Ventromedial Hypothalamic Nucleus-Specific Enhancer of Ad4BP/SF-1 Gene
Yuichi Shima,
Mohamad Zubair,
Satoru Ishihara,
Yuko Shinohara,
Sanae Oka,
Shioko Kimura,
Shiki Okamoto,
Yasuhiko Minokoshi,
Sachiyo Suita and
Ken-ichirou Morohashi
Division for Sex Differentiation (Y.S., M.Z., S.I., Y.S., S.Oka, K-i.M.), National Institute for Basic Biology, National Institutes of Natural Sciences, Myodaiji-cho, Okazaki 444-8787, Japan; Core Research for Evolutional Science and Technology (CREST) (Y.S., M.Z., S.I., Y.S., S.Oka, K.-i.M.), Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan; Department of Pediatric Surgery (Y.S., S.S.), Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; Laboratory of Metabolism (S.K.), National Institutes of Health, Bethesda, Maryland 20892; and Division of Endocrinology and Metabolism (S.Okam., Y.M.), Department of Developmental Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Myodaiji-cho, Okazaki 444-8585, Japan
Address all correspondence and requests for reprints to: Prof. Ken-ichirou Morohashi, PhD., Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Myodaiji-cho, Okazaki 444-8787, Japan. E-mail: moro{at}nibb.ac.jp.
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ABSTRACT
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Ad4BP/SF-1 [Ad4 binding protein/steroidogenic factor-1 (designated NR5A1)] is a transcription factor essential for animal reproduction. Based on the phenotypes observed in gene-disrupted mice, Ad4BP/SF-1 is thought to be involved in establishment of the hypothalamic-pituitary-gonadal axis. However, the mechanisms underlying tissue-specific expression of Ad4BP/SF-1 are largely unknown. Here, we investigated the cis-regulatory regions of the mouse Ad4BP/SF-1 gene by transgenic mouse assays, and identified a ventromedial hypothalamic nucleus (VMH)-specific enhancer. The enhancer localized in intron 6 is highly conserved between mouse, human, and chick. The enhancer has the potential to reproduce endogenous gene expression from the fetal ventromedial diencephalon to the adult VMH. The VMH enhancer was characterized by the presence of suppressive and activating elements. Mutation of the former element resulted in ectopic lacZ reporter gene expression in an area dorsal to the intrinsic expression domain and in the ventricular zone, whereas mutations in the latter containing ATTA motifs led to the disappearance of the reporter gene expression, suggesting the involvement of homeobox proteins. Using nuclear extracts prepared from the adult hypothalami, EMSAs identified specific protein binding to the activating elements but not to the suppressive element.
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INTRODUCTION
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Ad4BP/SF-1 [Ad4 BINDING PROTEIN (1)/steroidogenic factor-1 (2), officially designated NR5A1 (3)] belongs to an orphan nuclear receptor superfamily. The factor was initially identified as a steroidogenic tissue-specific transcription factor regulating steroidogenic P450 genes (4), and was shown to be expressed not only in steroidogenic tissues such as the adrenal cortex and gonads but also in the ventromedial hypothalamic nucleus (VMH), pituitary gonadotrope (5, 6, 7, 8, 9), and spleen (10). In addition to regulation of steroidogenic gene transcription, Ad4BP/SF-1 was shown to be essential for the development of these tissues (5, 6, 11, 12).
Studies involving physical or chemical ablation have shown that the VMH is the center of homeostatic behavioral responses, such as food intake, locomotion, and female sexual behavior (13, 14, 15). Indeed, VMH-lesioned mice exhibited hyperphagia and severe obesity (16). To understand the physiological functions of the VMH, genes showing VMH-specific expression have been studied, and recently, brain-derived neurotrophic factor expressed specifically in the VMH was found to control energy intake and expenditure downstream of the melanocortin-4 receptor (17). Induction of late-onset obesity, decrease of activity, and markedly increased leptin levels were observed in Ad4BP/SF-1 gene-disrupted mice of which adrenal function was rescued by adrenal transplantation (18). These findings suggest the involvement of Ad4BP/SF-1 in VMH functions such as regulation of appetite and body weight. With respect to female sexual behavior, the VMH neurons are known to project predominantly to the periaqueductal gray and bed nucleus of the terminal striatum, and thereby to promote female sexual behavior, represented by lordosis (19, 20, 21). In addition to the above functional studies, elucidation of the molecular mechanisms underlying establishment of the functional VMH is important for clarifying physiological functions of the tissue. In this regard, it has been crucial to establish that Ad4BP/SF-1 is required for terminal differentiation and normal projections of the VMH neurons (22). However, transcriptional targets of Ad4BP/SF-1 in the VMH are still unknown. Moreover, considering the functions of Ad4BP/SF-1 in the VMH, it is important to elucidate the mechanism driving VMH-specific expression of the gene.
