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Originally published In Press as doi:10.1074/jbc.M311740200 on February 4, 2004

J. Biol. Chem., Vol. 279, Issue 16, 16356-16367, April 16, 2004
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Helt, a Novel Basic-Helix-Loop-Helix Transcriptional Repressor Expressed in the Developing Central Nervous System*

Tomoya Nakatani, Eri Mizuhara, Yasuko Minaki, Yoshimasa Sakamoto, and Yuichi Ono{ddagger}

From the KAN Research Institute Inc., 93 Chudoji-Awata-cho, Shimogyo-ku, Kyoto 600-8815, Japan

Received for publication, October 27, 2003 , and in revised form, January 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuronal differentiation is regulated by many basic-helix-loop-helix (bHLH) family transcriptional activators and repressors, and the balance of activity between these factors is important for the differentiation process. Here, we report the identification of a novel transcriptional repressor, designated Helt. Helt encoded a Hey-related bHLH protein containing the bHLH and Orange domains. Helt could homodimerize, and heterodimerize with Hes5 or Hey2. Both the bHLH and Orange domains were involved in the homodimerization. In contrast, only the bHLH domain was required for the heterodimerization with Hey2, whereas only the Orange domain mediated the interaction between Helt and Hes5. Thus, Helt has two dimerization domains, and these domains independently select a partner. Identification of preferred recognition sequences by CASTing experiments revealed that Helt bound to the E box, which was distinct from the Hes1 optimal sequence around the E box core. Not only the core sequence but also sequences flanking the E box were essential for the recognition by Helt and Hes1. Furthermore, Helt repressed transcription from an artificial promoter through binding to the optimal E box elements, as well as transcription from its own promoter. Using in situ hybridization and immunohistochemistry, Helt expression in embryos was investigated. Helt was mainly expressed in undifferentiated neural progenitors in some of the developing brain regions, including the mesencephalon and diencephalon, at the neurogenesis stage. These results suggest that Helt acts as a transcriptional repressor to regulate neuronal differentiation and/or identity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcription factors of the basic-helix-loop-helix (bHLH)1 family play important roles in lineage restriction and differentiation in many developmental processes. In the central nervous system, many bHLH factors, which are conserved from flies to vertebrates, have been identified and shown to regulate neurogenesis (1, 2). Some of these bHLH factors, such as Mash1, regulate neurogenesis positively by activating downstream target gene expression, whereas others, such as Hes1, inhibit neuronal differentiation by acting as transcriptional repressors (3, 4). The balance of activity between activators and repressors is thought to determine the differentiation status.

The bHLH repressors were originally identified in Drosophila as Hairy and E(spl). Subsequently, many vertebrate homologues and additional related proteins were identified (5–9). These transcription factors share a common structure, the Orange domain, located just C-terminal to the bHLH domain, and are referred to as the bHLH-Orange (bHLH-O) proteins (10). The members of the bHLH-O family are grouped into four distinct subfamilies, Hairy, E(spl), Hey, and Stra13, based on their primary structures (10). Most of the known members of these subfamilies act as transcriptional repressors.

As a general feature among bHLH and bHLH-O proteins, the basic region has been shown to mediate DNA binding, whereas the HLH domain is necessary for dimerization (10). On the other hand, the role of the Orange domain is not well understood. Domain swapping experiments of Hairy and E(spl) in Drosophila revealed that the Orange domain functions in subfamily specificity (11). In vertebrates, roles for the Orange domain in transcriptional repression and dimerization have been reported (12, 13), but the molecular basis of these observations has not been clarified.

An additional characteristic structure, the WRPW motif, in the C terminus is conserved among the Hairy and E(spl) subfamilies in vertebrates and flies (10). This motif is involved in the recruitment of the TLE/Groucho corepressor and required for the transcriptional repression activity (14, 15). Related sequences are present near the C terminus of the Hey subfamily members. However, this region is not required for the repression activity (7), although a zebrafish Hey2 mutant lacking this region could not rescue the gridlock mutant phenotype (16). Rather, the bHLH domain of the Hey family mediates recruitment of other corepressors, N-CoR and mSin3A, to repress the transcription (7). In the case of the Stra13 subfamily, histone deacetylase-dependent and -independent transcriptional repression through their C terminus regions has been reported (17). However, which corepressor mediates this repression has not been determined.

Hes1 and Hes5 have been shown to act as downstream effectors for Notch signaling (18–20). When the neural progenitors exit the cell cycle in the ventricular zone of the neural tube, these cells express Notch ligands, Dll1 or Jagged, to activate the Notch receptors expressed on their neighboring progenitor cells. In the Notch-activated cells, Hes1 and Hes5 are induced and repress both the expression and activity of Mash1, and then neuronal differentiation is inhibited. This signaling process, so-called "lateral inhibition," maintains the number and status of undifferentiated neural progenitors and neural stem cells in the developing central nervous system (21). In this process, Hes6 can antagonize the activity of Hes1 or Hairy 1/2 to promote neurogenesis (22–24). Thus, a balance between the bHLH-O transcriptional repressors is essential for the proper development of the central nervous system. Expression of the Hey family members is also known to be activated by Notch signaling in cultured cells (25–27). Although it has recently been reported that Hey1 and Hey2 regulate the maintenance of neural progenitors in the developing brain (28), the significance of the Hey family downstream of Notch signaling is not evident. Other members of the bHLH-O family, such as Hes3, Hes6, Stra13, and SHARP1, do not respond to Notch activation but are up-regulated by many other signals (24, 29–32). Thus, it is not a general feature that bHLH-O proteins are downstream targets for Notch signaling

Here, we report the identification of a novel transcriptional repressor, designated Helt. Helt encodes a Hey-related bHLH protein, which can homodimerize, as well as heterodimerize with Hes5 or Hey2. Helt homodimers can bind to E box elements and repress transcription through binding to these elements. Identification of the preferred recognition sequences by CASTing experiments revealed that Helt binds to the E box, which is distinct from the Hes1 optimal sequence. Furthermore, sequences flanking the E box are essential for the recognition by Helt and Hes1. Helt is mainly expressed in undifferentiated neural progenitors in the developing central nervous system, suggesting that it functions in the regulation of neuronal differentiation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Helt—Brain regions including the mesencephalon and metencephalon were dissected from E10.5 mouse embryos and divided into two portions, the ventral and dorsal regions. Total RNA was isolated using an RNeasy mini kit (Qiagen). Subtractive PCR was performed as described (33). After four rounds of subtractive hybridization, the amplified cDNA fragments were cloned and sequenced. Differential expression of the obtained clones was confirmed by PCR using cDNA amplicons as templates. Full-length cDNAs were screened from an E12.5 mouse brain cDNA library. Four independent clones were obtained and sequenced.

