site is located in S1.1C (Fig. (Fig.7B).7B). Both Lef-1 and Tcf-4 mutant mice show postnatal lethality with no obvious phenotype in facial or limb bud development, whereas Gata3 mutant mice die at midgestation and show craniofacial abnormalities (30, 36, 50, 64). Gata3 is expressed in the branchial arches, and its expression is under the control of an enhancer containing GATA binding sites (35).

To determine if GATA factors can bind S1.1B, competition with S1.1B containing a mutation in the GATA binding site (S1.1B*) was tested in an EMSA (Fig. (Fig.7D).7D). S1.1B* could not compete with S1.1B for binding, confirming the presence of the GATA binding site. To further confirm the implication of GATA factors, EMSA were performed with recombinant GATA proteins (Fig. (Fig.7E).7E). All the GATA proteins tested were able to specifically interact with S1.1B. Moreover, GATA3 did not bind the S1.1B* mutant probe (Fig. (Fig.7E,7E, lane 25). Altogether these results indicate the presence of a genuine GATA binding site.

A functional GATA binding site is present in the R3 branchial arch enhancer.

To test whether the GATA binding site identified in the R3 domain is functional and to determine if GATA can bind this site to activate N-myc expression, a transfection reporter assay was performed. HEK293 human cells were cotransfected with a reporter construct in which the luciferase reporter gene was placed under the control of the murine N-myc BA enhancer (600-bp R3 region, BA/enh-pGL3) in the presence or absence of various GATA expression vectors (Fig. (Fig.88 A). In the presence of GATA3, -4, -5, or -6 proteins, a statistically significant increase in relative luciferase activity was observed. These data show that GATA proteins can bind to the R3 branchial arch enhancer and activate transcription.

FIG. 8.
GATA binds to the BA enhancer in vivo and can activate transcription in trans-activation assays. (A) In a transient-transfection assay, mouse GATA1, -3, and -5 or rat GATA4 and -6 proteins upregulated transcription of a luciferase reporter construct containing ...

To establish whether GATA3 is able to bind to the 230-bp BA enhancer in vivo, a ChIP assay was performed on cross-linked chromatin isolated from the neuroblastoma cell line NBA2 overexpressing N-myc without N-myc locus amplification. DNA in the immunoprecipitate was subjected to qPCR analyses with specific primer pairs for the 230-bp BA enhancer and the first and third exons, as well as a downstream region located at kb 160 of the N-myc gene. As shown in Fig. Fig.8B,8B, GATA3 was recruited to the BA enhancer and the third exon, whereas no binding was observed on the first exon and the downstream sequences. As expected, recruitment of the histone H3 was observed for all the regions tested. These results indicate that GATA3 is able to bind to the 230-bp BA enhancer both in vitro and in vivo. The GATA3 interaction with the third exon may be explained by the fact that the N-myc 3′-untranslated region of exon 3 is highly conserved. It corresponds to the R4 domain, which contributes to BA expression in the N-myc gene context. Moreover, a potential GATA binding site is present in the vicinity of the exon 3 amplified fragment.

To determine the contribution of the GATA binding site to N-myc BA expression via the 230-bp BA enhancer, transgenic embryos were generated with construct 1 carrying a deletion of the 230-bp BA enhancer (construct 29). Construct 29 was able to direct expression in the LB, but no expression was observed in the BA (Fig. (Fig.99 A to C). Similarly, a point mutation into the GATA binding site (construct 30) also directed expression in the LB but not in the BA. Together these data indicate the crucial role played by the GATA binding site in N-myc BA expression.

FIG. 9.
The GATA binding site in the 230-bp BA enhancer is essential for the enhancer activity. (A) Schematic representation of N-myc-lacZ transgenes used to generate F0 transgenic embryos. The 230-bp BA enhancer was deleted in construct 29, whereas point mutations ...

Finally, we directly assessed the in vivo requirement of Gata3 gene function in N-myc BA expression by examining whether the expression of the N-myc 41S transgene, used as reporter of N-myc expression in the BA (10), could be altered in Gata3 mutant embryos. To do so, we introduced the N-myc 41S transgene into the Gata3 mutant background, and Gata3+/ Tg+/N-myc 41S males were mated with Gata3+/ females. E9.25 transgenic embryos carrying the N-myc 41S transgene and various combinations of the Gata3 mutant alleles were generated and stained for β-galactosidase activity. In Gata3+/+ Tg+/N-myc 41S transgenic embryos, X-Gal staining was detected in the BA and the LB as previously described (Fig. (Fig.9D)9D) (10). However, in the absence of Gata3 function (Gata3/ Tg+/N-myc 41S embryos), decreased lacZ expression was observed in the BA (Fig. (Fig.9D)9D) but not in the LB compared to wild-type or Gata3+/ embryos. Altogether these experiments demonstrate the involvement of GATA3 in N-myc BA expression.