In the present study, we investigated cis-regulatory regions of Ad4BP/SF-1 by transgenic mouse assays and identified a VMH-specific enhancer. This enhancer contains elements for suppressive and activating regulation, and interestingly, the activating elements contain ATTA motifs. Mutations in all the activating elements dismissed the VMH enhancer function completely. Meanwhile, mutation of the suppressive element expanded the reporter gene expression ectopically. These observations strongly suggest that both positive and negative regulation is required cooperatively for VMH-specific expression of the Ad4BP/SF-1 gene.
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RESULTS
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Localization of VMH-Specific Enhancer by Transgenic Mouse Assays
To identify tissue-specific enhancers of the mouse Ad4BP/SF-1 gene by transgenic mouse assay, we constructed a basal cosmid vector containing the 5' upstream region of the gene. When constructing the basal vector, it was considered that the Ad4BP/SF-1 gene has multiple noncoding first exons (Ia, Io, Ig, and Ix) of which sequences are conserved among mouse, rat, and human (23, 24). Because these noncoding first exons are used preferentially among tissues, the vector was constructed to contain all first exons. As shown in Fig. 1A
, all first exons and the noncoding region of the second exon (6-kb DNA fragment) were placed 5' upstream of a bacterial lacZ reporter gene. The simian virus 40 (SV40) polyadenylation [poly (A)] signal was placed 3' downstream of the reporter gene (Ad4BP-lacZ cassette in Fig. 1A
).

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Fig. 1. Identification of the VMH-Specific Enhancer of the Mouse Ad4BP/SF-1 Gene
A, The Ad4BP/SF-1 gene (Gene ID: 26423) consists of seven exons (boxes). Among the noncoding multiple first exons, exon Io is indicated by an open box because exon Io is not active in the mouse gene. Two other genes, Nr6a1 (Gene ID: 14536) and Gpr144 (Gene ID: 381361), are localized 5' upstream and 3' downstream of the Ad4BP/SF-1 gene, respectively. Positions and structures of these genes were obtained from the National Center for Biotechnology Information database (Entrez Gene). A 6-kb fragment of the Ad4BP/SF-1 promoter region from the initiation methionine in exon 2 was used to generate the basal vector by ligation to the bacterial lacZ gene followed by SV40 poly (A) signal (Ad4BP-lacZ cassette). Various DNA fragments were inserted downstream of Ad4BP-lacZ, and subjected to transgenic mouse assays. Among the cosmid clones covering the Ad4BP/SF-1 locus, cIA3 induced lacZ expression in fetal adrenal primordia, ventral diencephalon (vd), and Rathkes pouch (Rp), whereas cGcnf5 induced lacZ expression only in adrenal primordium. cIVC6 induced no lacZ expression. B, A 21-kb fragment in cIA3 was released by EcoRI digestion [E1-E2 (21.0)]. Sequentially truncated constructs from the 21-kb fragment were used for transgenic mouse assays. E14.5 fetuses were evaluated for lacZ expression. C, Because the VMH-specific enhancer was localized in the 3.2-kb BamHI-BamHI fragment [B1-B2 (3.2)], the 3.2-kb fragment [B1-B2 (3.2)] was used for further truncation. E14.5 fetuses were also used for evaluation. The VMH enhancer was assumed to be in the region indicated by an oval. Restriction enzymes are abbreviated as follows: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; N, NotI; Sc, ScaI; Sn, SnaBI. D, A representative lacZ expression in vd induced by pAd4BP-lacZ-[B1-B2 (3.2)]. E and F, A representative lacZ expression in the ventral part of Rp induced by pAd4BP-lacZ-[B2-B3 (5.8)].