Construction of Plasmids—pcDNA-FLAG-NII was constructed by ligating the oligonucleotides 5'-GAT CGG CCA CCA TGG ATT ACA AGG ATG ACG ACG ATA AGG TCG ACC TCG AGG GAT CCG AAT TCG CGG CCG CG-3' and 5'-TCG ACG CGG CCG CGA ATT CGG ATC CCT CGA GGT CGA CCT TAT CGT CGT CAT CCT TGT AAT CCA TGG TGG CC-3' into the BamHI/XhoI site of pcDNA3.1+ (Invitrogen). pcDNA-HA-NII was constructed by ligating the oligonucleotides 5'-GAT CGG CCA CCA TGG ACT ACC CAT ACG ACG TCC CAG ACT ACG CTG TCG ACC TCG AGG GAT CCG AAT TCG CGG CCG CG-3' and 5'-TCG ACG CGG CCG CGA ATT CGG ATC CCT CGA GGT CGA CAG CGT AGT CTG GGA CGT CGT ATG GGT AGT CCA TGG TGG CC-3' into the BamHI/XhoI site of pcDNA3.1+. pMXIII was constructed by ligating the oligonucleotides 5'-GAT CGA AGC TTA CGC GTG TCG ACC TCG AGG GAT CCG AAT TCG CGG CCG CG-3' and 5'-TCG ACG CGG CCG CGA ATT CGG ATC CCT CGA GGT CGA CAC GCG TAA GCT TC-3' into the BamHI-XhoI site of pMXII (34). pMX-GAL4 was constructed by ligating the cDNA encoding the GAL4 DNA-binding domain amplified using primers 5'-GAG AAG CTT GCC ACC ATG AAG CTA CTG TCT TCT ATC GAA CA-3' and 5'-GAG ACG CGT CGG CGA TAC AGT CAA CTG TCT TTG AC-3' into the HindIII/MluI sites of pMXIII. The cDNAs of Helt, Hes1, Hey2, and Hes5 were amplified by PCR using the following primer sets: Helt, 5'-GAG GTC GAC ATG TCT GAC AGG CTC AAG GAA CGC AA-3' and 5'-GAG GCG GCC GCT CAG AGC ACC GGA GGG TGC TGG GGC G-3'; Hes1, 5'-GAG CTC GAG ATG CCA GCT GAT ATA ATG GAG AAA AA-3' and 5'-GAG GCG GCC GCT CAG TTC CGC CAC GGT CTC CAC ATG G-3'; Hey2,5'-GAG CTC GAG ATG AAG CGC CCT TGT GAG GAA ACG AC-3' and 5'-GAG GCG GCC GCT TAA AAG GCT CCA ACT TCT GTC CCC C-3'; and Hes5, 5'-GAG GTC GAC ATG GCC CCA AGT ACC GTG GCG GTG GA-3' and 5'-GAG GGA TCC TCA CCA GGG CCG CCA GAG GCC GCA GG-3'. The Helt fragment was digested with SalI/NotI and cloned into the XhoI/NotI sites of pcDNA-FLAG-NII, pcDNA-HA-NII, pMXIII, and pMX-GAL4. Hes1 and Hey2 fragments were digested with XhoI/NotI and cloned into the same sites of pcDNA-FLAG-NII and pcDNA-HA-NII and pMXIII. The Hes5 fragment was digested with SalI/BamHI and cloned into the XhoI/BamHI sites of pcDNA-FLAG-NII and pcDNA-HA-NII. Various mutant Helt constructs containing the following amino acid (aa) residues were made: Helt-WT, aa 1–240 (full-length); Helt{Delta}H, aa 76–240; Helt{Delta}O, aa 1–83 + 127–240; Helt{Delta}C, aa 1–140; Helt{Delta}C2, aa 1–207; Helt-H, aa 1–75; Helt-O, aa 76–140; and Helt-C, aa 141–240. A Helt-BM mutant substituting Lys-Arg-Arg (20–22) with Ala-Ala-Ala was constructed by PCR.

Multiple responsive elements containing reporters were constructed by ligating the following oligonucleotides into the KpnI/SacI sites of the pGL2-promoter (Promega): E box class A-luc, 5'-CGG CAG GTG CCG GCA GGT GCC GGC AGG TGC CGG CAG GTG CCG GCA GGT GCC GAG CT-3' and 5'-CGG CAC CTG CCG GCA CCT GCC GGC ACC TGC CGG CAC CTG CCG GCA CCT GCC GGT AC-3'; E box class B-luc, 5'-CGG CAC GTG CCG GCA CGT GCC GGC ACG TGC CGG CAC GTG CCG GCA CGT GCC GAG CT-3' and 5'-CGG CAC GTG CCG GCA CGT GCC GGC ACG TGC CGG CAC GTG CCG GCA CGT GCC GGT AC-3'; E box class C1-luc, 5'-CGG CAC GCG ACG GCA CGC GAC GGC ACG CGA CGG CAC GCG ACG GCA CGC GAC GAG CT-3' and 5'-CGT CGC GTG CCG TCG CGT GCC GTC GCG TGC CGT CGC GTG CCG TCG CGT GCC GGT AC-3'; E box class C1-1-luc, 5'-CCG CAC GCG ACC GCA CGC GAC CGC ACG CGA CCG CAC GCG ACC GCA CGC GAC GAG CT-3' and 5'-CGT CGC GTG CGG TCG CGT GCG GTC GCG TGC GGT CGC GTG CGG TCG CGT GCG GGT AC-3'; E box class C1-2-luc, 5'-CGG CAC GCG ACG GCA CGC GAC GGC ACG CGA CGG CAC GCG ACG GCA CGC GAC GAG CT-3' and 5'-CGT CGC GTG CCG TCG CGT GCC GTC GCG TGC CGT CGC GTG CCG TCG CGT GCC GGT AC-3'; E box class C1-5-luc, 5'-CGG CAC GCG CCG GCA CGC GCC GGC ACG CGC CGG CAC GCG CCG GCA CGC GCC GAG CT-3' and 5'-CGG CGC GTG CCG GCG CGT GCC GGC GCG TGC CGG CGC GTG CCG GCG CGT GCC GGT AC-3'; E box class C1-6-luc, 5'-CAA CAC GCG TTA ACA CGC GTT AAC ACG CGT TAA CAC GCG TTA ACA CGC GTT GAG CT-3' and 5'-CAA CGC GTG TTA ACG CGT GTT AAC GCG TGT TAA CGC GTG TTA ACG CGT GTT GGT AC-3'; and UAS-SV40-luc, 5'-CCG GAG TAC TGT CCT CCG AGC GGA GTA CTG TCC TCC GAG CGG AGT ACT GTC CTC CGA GCG GAG TAC TGT CCT CCG AGG AGC T-3' and 5'-CCT CGG AGG ACA GTA CTC CGC TCG GAG GAC AGT ACT CCG CTC GGA GGA CAG TAC TCC GCT CGG AGG ACA GTA CTC CGG GTA C-3'.

Helt promoter reporter constructs were constructed by ligating the genomic fragments amplified using the following primers into the KpnI/SacI sites of pGL2-basic (Promega): -934-luc, 5'-GAG GGT ACC ATA GGC TAC AAA TTG AGC CTC TTC CC-3' and 5'-GAG GAG CTC CGT TCC ACC TCG TGT GCC TCT TCA AG-3'; -780-luc, 5'-GAG GGT ACC TGC CAG CCG CAG CCC CAC ACC TGC CG-3' and 5'-GAG GAG CTC CGT TCC ACC TCG TGT GCC TCT TCA AG-3'; and -934{Delta}27-luc, 5'-GAG GGT ACC ATA GGC TAC AAA TTG AGC CTC TTC CC-3' and 5'-GAG GAG CTC TTC CAG GTA GAC TGC AGC CTC CCA GG-3'. -934M-luc was constructed by mutagenesis of two E box class B, CACGTG (-304 and -323) to GTCGTG.

Immunoprecipitation—293E cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. 293E cells (2 x 106) were transfected with the indicated combinations of 5 µg of each plasmid using TransIT LT1 reagent (Mirus Corp.). Immunoprecipitation experiments and detection by Western blotting were performed as described (33).