DISCUSSION

The regulation of N-myc gene expression requires a number of cis-acting elements, all necessary to obtain a precise modulation of expression. In vitro and in vivo assays using tissue culture systems and transgenic mice have identified DNA regions involved in N-myc gene regulation. Such experiments have revealed that the sequences able to direct N-myc expression in transfection assays cannot entirely reconstitute its embryonic expression pattern when tested in transgenic mice (10). Only a restricted number of N-myc expression sites, such as the LB and the BA, were reconstituted with the sequences identified in vitro (Fig. (Fig.22 and and3)3) (10). These results indicated that the regulation of N-myc developmental expression requires additional control DNA elements. In the present study, we aimed to identify the N-myc regulatory elements involved in LB and BA expression and to understand their mechanisms of action.

N-myc expression in LB involves a tissue-specific enhancer.

By transgenic analyses, we have defined a DNA region located between positions kb −4.4 and kb −2.0 that is essential to confer limb bud expression (named the R1 domain in Fig. 10). When tested in the context of a heterologous promoter, staining in the LB was retained, indicating that the R1 domain contains tissue-specific enhancer sequences sufficient to direct expression in LB. Our previous study, based on stable transgenic mouse lines, indicated that sequences up to kb −2.0 (construct 2) were sufficient to direct LB expression. The discrepancy between the results obtained in F0 transgenic embryos versus stable transgenic mouse lines suggests that other DNA sequences residing in construct 2 may be involved in LB expression, but they are sensitive to the integration site of the transgene. Alternatively, the integration of the transgene into the mouse genome and its modification via epigenetic events in the germ line and during early development may participate in correcting N-myc expression. Such a mechanism was previously proposed to explain why a N-myc construct that failed to reproduce the endogenous N-myc expression profile in transfection assays closely recapitulated it in newborn transgenic mice (72).

The R4 domain acts as a somite enhancer and contributes to BA expression.

N-myc gene expression in the BA is first detected in the mesenchyme of the first and second BA at E9 (10, 26). In the present study, we showed that BA expression is supported by sequences encompassing the N-myc gene and located between kb −0.9 and kb +6.1, which includes less than 350 bp of sequence downstream of the polyadenylation signal. At least three DNA domains, in addition to the promoter region, are located within the intragenic sequences of the gene and are involved in N-myc expression in the BA.

One of these domains, R4, is located in the 3′ region of the N-myc gene, and it is required for BA expression in the context of the N-myc gene, but it cannot direct BA expression by itself. The R4 domain, rather, acts as a somite enhancer when linked to a heterologous promoter, as shown with constructs 12 and 13 (Fig. (Fig.3).3). However, the R4 domain does not have any somite transcriptional activity when tested in the context of the N-myc gene, suggesting that inhibitory sequences may repress the enhancer activity in the somites. Consistent with this, previous in vitro studies in cell culture have identified negative transcriptional regulatory elements upstream of the transcription initiation site and within the first intron sequences of the human and mouse N-myc genes (21, 56, 66, 68, 71). In agreement with these findings, one-third of the transgenic embryos obtained with constructs 14 and 19, which both contain the R4 region but lack the intragenic sequences located between kb +0.2 and kb +4.6 or between kb +0.2 and kb +1.8, respectively, showed a faint X-Gal staining in somites (Fig. (Fig.4).4). The weak somite signal obtained with construct 14 compared to that of constructs 12 and 13 indicates that a negative element(s) may also be present in the promoter region. Deletion of sequences from the N-myc promoter region supports this possibility and localized one of these elements between positions kb −1.6 and kb −0.9, the X-Gal staining in the somites being more intense and more frequent in transgenic embryos (data not shown). Moreover, the R4 domain can also contribute to branchial arch expression in the context of the minimal N-myc promoter (kb −0.9 to kb +0.2) in the absence of intragenic N-myc sequences, including the BA enhancer (data not shown). Together, these data demonstrate that the R4 domain positively contributes to N-myc expression in branchial arches and somites. However, more positive regulatory sequences that remain to be identified are required for somite expression to circumvent the negative sequences present in the N-myc genomic regions analyzed in the present study.

The BA enhancer in the second N-myc intron is regulated by inhibitory sequences located in the first intron.