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Partially digested mouse genomic DNA fragments were inserted downstream of the SV40 poly (A) signal of the basal cosmid vector (cAd4BP-lacZ). Clones carrying DNA fragments between genes localized 5' upstream (Nr6a1, Gene ID: 14536) and 3' downstream (Gpr144, Gene ID: 381361) of the Ad4BP/SF-1 were isolated and subjected directly to transgenic mouse assays. One clone, cIA3, induced lacZ expression in the fetal adrenal and ventral diencephalon (vd, future VMH) at embryonic day (E) 12.5, whereas another clone, cGcnf5, induced lacZ expression only in the adrenal (Zubair, M., S. Ishihara, S. Oka, K. Okamura, and K.-i. Morohashi, manuscript in preparation). cIVC6 did not induce any lacZ expression. At E14.5 or later, lacZ expression driven by cIA3 was observed at the ventral part of Rathkes pouch (Rp, pituitary anlage). Because a 21-kb fragment released by EcoRI digestion from cIA3 ([E1-E2 (21.0)] in Fig. 1B
) was confirmed to reproduce lacZ expression of the original cIA3 in E14.5 fetuses, the fragment was truncated sequentially to localize the region necessary for the vd-specific expression. As summarized in Fig. 1B
, Ad4BP-lacZ-[E1-Bg(14.0)] containing a 14.0-kb fragment of the 5' side of the 21-kb fragment was still able to induce lacZ expression at both vd and Rp, whereas Ad4BP-lacZ-[E1-B2(10.2)] induced lacZ expression in vd but not in Rp. Further deletion, Ad4BP-lacZ-[E1-B1(7.0)], resulted in disappearance of lacZ expression from both vd and Rp. Consistent with this, Ad4BP-lacZ-[B2-E2(10.8)] induced the reporter gene expression in Rp, whereas Ad4BP-lacZ-[B3-E2(5.0)] failed to induce it in either vd or Rp. Thus, it was highly likely that the vd-specific enhancer (VMH enhancer) and Rp-specific enhancer were located in the 3.2-kb [B1-B2(3.2)] and 5.8-kb [B2-B3(5.8)] BamHI-BamHI fragments, respectively. In fact, transgenic studies with pAd4BP-lacZ-[B1-B2(3.2)] and pAd4BP-lacZ-[B2-B3(5.8)] clearly showed their distinct abilities to induce the expression of reporter gene at vd and Rp, respectively (Fig. 1
, DF). To localize precisely the vd-specific enhancer, this 3.2-kb fragment was further truncated. As shown in Fig. 1C
, Ad4BP-lacZ[B1-N2(1.4)], carrying a 1.4-kb BamHI-NotI fragment, did not induce lacZ expression in vd, whereas Ad4BP-lacZ[N2-B2(1.9)], Ad4BP-lacZ[K-B2(1.4)] and Ad4BP-lacZ[K-Sc(0.9)] were able to drive the reporter gene expression in vd. A smaller KpnI-HindIII fragment (Ad4BP-lacZ[K-H (0.5)]) failed to reproduce lacZ expression. Thus, we tentatively concluded that the 0.9-kb KpnI-ScaI fragment has potential to drive vd-specific expression.
Because tissue-specific enhancers are frequently conserved among animal species, the nucleotide sequence of the mouse genomic region capable of driving vd-specific (VMH-specific) expression was compared with the corresponding region of the human gene. As shown in Fig. 2
, the nucleotide sequence of the enhancer region was highly conserved between the two animals. Interestingly, homology of the enhancer region appeared to be higher than that of the exons indicated. The conserved sequence was found to a lesser extent even in the chicken Ad4BP/SF-1 gene (data not shown).

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Fig. 2. Homology Plot of Ad4BP/SF-1 Genes (Exon 4-Exon 7) in Mouse and Human
Genomic nucleotide sequences from exon 4 to exon 7 of the Ad4BP/SF-1 genes were compared between mouse and human. The VMH-specific enhancers were highly conserved, and relatively localized at the 5' part of the intron 6. A single dot indicates more than 14 matches in 15 nucleotides.
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Expression Profile of lacZ Driven by VMH Enhancer
To address whether the conserved region functions as the VMH-specific enhancer, the region of the mouse gene (503 bp) was amplified by PCR, and the fragment was analyzed by transient transgenic assays. Three of five transgenic mouse fetuses at each E11.5 and E14.5 showed vd-specific lacZ expression identical with that described above. Therefore, the 503-bp fragment was used for generating transgenic mouse lines. Ten lines of the transgenic mice were generated and the lacZ expressions were analyzed. More than two founders for each line were analyzed at E11.5, E14.5, and adulthood. Seven of 10 lines showed lacZ expression in the developing and adult VMH, although the intensities of the lacZ expression varied (data not shown). A mouse line showing moderate lacZ expression was then used for the following studies. LacZ expression in the transgenic mice was examined in coronally (Fig. 3
, AE) and sagittally (Fig. 3
, KO) sectioned specimens. In addition, lacZ and endogenous Ad4BP/SF-1 expression levels were compared on the same coronal (Fig. 3
, FJ) and sagittal (Fig. 3
, PT) sections.