EMSA—Cell lysates of transfected 293E cells were prepared as described. Biotinylated oligonucleotides were annealed to generate double-stranded probes. One microliter of each cell lysate was incubated with in the presence or absence of competitors (150 ng) or anti-FLAG monoclonal antibody (2.8 µg) for 5 min at room temperature and then with biotinylated probes (0.5 ng) for another 30 min at room temperature in 20 µl of a solution consisting of 25 mM HEPES, pH 7.5, 0.05% Nonidet P-40, 5 µM ZnSO4, 50 mM KCl, 0.5 mM dithiothreitol, 1 mM EDTA, 20% glycerol, 1 µg of poly(dI-dC). The samples were electrophoresed in a 5% polyacrylamide gel with Tris-borate-EDTA buffer (50 mM Tris-HCl, pH 7.5, 50 mM boric acid, and 1 mM EDTA) at 50 V for 2.0 h. Detection of the biotinylated oligonucleotides was carried out using a chemiluminescent EMSA kit, LightShift (Pierce) following the manufacturer's protocol. The oligonucleotide probes were as follows: E box class A, 5'-TCG AGG GTG GCA GGT GCC ATT-3'; E box class B, 5'-TCG AGG GTG GCA CGT GCC ATT-3'; E box class C1, 5'-TCG AGA GCC GGC ACG CGA CAG G-3'; E box class B mutant, 5'-TCG AGG GTG GAT CGT ACC ATT-3'; E box class C1 mutant, 5'-TCG AGA GCC GGG TCG CGA CAG G-3'; C1-1, 5'-CCT CGC ACG CGA CGG CTC TCG A-3'; C1-2, 5'-TCG AGA GCC GGC ACG CGA CAG G-3'; C1-3, 5'-CCT CTC ACG CGA CGG CTC TCG A-3'; C1-4, 5'-TCG AGA GCC GTC ACG CGA CAG G-3'; C1-5, 5'-TCG AGA GCC GGC ACG CGC CAG G; and C1-6, 5'-CCT AAC ACG CGT TGG CTC TCG A-3'.

Reporter Assays—NS20Y cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Recipient cells (5 x 104) were transfected with the indicated combinations of plasmids together with 0.005 µg of pRL-SV40 (Promega) using TransIT LT1 reagent. After 48 h, cell lysates were prepared and assayed using a dual-luciferase reporter assay system (Promega). Luciferase activity was normalized with Renilla luciferase activity.

CASTing—Double-stranded degenerate oligonucleotides were amplified using a random oligonucleotide, 5'-CAG CTC CAC AAC CTA CAT CAT TCC ACT TT(N)18TTT GTC AGA GTC TCA GAG AGA AGA TGG AC-3', as a template and the primer set 5'-CAG CTC CAC AAC CTA CAT CAT TCC AC-3' and 5'-GTC CAT CTT CTC TCT GAG ACT CTG AC-3'. Amplification was carried out by three cycles of denaturation at 94 °C for 30 s (2 min in the first cycle), annealing at 65 °C for 1 min, and extension at 72 °C for 1 min. Amplified DNA was purified using a Qiaquick PCR purification kit (Qiagen), and 50 ng of degenerate oligonucleotides was incubated with 25 µl of cell lysates of transfected 293E cells in an EMSA reaction buffer. Bound oligonucleotides were immunoprecipitated with anti-FLAG antibody beads and eluted with FLAG peptide. The eluted oligonucleotides were then amplified by 15 cycles of PCR. After four rounds of selection, the amplified oligonucleotides were cloned into pCRII (Invitrogen) and sequenced.

Chromatin Immunoprecipitation—NS20Y cells (1 x 106) were transfected with the indicated expression vector. After 24 h, cells were lysed on ice in 0.1% Nonidet P-40, 25 mM Hepes (pH 7.6), 50 mM KCl, 10 mM EDTA for 30 min. Nuclei were cross-linked on ice in 1% formaldehyde for 15 min and lysed on ice for 10 min in 1% SDS, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA. The lysates were sonicated on ice and diluted 1:10 in 1.1% Triton X-100, 16.7 mM Tris-HCl (pH 8.0), 167 mM NaCl, 1.2 mM EDTA buffer. Cross-linked protein/DNA complex was immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated DNA was purified with a DNeasy kit (Qiagen). PCR amplification of endogenous Helt promoter fragment and HNF3{beta} genomic fragment (as a control) was carried out by denaturation at 94 °C for 30 s (5 min in the first cycle), annealing at 65 °C for 30 s, and extension at 72 °C for 30 s (5 min in the last cycle) with ExTaq polymerase (Takara). The number of cycles was 30 for both genomic fragments. The primer sequences were as follows: Helt promoter, 5'-GCC TCC GTC TCC AGA AGC TCT CCC TT-3' and 5'-GCC TCT TCA AGT GTT CCA GGT AGA CT-3'; and HNF3{beta} genome, 5'-TGC ACC CAG ACT CGG GCA ACA TGT TC-3' and 5'-TCC TGA CAC GGA GAA GCG CTG GAA TG-3'.

RT-PCR—RT-PCR was performed essentially as described previously (35). ExTaq polymerase was used for amplification. Amplification was carried out by denaturation at 94 °C for 30 s (2 min in the first cycle), annealing at 65 °C for 30 s, and extension at 72 °C for 30 s (2 min in the last cycle). The numbers of cycles were 35 for Helt and 25 for G3PDH. The primer sequences were as follows: Helt, 5'-CTG CTT GAA CGA GCT GGG CAA GAC AG-3' and 5'-CTT GGA TTG CAA GAA GGC GAG GAT GC-3'; and G3PDH, 5'-ACG ACC CCT TCA TTG ACC TCA ACT AC-3' and 5'-CAA GTA GAC TCC ACG ACA TAC TCA GC-3'.

In Situ Hybridization—Mouse embryos were harvested at E12.5 and immersed in 4% paraformaldehyde in phosphate-buffered saline at 4 °C for 2 h. After fixation, the embryos were cryoprotected and sectioned into 16-µm coronal and sagittal sections for in situ hybridization. A DIG-labeled Helt probe was transcribed with digoxigenin-11-UTP according to the manufacturer's instructions (Roche Applied Science) from a 1.2-kb cDNA amplified using the following primers: 5'-CTT GAA GAG GCA CAC GAG GTG GAA CG-3' and 5'-TCG CAA GTA ATC TAG GCA GTG AGT GG-3'. For hybridization with Helt probes, cryosections were refixed in 4% paraformaldehyde for 30 min at room temperature and then washed with PBS. Tissue sections were hybridized with Helt probes diluted (final concentration 1 µg/ml) in hybridization buffer (50% formamide, 5x SSC, 1% SDS, 50 µg/ml heparin, 50 µg/ml tRNA, pH 4.5) for 40 h at 68 °C. After hybridization, the sections were washed three times with buffer 1 (50% formamide, 5x SSC, 1% SDS) at 68 °C for 20 min and then treated with 0.05 µg/ml RNase A in 1x RNase buffer (0.5 M NaCl, 10 mM Tris-HCl, 1 mM EDTA) at room temperature for 5 min followed by three 5-min incubations in 1x RNase buffer at room temperature. The sections were then washed three times with 0.2x SSC at 68 °C for 5 min and three times with 1x TBS-T (TBS containing Tween 20) at room temperature for 5 min. For immunological detection of DIG-labeled hybrids, the sections were first blocked in 1x blocking reagent (Roche Applied Science) at room temperature for 1 h and then incubated overnight at 4 °C in a solution containing alkaline phosphatase-conjugated rabbit anti-DIG Fab fragments (DAKO) diluted 1:80 in 1x TBS-T. The sections were then washed three times with 1x TBS-T containing 2 mM levamisole at room temperature for 20 min followed by equilibration with buffer 2 (0.1 M NaCl, 50 mM MgCl2, 0.1 M Tris-HCl, 0.1% Tween 20, 2 mM levamisole) at room temperature for 5 min. The alkaline phosphatase chromogen reaction was performed using nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (DAKO) as the substrate at room temperature for 20 h. The sections were then washed with distilled water, treated with xylene, and mounted in mounting medium.