The second intron of N-myc contains the R3 domain, which acts as a BA enhancer when linked to a heterologous promoter. In the context of the N-myc gene, the activity of the R3 domain is modulated by the R2 and R4 domains. In the presence of R2, R3 can direct expression in BA only if R4 is present. In contrast, in the absence of R2, R4 is not required for the BA enhancer activity of R3 (construct 19) (Fig. (Fig.4).4). Thus, R2 appears to be able to repress the enhancer activity of R3. The R2 domain includes the entire first intron of N-myc, a region that, according to data obtained from the human and the mouse genes, contains at least three negative regulatory elements (21, 56, 66, 68, 71). These elements include an attenuator of transcription localized at the 5′ end of the first intron and a tissue-specific element located in the first intron that represses N-myc expression when present in the transcription unit. This tissue-specific element most likely acts posttranscriptionally (56). A third element is also present in the first intron and acts as a tissue-specific silencer. The presence of the tissue-specific element and the silencer has not been confirmed for the murine gene. Moreover, the sequence of the silencer is not conserved between mouse and human (60). However, based on these data, two mechanisms can be envisaged to explain the requirement of R4 for N-myc BA expression (Fig. (Fig.10).10). In the first one, the R3 BA enhancer can direct expression in BA, but this expression is blocked by transcriptional attenuation or silencing mediated by the R2 domain, while the R4 domain can synergize with the R3 domain to overcome R2 repression. Alternatively, the R4 domain may directly act on the R2 domain to release the transcriptional inhibition on R3. The ability of the R4 domain to direct expression in the BA in the presence of the N-myc promoter favors the first model (data not shown).

FIG. 10.
Model for the regulation of N-myc expression in branchial arches, somites (Som), and limb buds. N-myc branchial arch expression involves at least three regulatory domains, identified as R2, R3, and R4. The R3 domain corresponds to the second intron of ...

Crucial role of GATA factors in N-myc BA expression.

GATA transcription factors have been shown to play key roles in regulating eukaryotic development by regulating the expression of mediators. The expression profile of the individual GATA family members are both diverse and developmentally dynamic (7). GATA1, -3, -4, and -6 have been shown to be expressed in the BA (19, 52). However, GATA1 is known for its role in erythropoiesis, whereas GATA6 was shown to be essential for the development of the visceral endoderm and in heart development (17, 51, 69). Even though endogenous Gata4 expression has never been reported, the knock-in of a reporter gene in the Gata4 locus indicates that the gene is transcribed in neural crest derivatives, including the BA. GATA3 is also expressed in the BA. Its expression is under the control of a BA enhancer that contains consensus GATA binding sites (13, 35). Moreover, Gata3−/− embryos show underdevelopment of the BA, as has been previously described for N-myc mutant embryos (50, 54, 59). By demonstrating a genetic interaction between GATA3 and the N-myc BA enhancer, our data provide a molecular basis for the previous findings. However, the GATA3 mutation does not completely eliminate N-myc-lacZ expression in the BA, suggesting that other members of the GATA family expressed in this structure can compensate for the absence of GATA3. This was supported by our in vitro trans-activation assay, which showed that GATA4, -5, and -6 can also activate transcription through the R3 branchial arch enhancer (Fig. (Fig.8A).8A). Gata3 expression is not restricted to the BA, whereas the N-myc-lacZ construct carrying the BA enhancer is confined to the BA, indicating that although GATA3 is essential for N-myc BA expression, GATA3 alone may not be sufficient to restrict expression in BA via the N-myc BA enhancer. Alternatively, other factors that remain to be identified may be also implicated in restricting the N-myc BA enhancer action to the BA in conjunction with GATA3. The conserved TCF/LEF binding site located in the S1.1C fragment (Fig. (Fig.7B)7B) next to the GATA binding site suggests that TCF and LEF proteins may be good candidates. Moreover, Lef1 and Tcf4 are expressed in the BA at E10.5 (19).

In summary, our characterization has allowed us to identify several DNA regions dispersed along the N-myc locus and involved in N-myc developmental expression. These regions include the R1 domain, a limb bud-specific enhancer and localized in N-myc upstream sequences, and three domains within the N-myc gene, the R2, R3, and R4 domains, which cooperate in a complex fashion to regulate N-myc expression in branchial arches. The characterization of cis-acting regulating sequences has led to the identification of GATA3 as being a trans-acting factor essential for the activity of the BA enhancer. The R4 domain also contains a somite enhancer. The precise roles of these domains and the regulatory mechanisms involved in N-myc developmental expression await further studies.

Acknowledgments

We thank Lucie Jeannotte, Josée Aubin, and Robert S. Viger for critical comments on the manuscript, Benoit Lachapelle, Julie Pageau, and Valérie Garceau for technical support, Hiroaki Taniguchi, Robert S. Viger, Nikita Avvakumov, and Jacques Coté for technical advice on ChIP assays, and Michael Parmacek for the GATA5 expression vector and James D. Engel for the Gata3 mutant mouse line.

J.-F.C.-G. held a studentship from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche. This work was supported by funding from NSERC and the Cancer Research Society Inc. to J.C.

Footnotes

[down-pointing small open triangle]Published ahead of print on 20 September 2010.

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