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Fig. 3. Comparison between lacZ Expression Induced by the VMH Enhancer and Endogenous Ad4BP/SF-1 Expression during Development of the VMH
LacZ expression driven by the 503-bp VMH enhancer was investigated in a transgenic mouse line. Using a vibratome, fetal and adult brains were sectioned coronally (AE) or sagittally (KO) followed by X-gal staining. To compare lacZ expression and endogenous Ad4BP/SF-1, fetal and adult brains were cryotome-sectioned coronally (FJ) or sagittally (PT). After detection of lacZ expression, the sections were subjected to immunohistochemical analyses using the antibody to Ad4BP/SF-1. Insets in FJ and PT show cellular colocalization of lacZ and Ad4BP/SF-1. Small arrows in F and P indicate weak lacZ expression at this stage. In I, J, Q, R, S, and T, the distribution of lacZ-positive cells is encircled by broken lines. Rathkes pouch is indicated by the asterisk in K, L, M, N, P, Q, R, and S. Arrowheads in G indicate Ad4BP/SF-1-positive but lacZ-negative cells in the dorso-medial part of the vd. Arrowheads in R and S indicate Ad4BP/SF-1-positive but lacZ-negative cells in the ventral part of Rp. Arrows in B, C, L, and M indicate ectopic lacZ expression in the choroid plexus. Bars, 200 µm.
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As described previously, the endogenous Ad4BP/SF-1 expression is initially recognized in ventral part of the diencephalon at E9.5 (8), and continued to be expressed in the ventral diencephalon through the embryonic period. Ad4BP/SF-1-expressing neurons then gradually aggregate and are observed as a coherent nucleus at approximately E17.0 (25). Consistent with the endogenous expression, lacZ expression was first observed in the outer layer of the ventral part of diencephalon at E10.5 or earlier (Fig. 3
, A and K). In the E10.5 fetuses, although the expression levels of both lacZ and endogenous Ad4BP/SF-1 were weak, cellular colocalization of the two was observed (Fig. 3
, F and P, arrows in insets). In E12.5 fetuses, lacZ expression was clear in the ventral diencephalon (Fig. 3
, B and L). As shown in Fig. 3
, G and Q, the distribution of endogenous Ad4BP/SF-1 overlapped with that of lacZ in the ventral part of the developing diencephalon (Fig. 3
, G and Q, insets). However, Ad4BP/SF-1-positive but lacZ-negative cells were observed in the dorsomedial region (arrowheads in Fig. 3G
). Neither lacZ- nor Ad4BP/SF-1-positive cells were detected in Rathkes pouch at this stage (asterisk in Fig. 3Q
). In E14.5 fetuses, lacZ-positive cells aggregated in the ventral part of the diencephalon (Fig. 3
, C and M) where endogenous Ad4BP/SF-1 was expressed (Fig. 3H
and encircled by broken line in R). By this stage, endogenous Ad4BP/SF-1 expression was initiated in the ventral part of Rathkes pouch (Rp) (arrowheads in Fig. 3R
), and continued thereafter at E16.5 (arrowheads in Fig. 3S
). LacZ expression was not observed in the Rp at all, indicating that the 503-bp fragment does not drive reporter gene expression in the Rp. In E12.5 and E14.5 fetuses, ectopic lacZ expression was observed in the choroid plexus (arrows in Fig. 3
, B, C, L, and M). This ectopic expression was also detected in all other lines of the transgenic mice (data not shown). By E16.5, lacZ-positive cells mostly overlapped with Ad4BP/SF-1-positive cells (Fig. 3
, I and S, encircled by broken lines), and the distribution pattern (Fig. 3
, D and N) was similar to that in the adult. Finally, in the adult, strong or weak expression of Ad4BP/SF-1 has been observed in the medial and central part of the VMH or in lateral quadrant, respectively (26). Consistent with the previous observation, LacZ was strongly expressed in the medial and central parts of the adult VMH (Fig. 3
, E and O), and weakly in the lateral part. Moreover, the distribution pattern of lacZ was almost identical with that of Ad4BP/SF-1 immunoreactive cells (Fig. 3
, J and T, encircled by broken lines). Cellular colocalization was clearly detected (insets in Fig. 3
, J and T). Taken together, these findings indicated that the 503-bp fragment was sufficient to reproduce the VMH-specific expression of Ad4BP/SF-1 from fetal stage to adult.