Immunohistochemistry—A polyclonal rabbit anti-Helt antibody was raised against glutathione S-transferase (GST)-Helt{Delta}bHLH (aa 76–240) and affinity-purified. 4% paraformaldehyde-fixed cryosections, as described above, were washed three times with PBS at room temperature for 5 min. The sections were blocked in 25% Block Ace (Dainipponseiyaku) at room temperature for 30 min and incubated with one of the following antibodies overnight at 4 °C: mouse anti-{beta}III-tubulin (TuJ1, Covance) or mouse anti-Lim1/2 (4F2, Developmental Studies Hybridoma Bank). The sections were washed three times with 0.05% Tween 20 in PBS (1x PBS-T) and then incubated in one of the following secondary antibodies: fluorescein- or Cy3-conjugated anti-rabbit or anti-mouse IgG (Jackson). For immunological reactions, the sections were incubated at room temperature for 1 h, washed three times with 1x PBS-T, and mounted in mounting medium.

BrdUrd (Sigma) was injected intraperitoneally into pregnant mice (50 µg/g of body weight) 2 h before isolation of embryos at E12.5. Embryos were harvested and analyzed for incorporation of BrdUrd using anti-BrdUrd antibodies (Roche Applied Science) in combination with anti-Helt antibodies.

Immunofluorescence analysis of the transfected NS20Y cells was performed as described (34). Nuclei were counterstained with TO-PRO-3 (Molecular Probes).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of Helt as a Novel bHLH-O Transcription Factor—To identify transcription factors regulating neuronal differentiation in the developing brain, we performed subtractive PCR using total RNA derived from E10.5 ventral and dorsal neural tubes as the tester and driver, respectively. One of the genes isolated, which encoded a novel protein, was confirmed to be specifically expressed in the ventral region of the E10.5 brain by RT-PCR (data not shown). To obtain the full-length cDNA, a mouse E12.5 brain cDNA library was screened. Four clones were obtained, and the longest clone, 1548 bp (hereafter called Helt: Hey-like transcriptional repressor), had a 240-amino-acid-long open reading frame. Computer data base searches revealed that Helt had a bHLH domain and an Orange domain, which are characteristic motifs among the bHLH-O family transcriptional repressors (Fig. 1A). To determine the homology of Helt to members of the bHLH-O families, protein alignment and phylogenetic analysis were performed (Fig. 1B and data not shown).



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  		FIG. 1.
Structure and phylogenetic analysis of Helt. A, schematic representation of the domain structure of Helt. The bHLH and Orange domains are indicated. The amino acid identities between Helt, mouse Hey1, and Drosophila Hesr1 are shown. B, phylogenetic analysis of bHLH-O proteins. Full-length amino acid sequences obtained from GenBankTM were used for the analysis. The phylogenetic analysis was performed using the ClustalW method. C, amino acid alignment of the basic regions of Helt and other bHLH-O proteins. Arrowheads indicate the positions of conserved proline and glycine residues. D, schematic representation of the Helt construct used in this study. WT, wild type.

 
The bHLH domain of Helt shows significant similarity to those of the Hes and Hey families. A characteristic feature of the Hes family is conservation of a WRPW motif at the C terminus, which is required for the recruitment of the TLE/Groucho corepressor, and a proline residue in the basic region, which is thought to be involved in the DNA binding specificity. Neither of these conserved motifs was present in the Helt sequence. Together with the higher homology to the Hey family than to the Hes family over the whole structure (Fig. 1B), Helt is distinct from the Hes family members. The mouse Hey family members are highly homologous, sharing 89–95% and 60–79% identities in the bHLH and Orange domains, respectively. There were only 55–56% and 25–26% identities for the same domains between Helt and the mouse Hey family members (Fig. 1A). On the other hand, Drosophila Hesr1 is more homologous to the mouse Hey family than Helt, sharing 78–82% and 55–72% identities in the same regions. The N-terminal half of the basic region is highly conserved among the Hey family and Drosophila Hesr1, whereas the same region of Helt is divergent (Fig. 1C). In addition, a glycine residue in the basic region is conserved between the mouse Hey family and Drosophila Hesr1 instead of a proline residue in the Hes family. However, this residue is replaced by a lysine residue in Helt. Thus, the mouse Hey family and Drosophila Hesr1 are classified into the evolutionally conserved Hey subfamily, but Helt is more distantly related. Furthermore, the divergence of the N-terminal half of the basic region suggests different target specificity and thus distinct functions for Helt when compared with the Hes and Hey families.

Helt Can Homodimerize and Heterodimerize with Other bHLH-O Repressors—In general, the bHLH transcription factors need to dimerize to bind to DNA. Thus, we first examined the dimerization property of Helt. FLAG-tagged Helt and HA-tagged Helt were transiently expressed in 293E cells and immunoprecipitated with an anti-FLAG antibody. As shown in Fig. 2A, HA-Helt was coimmunoprecipitated with FLAG-Helt, indicating that Helt is capable of homodimerizing in intact cells.



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  		FIG. 2.
Helt homodimerizes, and heterodimerizes with other bHLH-O proteins through the bHLH and Orange domains. A, dimerization properties of Helt. 293E cells were transiently transfected with an expression vector for each tagged protein as indicated. Cell lysates were immunoprecipitated (IP) with an anti-FLAG antibody. Immunoprecipitates and cell lysates were immunoblotted using an anti-FLAG or anti-HA antibody as indicated. WB, Western blotting; WT, wild type. B–E, analysis of the domains required for dimerization. Deletion mutants of Helt were transiently expressed in 293E cells, and immunoprecipitation experiments were carried out as described in panel A. F, Helt and Hey2 form a ternary complex through the Orange domain. Immunoprecipitation experiments were carried out as described above using anti-FLAG and anti-HA antibodies as indicated.

 
Since some bHLH-O factors are known to heterodimerize with other bHLH-O proteins, we examined whether Helt could form heterodimers. We chose Hes1, Hes5, and Hey2 as representatives of the Hairy, E(spl), and Hey subfamilies, respectively. HA-Helt was efficiently coimmunoprecipitated with FLAG-Hes5 and FLAG-Hey2, indicating that Helt was capable of heterodimerizing with Hes5 and Hey2 (Fig. 2A). In contrast, no interaction between FLAG-Hes1 and HA-Helt was observed. Thus, Helt dimerizes with selected members of the bHLH-O proteins. It has been reported that Hes1 can bind to the activator bHLH protein E47 in vitro (5), but we did not detect any interaction between Helt and E47 using an immunoprecipitation assay (data not shown).

Next, we analyzed the domain required for dimerization. The bHLH transcription factors are thought to dimerize through the HLH domain. However, a mutant lacking the bHLH domain (Helt{Delta}H) could still interact efficiently with full-length Helt (Fig. 2B). The C-terminal region was dispensable for homodimerization. In addition, a Helt mutant containing only the Orange domain (Helt-O) was capable of binding to full-length Helt, suggesting that the Orange domain was involved in the homodimerization. On the other hand, a mutant lacking the Orange domain (Helt{Delta}O) could still homodimerize, although the interaction was weaker than that of full-length Helt. These results strongly suggest that Helt homodimerizes through two distinct domains, the bHLH and Orange domains.

bHLH domains have been shown to interact with each other. To determine whether the Orange domains also interact with each other, we performed a set of immunoprecipitation experiments using a Helt mutant lacking the bHLH domain. As shown in Fig. 2C, HA-Helt{Delta}H was coimmunoprecipitated with FLAG-Helt{Delta}H but not with FLAG-Helt{Delta}O. Thus, the Orange domains can interact with each other but not with the bHLH and C-terminal regions. Furthermore, the bHLH domain is not necessary for the interaction between the Orange domains.