Identification of Functional Elements in the VMH-Specific Enhancer
To identify functional elements in the VMH-specific enhancer, the enhancer region was divided into five fragments, F1 to F5 (Fig. 4A
). These fragments were deleted alone or in combination from the original construct (Ad4-enV in Fig. 4B
). The lacZ expression patterns were analyzed with this series of deletion constructs. Deletion of F1 (Ad4-enV[
1]) affected lacZ expression at E11.5 as follows; lacZ expression expanded dorsally to the intrinsic expression domain at the anterior part (Fig. 4D
), whereas lacZ expression was observed ectopically in the ventricular zone (vz) at the posterior part (Fig. 4C
). These aberrant expression patterns suggested the presence of a putative element, which suppresses ectopic expression of Ad4BP/SF-1 beyond the intrinsic expression domain (Fig. 4B
). Deletion of F2 (Ad4-enV[
2]), F3 (Ad4-enV[
3]) or F5 (Ad4-enV[
5]) did not affect the lacZ expression at all (Fig. 4E
). Interestingly, however, simultaneous deletion of F2 and F5 (Ad4-enV[
2,5]) resulted in complete disappearance of lacZ expression (Fig. 4F
), suggesting the presence of compensatory elements between the two fragments. Likewise, deletion of F4 (Ad4-enV[
4]) also led to disappearance of lacZ expression.

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Fig. 4. LacZ Expression Patterns Affected by Partial Deletion of the VMH Enhancer
A, Nucleotide sequences of the VMH-specific enhancer regions were compared among mouse, human, and chicken. Asterisks indicate conserved nucleotides among the three animals. Conserved nucleotide clusters of more than consecutive 6 bp are enclosed by boxes. The enhancer region is divided into five fragments, designated F1 to F5. B, Deletion constructs of the VMH enhancer region and the effects of the deletion are indicated. Each fragment, F1 to F5, was deleted from the wild-type construct (Ad4enV) to generate Ad4-enV[ 1], Ad4-enV[ 2], Ad4-enV[ 3], Ad4-enV[ 4], and Ad4-enV[ 5]. F2 and F5 were deleted simultaneously to generate Ad4-enV[ 2, 5]. Transgenic mouse fetuses (Tg) were analyzed at E11.5 and E14.5 to E16.5. Numbers of the fetuses showing the lacZ expression in the ventral diencephalon (vd), ventricular zone (vz), and dorsal side of the vd (d) are summarized. C and D, Representative expression patterns with Ad4-enV[ 1] are indicated. The transgenic fetus at E11.5 was subjected to lacZ staining, and thereafter sectioned coronally. Ectopic lacZ expression in vz (indicated by black arrows) and d (encircled by broken line) in addition to normal expression in vd (indicated by a white arrow) was observed. E, The lacZ expression in the transgenic fetus at E11.5 with Ad4-enV[ 3] is shown. The staining pattern was almost the same as the wild type. F, Complete disappearance of lacZ expression in the transgenic fetus at E14.5 with Ad4-enV[ 2, 5] is shown. As encircled by the broken line, lacZ expression disappeared from vd completely.
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To identify nucleotide sequences responsible for the above activities, the enhancer regions were compared among the mouse, human and chick genes, and we identified the presence of multiple conserved nucleotide clusters. As indicated in Fig. 4A
, F1 contained four clusters, c1.1, c1.2, c1.3, and c1.4, each containing more than six consecutive nucleotides. To address which sequence was implicated in the suppressive regulation, the nucleotides in each cluster were substituted, and the mutated constructs were subjected to transgenic assays. Mutation at c1.3 induced ectopic lacZ expression at the dorsal side of the intrinsic expression domain (Fig. 5
, B and C, encircled by a broken line) and ventricular zone (Fig. 5B
, indicated by a black arrow) in addition to the intrinsic expression at the vd (Fig. 5
, B and C, indicated by a white arrow). Mutation at the other sites had no effect on lacZ expression (data not shown).

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Fig. 5. Identification of Suppressive and Activating Sequences by Mutated Constructs
A, The conserved clusters of nucleotides indicated in Fig. 5A were mutated as indicated. The conserved sequences, c1.1, c1.2, c1.3, and c1.4 in F1 were mutated to generate Ad4-enV[c1.1m], Ad4-enV[c1.2m], Ad4-enV[c1.3m], and Ad4-enV[c1.4m], respectively. The conserved sequences, c4.1, c4.2, and c4.3 in F4 were mutated to generate Ad4-enV[c4.1m], Ad4-enV[c4.2m], and Ad4-enV[c4.3m], respectively. Sequences containing putative binding sites for homeobox proteins, HD2 and HD3 in F2 and HD4 and HD5 in F5, were mutated to generate Ad4-enV[HD2/3m], and Ad4-enV[HD4/5m], respectively. All the putative binding sites, HD2 to HD5, were mutated to generate Ad4-enV[HD2/3/4/5m]. Transgenic mouse fetuses (Tg) were analyzed at E11.5 and E14.5 to E16.5. Numbers of the fetuses exhibiting lacZ expression in the ventral diencephalon (vd), ventricular zone (vz), and dorsal side of the vd (d) are summarized. B and C, A representative lacZ expression pattern in a transgenic fetus at E11.5 with Ad4-enV[c1.3m] is shown. In the coronal section (B), ectopic expression in d (encircled by a broken line) and vz (indicated by a black arrow) were observed in addition to vd (indicated by a white arrow). In the sagittal section (C), ectopic expression was observed in d (encircled by a broken line). D and E, Representative lacZ expression pattern in a transgenic fetus at E14.5 with Ad4-enV[HD2/ 3/ 4/ 5m] (E) was compared with that of wild-type (Ad4-enV) (D). Transgenic founders carrying the wild-type VMH enhancer induced lacZ expression in vd, whereas those carrying Ad4-enV[HD2/ 3/ 4/ 5m] failed to express lacZ in vd (encircled by a broken line). F, EMSAs with probes containing the activating core sequences, HD2/3 and HD4/5, were performed using nuclear extracts prepared from murine adult hypothalami. One hundred folds of the wild-type oligonucleotides (HD2/3 and HD4/5) or the mutated oligonucleotides (HD2/3m and HD4/5m) were added as the competitors. In addition, oligonucleotides, c1.3, containing the suppressive sequence was used as the competitor. The major DNA-protein complex is indicated by the arrow at the left of the panel. The asterisk shows additional DNA-protein complexes detected only when the HD4/5 probe was used.
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Simultaneous deletion of F2 and F5 strongly suggested that the same elements were localized in both fragments. Based on this observation, we found that two repeated ATTA motifs (consensus recognition sequence for homeodomain proteins) were present in common in F2 (HD2 and HD3) and F5 (HD4 and HD5) with a 7- and 12-nucleotide interval, respectively. All these ATTA motifs were conserved even in the chick gene (Fig. 4A
). Consistent with the deletion analyses above, lacZ expression was unaffected when either HD2 and HD3 or HD4 and HD5 were mutated. However, as expected, when all the ATTA motifs were mutated, the enhancer activity disappeared (Fig. 5E
). Finally, we found three conserved nucleotide sequences in F4 (c4.1, c4.2, and c4.3 in Fig. 4A
). Unfortunately, however, the lacZ signal was still evident, even with mutation at each conserved sequence.
To certify that these functional sequences are recognized by nuclear factors, we performed EMSAs using nuclear extracts prepared from the hypothalami of adult mice. As indicated in Fig. 5F
, the HD2/3-containing probe formed a DNA-protein complex. This complex was abolished by the wild-type competitor HD2/3, but not by c1.3 competitor or the mutant competitor HD2/3m carrying nucleotide substitution identical with that used in the transgenic mouse assay. Intriguingly, HD4/5 also competed with HD2/3 for the complex formation. Similarly, the HD4/5 probe formed a DNA-protein complex at a location similar to that of the HD2/3 probe. This complex was abolished by HD4/5 or HD2/3, but neither by c1.3 nor HD4/5m. In addition to the complex noted above, larger complexes were observed with HD4/5 probe (asterisk in Fig. 5F
). The formation of these complexes was influenced by both HD4/5 and partially by HD2/3, but not by c1.3 competitor. However, HD4/5m was still active as the competitor, suggesting that these complexes were formed on sequences other than the ATTA core regions. Taken together, these results indicated that the activating core sequences, HD2/3 and HD4/5, are occupied by the same factor, and that mutations impaired the enhancer function and ablated the nuclear factor binding simultaneously. With regard to the suppressive sequence c1.3, we could not detect any sequence-specific DNA-protein complex (data not shown). This is presumably because the suppressing function of the enhancer is required only when hypothalamic regionalization occurs in fetal stages, and thus the factor is not expressed in the adult hypothalamus. Another possible explanation is that the content of the factor is too low to be detected by EMSAs.
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DISCUSSION
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Identification of a VMH-Specific Enhancer in the Ad4BP/SF-1 Gene
It has been generally accepted that genes are divided into two types according to their expression profiles. Firstly, there are the so-called housekeeping genes with ubiquitous expression among tissues and constitutive expression during developmental processes. In contrast, other genes display tissue-specific and/or developmental stage-specific expression. Many of the latter genes have been revealed to contain enhancer sequences, which may explain the molecular basis of regulation of these restricted expressions. Ad4BP/SF-1 is classified into the latter group of genes because of its strict tissue-specific expression (27, 28). In the present study, we identified a VMH-specific enhancer in intron 6 of the Ad4BP/SF-1 gene. Functionally, the enhancer was characterized by driving VMH-specific expression of the lacZ reporter gene when the enhancer was localized with the basal promoter of Ad4BP/SF-1. Moreover, the VMH-specific expression was also reproduced with the hsp68 basal promoter (data not shown), indicating that the VMH-specific expression can be driven primarily by this enhancer. Structurally, the sequence is conserved among animal species (mouse, human, and chicken), thus strongly indicating that the conserved sequences probably function as a VMH-specific enhancer beyond animal species.