Next, we determined whether the bHLH and Orange domains were also involved in heterodimerization. As shown in Fig. 2D, heterodimerization between Helt and Hey2 required the bHLH domain of Helt, but the Orange domain was dispensable, suggesting that Helt/Hey2 heterodimers are only formed through the bHLH domain. In contrast, the Orange domain, but not the bHLH domain, was required for heterodimerization between Helt and Hes5 (Fig. 2E). Taken together, these results suggest that both the bHLH domain and the Orange domain of Helt are independently involved in the selection of dimerization partners.

The interaction between Helt and Hey2 did not require the Helt Orange domain, suggesting the possibility that the Orange domains do not interact with each other in this heterodimer. If this was the case, the Orange domain of Helt in the Helt/Hey2 heterodimer could interact with another molecule to form a ternary complex. To test this possibility, we performed immunoprecipitation experiments using three tagged proteins (Fig. 2F). As mentioned above, Helt{Delta}H could not interact with Hey2. When Myc-tagged full-length Helt was co-expressed, HA-Hey2 was coimmunoprecipitated with FLAG-Helt{Delta}H. Thus, in this case, the full-length Helt acted as a bridging molecule to form a ternary complex. To examine which domain was required for the bridging activity, deletion mutants were used as the bridging molecules. Myc-Helt{Delta}C mediated the interaction between FLAG-Helt{Delta}H and HA-Hey2, similar to full-length Helt. In contrast, Myc-Helt{Delta}O could not act as a bridging molecule, although this mutant could interact with Hey2 (Fig. 2D), indicating that the Orange domain was necessary for the bridging activity. Thus, the Orange domain of Myc-Helt in Myc-Helt/HA-Hey2 heterodimers appeared to mediate interaction with FLAG-Helt{Delta}H. These results suggest that the Orange domain may play a role in the multimerization of bHLH-O proteins.

Helt Homodimers Bind to E Box Elements—The bHLH transcription factors have been shown to bind to DNA through the basic region (10). Helt contains a basic residue stretch immediately N-terminal to the HLH domain, as found in other bHLH-O repressors, prompting us to examine whether Helt could bind to DNA. We performed EMSA using various bHLH-binding E box elements (classes A, B, C1) (7) as probes. As shown in Fig. 3A, Helt did not bind to E box class A, a commonly recognized sequence for bHLH activators. In contrast, binding of FLAG-Helt to E box class B and C1 sequences, which are typical recognition sequences for bHLH-O repressors, was observed. The binding was competed out by a 300-fold excess of the wild type cold probe but not by the same amount of the mutant cold probe, indicating that this binding was specific for the E box elements. Since a crude cell lysate containing FLAG-Helt was used for the EMSA experiments, we could not determine whether Helt bound to DNA as a homodimer, or as a heterodimer with another factor expressed endogenously in 293E cells. To distinguish between these possibilities, we constructed a Helt mutant lacking the C-terminal region that could still bind to DNA (Helt{Delta}C2). The shifted band containing Helt{Delta}C2 migrated faster than that containing full-length Helt (Fig. 3B). When full-length Helt and Helt{Delta}C2 were mixed and used for EMSA, intermediate-sized bands were observed, clearly demonstrating that Helt bound to the E box elements as a homodimer.



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  		FIG. 3.
Helt binds to the E box elements as a homodimer. A, Helt binds to the typical E box elements. EMSA was carried out using cell lysates from Helt-expressing 293E cells and labeled oligonucleotides containing E box elements. The sequences of the E box probes were derived from those described previously (7). Cold competitors at 300-fold excess or an anti-FLAG monoclonal antibody were added as indicated. WT, wild type. B, Helt binds to DNA as a homodimer. EMSA was performed using cell lysates from 293E cells expressing full-length or {Delta}C2 Helt. The E box C1 sequence was used as a probe. C, Helt-BM is capable of homodimerization. Immunoprecipitation (IP) experiments were performed as described in the legend for Fig. 2. WB, Western blotting. D, Helt cannot bind to DNA. EMSA was performed as described above using the E box C1 sequence as a probe. Expression of Helt-BM was confirmed by Western blotting using an anti-FLAG antibody.

 
To determine the requirement for the basic region for DNA binding, we mutated three amino acids, KRR, in the basic region of Helt, which are highly conserved between bHLH-O family members (Fig. 1C), to alanines (Helt-BM). This mutant could still form homodimers, as revealed by a coimmunoprecipitation assay (Fig. 3C). To test whether Helt-BM had DNA binding activity, EMSA was performed using E box class C1 as a probe. As shown in Fig. 3D, no specific binding of Helt-BM was observed, although the protein expression was confirmed by Western blotting. Taken together, these results indicate that Helt binds to the E box elements through the basic region.

Helt Is a Transcriptional Repressor—Given the fact that Helt binds to DNA as a homodimer, we next asked whether Helt acted as a positive or negative regulator of transcription. To determine whether Helt had an intrinsic transcriptional repression domain, we constructed a Helt fusion protein containing the GAL4 DNA-binding domain. This chimeric protein was expressed in a neuroblastoma cell line, NS20Y, with a reporter gene containing GAL4-binding sites located upstream of the SV40 promoter and luciferase gene (UAS-SV40-luc). As shown in Fig. 4A, GAL4-Helt strongly repressed transcription from the reporter. When a reporter without GAL4-binding sites was used, transcriptional repression was not observed (data not shown). Thus, GAL4-Helt showed transcriptional repression activity only when the protein was tethered to the promoter through the GAL4 DNA-binding domain, indicating that Helt has an intrinsic repression domain.



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  		FIG. 4.
Helt is a transcriptional repressor. A, NS20Y cells were transfected with UAS-SV40-luc (0.2 µg) and the indicated expression vectors for GAL4-fusion proteins (0.2 µg). The results show the fold repression of the luciferase activity from the reporter with the GAL4 DNA-binding domain alone. The means ± S.D. of four independent experiments are shown. Expression of the GAL4-fusion proteins was confirmed by Western blotting using an anti-GAL4 antibody. WT, wild type. B, Helt represses transcription through the E box. NS20Y cells were transfected with a reporter plasmid containing multiple E boxes (0.2 µg) and the indicated expression vectors (0.2 µg). The results show the fold repression of the luciferase activity from the reporter alone. The means ± S.D. of four independent experiments are shown. C, nuclear localization of Helt-WT and Helt-BM. NS20Y cells were transiently transfected with the expression vector for FLAG-Helt or FLAG-Helt-BM and immunostained with an anti-FLAG antibody. The staining was visualized with a fluorescein isothiocyanate-conjugated secondary antibody, and the nuclei were counterstained with TO-PRO-3.

 
To compare the repression mechanism of Helt to that of other bHLH-O repressors, we mapped the repression domain of Helt. In the case of the Hes family, the C-terminal WRPW motif recruits the TLE/Groucho corepressor and is required for the transcriptional repression (15). On the other hand, the Hey family lacks a canonical WRPW motif, and the bHLH domain can recruit the corepressor components, mSin3A and N-CoR (7). As mentioned above, Helt did not possess the WRPW motif and could not interact with TLE/Groucho (data not shown). Deletion of the bHLH domain had little effect on the repression activity, in contrast to the case of the Hey family (7). On the other hand, deletion of the Orange domain resulted in complete loss of the repression activity, and a deletion mutant lacking the C-terminal region showed weak repression activity. Confirmation of the protein expression by Western blotting indicated that both the Orange domain and the C-terminal region were required for the transcriptional repression activity. Using coimmunoprecipitation experiments, we did not detect any interactions between Helt and any corepressors, including TLE/Groucho, N-CoR, mSin3A, and CtBP (data not shown), suggesting that Helt represses transcription through a mechanism distinct from those of the Hes and Hey families.