Possible Implication of Basal Promoter of Ad4BP/SF-1 into Tissue-Specific Gene Expression
Previously, Stallings et al. (29, 30) generated transgenic mice expressing enhanced green fluorescent protein using a 5' upstream fragment of the Ad4BP/SF-1 gene. Because the fragment used in that study spanned a segment 50 kb upstream from the second exon, the VMH enhancer located in intron 6 was excluded from the transgene construct. However, interestingly, the mice displayed enhanced green fluorescent protein expression in the VMH as well as the adrenal gland, spleen and gonads. Although the VMH enhancer identified in the present study contributed predominantly to the VMH-specific expression, their observations implicated the basal promoter region in the tissue-specific expression.
This assumption is supported by several lines of evidence as described below. As reported previously, the Ad4BP/SF-1 gene carries multiple noncoding first exons, Ia, Io, Ig, and Ix (23, 24). Interestingly, these first exons are used in a tissue-preferential manner. Indeed, exons Ia and Ig were transcribed dominantly in the spleen and pituitary, respectively. Although it has not been determined which first exon is used in the VMH, this tissue-selective usage of the first exon possibly corresponds to the presence of a structural basis for the tissue-specific transcription lying at the upstream from the common second exon. Moreover, Wilhelm et al. (31) reported that the region from 589 to +85 (transcription initiation site for exon Ia is numbered as +1) of Ad4BP/SF-1 gene was sufficient to drive gonad-specific expression. They also showed that the binding sites for Wt1 and Lhx9 on the promoter were indispensable for gonad-specific expression. Considering the crucial function of the two factors in the gonadal development revealed by gene disruption studies (32, 33), it is feasible that the basal promoter of Ad4BP/SF-1 containing the Wt1 and Lhx9 binding sites potentially drives gonad-specific expression. Recently, the activity of the basal promoter to drive gonadal expression was confirmed by generating a transgenic mouse in which Cre recombinase was expressed under the control of the basal promoter. Conditional deletion of a gene from the gonad was achieved with the transgenic mouse (34). Taken together, these findings indicate that the basal promoter region of Ad4BP/SF-1 gene potentially regulates VMH-specific expression cooperatively with the intronic VMH-specific enhancer.
It is intriguing that our basal construct for the transgenic study failed to display gonadal expression even though the construct contained 6.0-kb upstream region from the second exon. As described by Wilhelm et al. (31), the region from 589 to +85 could induce lacZ reporter gene expression in the fetal gonad. Because the region is included in our basal reporter construct, our construct would be predicted to produce lacZ expression in the fetal gonad. However, we could not detect it (data not shown). Although we do not have a rational explanation for the discrepancy at present, it should be noted that the two constructs are not identical; our construct carries 2.0 kb upstream from exon Ia, and carries the downstream first exons (Io, Ig, and Ix) and introns localized upstream from the second exon. The region between Ia and the second exon contains CpG island-like sequence (23). CpG island has been frequently found to be localized around the regulatory regions of housekeeping genes or tissue-specific genes, and methylation of CpG island is implicated in gene silencing (35). Moreover, using the chromatin immunoprecipitation assay, we recently elucidated the role of the polycomb component, M33, is implicated in Ad4BP/SF-1 gene regulation through direct binding (36). Because the M33 binding region is localized at 0.7 kb upstream from exon Ia, our construct but not Wilhelms construct contains the binding region. Considering these distinct structural features, it is reasonable that the two constructs differentially regulate the reporter gene expression.
Activating Core Sequences in the VMH-Specific Enhancer and Nkx2.1
Growth factors are known to contribute to cellular specification in the process of multiple tissue differentiation. Brain regionalization is also regulated primarily by the functions of growth factors together with regionally expressed homeobox proteins (37, 38). Moreover, it has been shown that combined expression of these homeobox proteins is essential to establish progenitor cell identity and neuronal cell subtypes during forebrain patterning (39, 40, 41). In the present study, we identified activating elements, HD2/3 and HD4/5, in the VMH enhancer. The primary sequence of these elements strongly suggested that they are recognized potentially by homeobox proteins. Indeed, EMSAs revealed that the activating elements are recognized by the same nuclear protein.