The findings that Helt homodimers bind to E box elements and that Helt has an intrinsic repression domain led us to examine whether Helt represses transcription through binding to the E box sites. Helt was expressed in NS20Y cells with a reporter gene containing multiple E box sequences located upstream of the SV40 promoter and a luciferase gene. As expected, Helt did not affect the transcriptional activity of the reporter gene without the E box (Fig. 4B). Similarly, Helt did not repress the E box class A-containing reporter. In contrast, the transcriptional activities of the E box class B- and class C1-containing reporters were efficiently repressed. Similar results were obtained using C3H10T1/2 cells (data not shown). The DNA binding activity of Helt was required for this effect since Helt-BM, which cannot bind to DNA but is still localized in the nucleus, did not repress the promoter activity (Fig. 4, B and C). Thus, Helt is a transcriptional repressor, which can repress transcription through binding to E box elements. This idea is further supported by the observation that Helt was localized in nuclear dot-like structures in NS20Y cells (Fig. 4C) since many transcriptional repressors and corepressors have been reported to show a similar localization pattern (36–39).

Preferred Recognition Sequence for Helt and Hes1—The observation that Helt homodimers can bind to E box class B and class C1, which are also recognized by Hes1 and Hey2 (7), prompted us to examine whether Helt preferentially recognizes the same target sequences as the other bHLH-O proteins. We determined the preferred recognition sequences for Helt and Hes1 by the CASTing procedure. Double-stranded random oligonucleotides were incubated with FLAG-Helt or FLAG-Hes1 expressed in 293E cells followed by affinity purification with an anti-FLAG antibody. Bound oligonucleotides were then amplified by PCR. After four rounds of selection, the obtained oligonucleotides were cloned, and about 160 clones were sequenced. A summary of the results and the aligned sequences are shown in Fig. 5.



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  		FIG. 5.
Preferred recognition sequences for Helt and Hes1. CASTing experiments were performed using FLAG-Helt or FLAG-Hes1 proteins expressed in 293E cells (see "Experimental Procedures"). A, a summary of the CASTing experiments. The percentages of the obtained sequences containing the E box or N box are shown. B, the nucleotides present at each position of the sequences obtained by CASTing. Nucleotides selected with a frequency of 40% or higher are highlighted.

 
Most of the sequences obtained by CASTing using FLAG-Helt contained E box class B (37%), E box class C1 (35%), or the N box (15%)(Fig. 5A). Thus, at least under these conditions, Helt preferentially bound to the typical sequences recognized by bHLH repressors. In contrast, all the FLAG-Hes1-bound sequences contained the E box class B (24%) or E box class C1 (87%), but not the N box, suggesting a difference in the preference for the core sequence between the bHLH-O repressors.

The sequences containing E box class C1 obtained by CASTing with FLAG-Helt can be combined to give a consensus sequence (5'-CGCACGCGAC-3', underlining indicates E box core sequence) (Fig. 5B). Similarly, alignment of the sequence containing the N box gave a consensus sequence (5'-CGCACGAGAC-3') (Fig. 5B). The flanking sequences around these E box class C1 and N box consensus sequences were identical. This strong selection of the bases flanking the core E box or N box suggests their requirement for target recognition by Helt. However, the flanking sequences of the consensus sequence containing E box class B were quite different and rather variable (Fig. 5B). Thus, in the case of E box class B, either Helt recognizes only the core E box sequence (5'-CACGTG-3') or many recognizable flanking sequences exist.

The sequences obtained using FLAG-Hes1 were aligned to give consensus sequences 5'-GGCACGCGCC-3' and 5'-GGCACGTGGC-3' for E box class C1 and class B, respectively (Fig. 5B). In contrast to the case of Helt, the flanking residues of Hes1 class B consensus sequences were highly selected. Together with the fact that the N box-containing sequence was not obtained by CASTing with FLAG-Hes1, Hes1 may be able to recognize highly restricted target sequences, when compared with Helt.

To confirm the binding of Helt and Hes1 to the sequences obtained from CASTing and the involvement of the flanking sequences around the core E box in the recognition, EMSA was performed using representative E box C1 sequences as probes. Four sequences from CASTing with Helt (5'-CGCACGCGAC-3', 5'-GGCACGCGAC-3', 5'-CTCACGCGAC-3' and 5'-GTCACGCGAC-3', designated C1-1 to C1-4, respectively), one sequence from CASTing with Hes1 (5'-GGCACGCGCC-3', C1-5), and one sequence that was not obtained from CASTing with either Helt or Hes1 (5'-AACACGCGTT-3', C1-6) were chosen (Fig. 6A). As shown in Fig. 6B, FLAG-Helt bound strongly to C1-1, C1-2, C1-3, and C1-4, confirming that FLAG-Helt could bind to the sequences obtained by CASTing. On the other hand, FLAG-Helt bound to C1-5, which was obtained from CASTing using FLAG-Hes1 but not that using FLAG-Helt, with relatively lower affinity. Furthermore, binding of FLAG-Helt to C1-6, which was not obtained by CASTing, was not observed, indicating that residues flanking the E box core are important for target recognition by Helt. Similarly, FLAG-Hes1 could bind to C1-2 and C1-5, both of which were obtained from CASTing with FLAG-Hes1, but not to the other E box sequences (Fig. 6C). These results clearly demonstrate that Helt and Hes1 recognize E box elements flanked by distinct sequences.



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  		FIG. 6.
Sequences around the E box core are important for recognition by Helt and Hes1. A, the E box sequences used for the EMSA and reporter assays. The representative E box sequences and a summary of the results of CASTing and EMSA are shown. B and C, binding to the preferred recognition sequences. EMSA was performed as described in the legend for Fig. 4 using FLAG-Helt (B) or FLAG-Hes1 (C) and E box C1-1 to C1-6 as probes. ChIP, chromatin immunoprecipitation. D, Helt represses transcription through binding to preferred E boxes. NS20Y cells were transfected with a reporter plasmid containing multiple E boxes (0.2 µg) and the indicated expression vectors (0.2 µg). The results show the fold repression of the luciferase activity from the reporter alone. The means ± S.D. of four independent experiments are shown.

 
Next, we examined whether the preferred sequences obtained by CASTing could be recognized in intact cells and mediate transcriptional repression by Helt. Reporter plasmids were constructed by insertion of multimerized C1-1, C1-2, C1-5, or C1-6 upstream of the SV40 promoter and transfected into NS20Y cells with a Helt expression plasmid. As shown in Fig. 6D, Helt efficiently repressed transcription from the C1-1- or C1-2-containing reporter genes. The C1-5-containing reporter gene, whose element was bound by Helt with lower affinity, was also repressed. In contrast, transcriptional activity of the C1-6-containing reporter gene was not affected. Similar results were obtained using C3H10T1/2 cells (data not shown). Thus, the binding specificity and ability to repress transcription through the optimal recognition sequences were confirmed in vivo. Taken together, these results strongly suggest that Helt and Hes1 regulate transcription through binding to different elements.

Helt Negatively Regulates Its Own Promoter—Given that many bHLH transcription factors regulate their own expression (17, 40), we tested this for the case of Helt. We cloned 881-bp Helt genomic fragments from positions -934 to -53 (first ATG codon as +1) into a promoter-less luciferase vector (-934-luc). When transfected into NS20Y cells, this reporter construct exhibited basal promoter activity (Fig. 7A). Transcriptional activity was significantly reduced by cotransfection of Helt. Thus, Helt can repress transcription from not only the E box-containing artificial promoter but also the native promoter. In addition, FLAG-Hes1 could not repress the transcriptional activity of the -934-luc reporter, suggesting that Helt specifically represses its own promoter. Furthermore, DNA binding of Helt was required for the repression since the DNA binding-deficient Helt-BM did not affect the promoter activity, suggesting that Helt directly bound to the promoter to repress it. To test this, we performed chromatin immunoprecipitation analysis. As shown in Fig. 7B, endogenous Helt promoter fragment was efficiently coimmunoprecipitated with FLAG-Helt. In contrast, FLAG-Helt-BM could not coimmunoprecipitate the promoter fragment, suggesting that Helt directly binds to its own promoter in intact cells. The binding of Helt to its promoter was specific since an unrelated genomic region (HNF3{beta} genome) was not coimmunoprecipitated with FLAG-Helt. Taken together, these results strongly suggest that Helt directly binds to its own promoter to repress it.



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  		FIG. 7.
Helt negatively regulates its own expression. A, Helt specifically represses its own promoter activity. NS20Y cells were transfected with a reporter plasmid containing the Helt promoter (0.2 µg) and the indicated expression vectors (0.2 µg). The relative luciferase activity when compared with that for the reporter plasmid alone was obtained. The means ± S.D. of four independent experiments are shown. WT, wild type. B, Helt binds to its own promoter in intact cells. NS20Y cells were transfected with the indicated expression vector. Cross-linked protein/DNA complex was immunoprecipitated with an anti-FLAG antibody, and the immunoprecipitated DNA was subjected to PCR analysis for the endogenous Helt promoter. genome indicates a control PCR reaction using NS20Y genomic DNA as a template. HNF3{beta} genomic fragment was used as an unrelated control. C, multiple elements are involved in the autoregulation of the Helt promoter. A schematic representation of the various E box and N box elements in the Helt promoter is shown. Deletion and point mutants of the reporter construct are indicated. NS20Y cells were transfected with the indicated reporter plasmid (0.2 µg) and indicated expression vectors (0.2 µg). The results show the fold repression of the luciferase activity from the reporter alone. The means ± S.D. of four independent experiments are shown.

 
There are two E box class C1 sequences, two E box class B sequences, and two N boxes in the Helt promoter, prompting us to examine which elements were involved in the repression by Helt. We constructed 5' deletion mutants and point mutants lacking the candidate Helt-binding elements as shown in Fig. 7C. Deletion of the 5' region including E box class C1, the flanking sequences of which match well with the Helt consensus-binding sequence, and the N box (-780-luc) abrogated the response to Helt, whereas 3' deletion including the second N box (-934{Delta}27-luc) had no effect. Similarly, mutation in the two E box class B (-934M-luc) sites abrogated the response. These results suggest that multiple elements including the E box and the N box are involved in the autoregulation by Helt.

Helt Is Expressed in Neural Progenitors in the Developing Central Nervous System—Next, we investigated the tissue distribution of Helt by RT-PCR analysis (Fig. 8A). Helt mRNA was detected in the embryonic brain, but not in the adult brain, suggesting a role in the regulation of central nervous system development. In other adult tissues, Helt mRNA was detected in the testes at high levels. Lower expression was also observed in the heart. Taken together, Helt is expressed in a highly restricted manner in adult mouse tissues.



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  		FIG. 8.
Expression of Helt in adult mouse tissues and the developing brain. A, RT-PCR analysis of Helt expression in adult mouse tissues. Total RNA from E12.5 or adult tissues was subjected to RT-PCR analysis for Helt and G3PDH (control). The amplified products were electrophoresed in 2% agarose gels and stained with ethidium bromide. B–E, in situ hybridization analysis of Helt expression in a E12.5 mouse embryo. A sagittal section (B and C), para-sagittal section (D), and coronal section (E) of the mesencephalon hybridized with a DIG-labeled Helt antisense riboprobe is shown. F, immunohistochemical analysis of Helt. A coronal section of E12.5 mesencephalon stained using a polyclonal anti-Helt antibody is shown. G–I, Helt is expressed in mitotically active neuronal progenitors. Coronal sections of E12.5 mesencephalon stained with anti-Helt (G–I, green), anti-{beta}III-tubulin (G, red), anti-Lim1/2 (H, red) or anti-BrdUrd (I, red) antibodies. Arrowheads indicate double-positive cells.

 
We focused on the embryonic expression and performed an in situ hybridization analysis. In E12.5 embryos, Helt expression was restricted to the neural tube (Fig. 8B). Strong expression of Helt was observed in the mesencephalon, diencephalon, and zona limitans intrathalamica regions, and a weak signal was detected in the telencephalon. Helt mRNA was not detected in the caudal neural tube posterior to the midbrain-hindbrain border (Fig. 8, C and D). As revealed in a coronal section of the E12.5 mesencephalon, the Helt signal was only detected in the ventricular layer of the neural tube (Fig. 8E). Many Helt-expressing cells were observed in the ventral region. Some dorsal cells also expressed Helt in this stage. Protein expression in the nucleus was detected using an antibody directed against Helt in a similar pattern to the in situ hybridization (Fig. 8F), confirming the specificity of the signals in both in situ hybridization and immunohistochemistry.

Next, we examined the expression profile of Helt in the course of neuronal differentiation. Helt expression was compared with the expression of {beta}III-tubulin and Lim1 as a postmitotic neuronal marker (41, 42). As shown in Fig. 8G, Helt was not coexpressed with {beta}III-tubulin. Similarly, most of the Helt-expressing cells did not coexpress Lim1 (Fig. 8H), indicating that Helt was mainly expressed in undifferentiated progenitors. However, some cells coexpressing Helt and Lim1 were observed, suggesting that Helt expression persisted after the final cell division and was down-regulated before neuronal differentiation. Expression of Helt in the neural progenitors was confirmed by BrdUrd-labeling experiments. As shown in Fig. 8I, Helt-expressing cells incorporated pulse-labeled BrdUrd. Taken together, these results strongly suggest that Helt is mainly expressed in the mitotically active neural progenitors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Helt Is a Novel Member of the bHLH-O Repressor Family—We have identified a novel transcriptional repressor, Helt. Helt contains the bHLH and Orange domains, both of which are characteristic among the bHLH-O transcriptional repressors (10). The bHLH-O family is subdivided into four classes, the Hairy, E(spl), Hey, and Stra13 subfamilies, by overall homology and conservation of characteristic motifs. The bHLH domain of Helt is most similar to that of the Hey family members, but the Orange domain is highly divergent. The Orange domain of the Hey family is well conserved, even in the Drosophila homologue, indicating that Helt has a relatively distant relationship to the Hey family. Furthermore, a glycine residue in the basic region, which is conserved in the Hey family, is substituted to lysine in Helt, suggesting that Helt has a distinct DNA binding specificity and plays a different role in transcriptional regulation.

Most of the members of the bHLH-O factors have been reported to be transcriptional repressors. Consistent with this, Helt has an intrinsic transcriptional repression domain and is capable of repressing transcription from not only E box elements containing artificial promoters but also from its native promoter. Thus, Helt functions as a transcriptional repressor. However, each subfamily uses a different system to repress transcription. The Hes family members recruit the TLE/Groucho corepressor through the C-terminal WRPW motif, whereas the Hey family members recruit mSin3A and N-CoR through the bHLH domain (7, 14, 15). In the case of Helt, the WRPW motif is not present, and therefore, TLE/Groucho cannot bind to Helt (data not shown). The bHLH domain of Helt is not required for repression, as shown by the GAL4 fusion assay, and no interactions between Helt and mSin3A or N-CoR were detected in immunoprecipitation assays (data not shown), strongly suggesting that the mechanism of transcriptional repression by Helt is different from those of the other bHLH-O factors.

Dimerization Mechanism—In general, the HLH-containing transcription factors form homo- or heterodimers through the HLH domain (10). In agreement with this, the bHLH domain of Helt can form homodimers. It has been reported that the Orange domain of chick Hairy enhances homodimerization, and heterodimerization with chick Hey1 or Hey2 (13). These observations are consistent with our observations for Helt. However, a deletion mutant lacking the bHLH domain and a mutant containing only the Orange domain could still form dimers, indicating that the Orange domain of Helt functions as a dimerization domain independently of the bHLH domain. Interestingly, the Helt Orange domain is required for heterodimerization with Hes5 but is not involved in the interaction with Hey2. Conversely, the bHLH domain is required for the dimerization with Hey2 but not that with Hes5. Thus, Helt has two distinct dimerization domains, the bHLH and Orange domains, and these domains can function independently in the selection of a partner. In Drosophila Hairy and E(spl), domain-swapping experiments revealed that the Orange domain confers specificity among the members of the Hairy and E(spl) families (11). In this case, the Orange domain may be involved in the selection of a partner to form proper homo- or heterodimers to regulate the transcription of the target genes. Thus, in general, the Orange domain may act as a dimerization domain to select the partner.

Our observations suggest an alternative function of the Orange domain. The Orange domains may not interact with each other in Helt/Hey2 heterodimers since these heterodimers are only formed through their bHLH domains. Conversely, the bHLH domains may not interact with each other in the Helt/Hes5 heterodimers. In agreement with this, we could not detect any DNA binding activity of Helt/Hes5 heterodimers (data not shown), although we cannot rule out the possibility that the probe sequences used were not a match for the preferred recognition sequence of the heterodimer. If this is the case, interaction through the Orange domain may not be required for dimer formation to bind to DNA. Rather, the interaction between the Orange domains may be involved in ternary complex formation for synergistic transcriptional regulation. Consistent with this hypothesis, complexes containing at least three bHLH-O monomers formed through the Orange domain were detected. At present, it is not clear whether the multimerized bHLH-O protein complexes have two or more DNA-binding interfaces. Further studies are needed to define the function of the Orange domain in the complex formation and transcriptional regulation.

DNA Recognition by Helt—Helt contains a basic amino acid stretch immediately N-terminal to the HLH domain. Mutation in this region abolished the DNA binding activity, indicating that this region is a functional basic domain for DNA binding. The C-terminal half of the basic region is highly conserved among bHLH-O factors including Helt, suggesting that Helt recognizes a similar sequence to that bound by the other bHLH-O proteins. In fact, Helt can bind to E box class B, E box class C1, or the N box, which are the typical target elements recognized by bHLH-O transcriptional repressors. Do all members of the bHLH-O proteins recognize the identical target sequence? The N-terminal half of the basic regions are divergent among the bHLH-O factors. In particular, the proline residue conserved among the Hes family, which is thought to be involved in the target recognition specificity, is replaced by a lysine residue in Helt. This suggests distinct target specificity. Consistent with this, the preferred sequence of Helt is distinct from that of Hes1 around the core E box. Flanking sequences around the E box have been shown to be important for binding by E(spl) proteins in Drosophila, both in vitro and in vivo (43). Our present observations reveal the involvement of the flanking sequences around the E box core in the target specificity for vertebrate bHLH-O proteins. Thus, each member of the bHLH-O family may regulate the transcription of distinct target genes. The diversity of the N-terminal portion of the basic region seems to parallel the differences in their target sequences. The N-terminal region may decide the sequence specificity flanking the E box core.

The bHLH-O factors form not only homodimers but also heterodimers with other bHLH-O proteins. DNA binding of Helt/Hey2 heterodimers to the E box was not observed, at least under our experimental conditions, in EMSA (data not shown). This might indicate that Helt homodimers and Helt/Hey2 heterodimers recognize different sequences. Thus, the homodimers and heterodimers of bHLH-O proteins may regulate the expression of different sets of downstream target genes.

Helt Functions in Central Nervous System Development— Helt is expressed in some restricted tissues in adults and embryos. In embryos, in particular, Helt expression is highly restricted to the ventricular zone of the developing central nervous system. Helt is not expressed in the adult brain, suggesting that Helt is only expressed at the neurogenesis stage. Comparison with the expression of growth or differentiation markers revealed that Helt is mainly expressed in the mitotically active undifferentiated neuronal progenitors, suggesting the involvement of Helt in the regulation of neuronal differentiation. Among the bHLH-O family transcriptional repressors, Hes1, Hes5, and Stra13 have been reported to be expressed in the ventricular zone of the developing central nervous system (19, 20, 29). Hes1 and Hes5 act as downstream effectors of Notch signaling and repress neuronal differentiation in vitro and in vivo (19, 20). On the other hand, Stra13 is thought to act as a neuronal inducer since it promotes neuronal differentiation in P19 embryonal carcinoma cells (29). Does Helt regulate neurogenesis positively or negatively? Helt could not induce neuronal differentiation in P19 cells nor affect Mash1-induced neuronal differentiation in P19 cells (data not shown). Thus, at present, Helt function in the regulation of neuronal differentiation is unknown. The capability of Helt to heterodimerize with Hes5 suggests a possible role in the inhibition of Hes5 function since Hes6 inhibits Hes1 function to promote neurogenesis (22, 23, 24). Alternatively, Helt may inhibit glial differentiation since Hey2 regulates gliogenesis in the developing retina (44). Identification of the downstream target genes of Helt may help to elucidate the role of Helt in central nervous system development.

In contrast to the broad expression of bHLH-O factors mentioned above, Helt expression is restricted to specific brain regions. At least at E12.5, Helt expression was not observed in the caudal neural tube posterior to the midbrain-hindbrain border. In the anterior neural tube, Helt is strongly expressed in the ventral mesencephalon, dorsal diencephalon, and zona limitans intrathalamica. Thus, the function of Helt may not be in the general regulation of neurogenesis. Rather, Helt may repress the expression of regionally restricted factors, such as bHLH activators or the LIM homeobox to regulate the neuronal identity. Loss-of-function experiments by targeted disruption may reveal the role of Helt.

We have found that Helt dimerizes with Hes5 or Hey2. It has been reported that Hes5 is an indispensable Notch effector in neuronal differentiation (19). Hey2 is also induced by Notch signaling in many cell types (25, 26). These observations suggest the possibility that Helt expression is also regulated by Notch signaling. However, a constitutively active mutant of Notch did not induce the activity of a reporter construct containing the Helt promoter or the expression of the endogenous Helt gene in several cell lines (data not shown). Thus, Helt may not be a downstream effector for Notch signaling. If this is true, Helt/Hes5 or Helt/Hey2 heterodimer formation suggests a link between Notch signaling and other signals. Although Helt and Hes5 show overlapping expression patterns in the ventricular zone region of the developing central nervous system, further studies are needed to define the significance of the heterodimerization between Helt and other bHLH-O proteins.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondene should be addressed. Tel.: 81-75-315-7570; Fax: 81-75-325-5130; E-mail: y-ono{at}kan.gr.jp.

1 The abbreviations used are: bHLH, basic-helix-loop-helix; bHLH-O, bHLH-Orange; BrdUrd, bromodeoxyuridine; aa, amino acids; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase; HA, hemagglutinin; luc, luciferase; DIG, digoxigenin; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; E, embryonic day; TLE transducin-like enhancer of split. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Y. Takai (Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine) for constant support and fruitful discussions. We also thank Dr. T. Imai (KAN Research Institute Inc.) for helpful comments and encouragement. We thank Dr. H. Hirata and Dr. R. Kageyama (Institute for Virus Research, Kyoto University) for advice regarding the in situ hybridization experiments. The monoclonal anti-Lim1/2 antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institutes of Health, NICHD, and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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