Several homeobox proteins are known to be expressed in various regions of the developing brain. Among these, it is noteworthy that the expression domain of Nkx2.1 (also designated TTF-1, T/ebp) overlaps with that of Ad4BP/SF-1 until E18.5 (22). Nkx2.1 is a member of the Nkx homeoprotein family, and plays important roles in ventral forebrain development in addition to the lung, thyroid gland, and pituitary (42, 43, 44, 45). In particular, in Nkx2.1 null mice (44), the ventromedial and dorsomedial nuclei were underdeveloped and fused in the midline. The expression profile of Nkx2.1 and the phenotypes of the gene-disrupted mice prompted us to analyze the effect of gene disruption on the VMH enhancer function. Thus, the Nkx2.1 gene-disrupted mice were crossed with the VMH enhancer transgenic mice and the effect of Nkx2.1 disruption on the lacZ expression driven by the enhancer was investigated in E11.5, E14.5, and E18.5 fetuses. Disappointingly, however, expression of the lacZ reporter gene driven by the VMH enhancer persisted even in the Nkx2.1 knockout background, suggesting that Nkx2.1 is not essential for the enhancer function (data not shown). This is supported by an observation that expression of Ad4BP/SF-1 was weak but still detectable in the affected ventral diencephalon of Nkx2.1 null mice (42). In this context, it is quite interesting to consider the potential redundant function of another family member, Nkx2.4, of which expression is overlapped with Nkx2.1 in the developing forebrain. Meanwhile, considering that the lacZ-positive cells in the ventral diencephalon of the Nkx2.1 knockout mice were fewer in number than in the wild type from the early developmental stage of VMH, Nkx2.1 could be partially involved in the VMH-specific enhancer function. Through the function of the VMH enhancer, Nkx2.1 might regulate the amount of Ad4BP/SF-1 expressed, and thereby stimulate VMH cell proliferation. Indeed, it was recently reported that cell numbers in adrenocortical primordium are tightly dependent on the amount of Ad4BP/SF-1 (46).
Suppressive Core Sequence in the VMH Enhancer
Notably, we have identified another element in the VMH enhancer of which mutation led to ectopic expression of the lacZ reporter gene. Because the intrinsic expression domain of Ad4BP/SF-1 is strictly restricted to the VMH, this element, together with a putative binding factor, is likely to suppress ectopic expression of Ad4BP/SF-1. This observation paradoxically indicated that the VMH enhancer has the potential to drive transcription beyond the VMH, and therefore that the suppressive element is needed for the regionally restricted expression of Ad4BP/SF-1 in the VMH. Considering this possibility, it is reasonable to assume that the intrinsic expression domain is defined cooperatively by the two reciprocal actions driven by the activating and suppressive elements in the VMH enhancer. Although the factors capable for binding to the active and suppressive elements remain to be identified, these factors are expected to satisfy these functions.
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MATERIALS AND METHODS
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Reporter Gene Construction
The bacterial lacZ gene followed by SV40 poly (A) signal was ligated to the SplI site downstream of the initiation methionine in the second exon of Ad4BP/SF-1 to construct the Ad4BP-lacZ cassette. Because a 6-kb fragment upstream from the initiation methionine was used, this cassette contained all the first exons (Ia, Io, Ig, and Ix) and more than 3 kb upstream from the exon Ia. To clone genomic DNA fragments, cosmid (cAd4BP-lacZ) and plasmid (pAd4BP-lacZ) vectors containing the Ad4BP-lacZ cassette were constructed with SuperCosI and pBluescriptSK () (Stratagene Corp., La Jolla, CA), respectively. After genomic DNA was partially digested by Sau3AI, the resulting fragments of approximately 30 kb in length were inserted into the BamHI site of the cosmid vector, cAd4BP-lacZ (Fig. 1
). Clones covering the Ad4BP/SF-1 gene locus were isolated and used for transgenic mouse assays. After identification of clones showing enhancer activity, the insert DNAs were truncated and recloned into the plasmid vector, pAd4BP-lacZ. The VMH enhancer region was amplified by PCR using the following primers: V-UP1; 5'-GGACTAGTCCTCTTTCTCGCTTGTCTCTG-3', and V-LP1; 5'-ACGCGTCGACGAGGCAGAAGGGTGGGAGATG-3'. SpeI and SalI adapter sequences were added to the 5' end of V-UP1 and V-LP1, respectively. The amplified fragment (503 bp) was inserted into SpeI-SalI site downstream of lacZ in pAd4BP-lacZ. To assay the activity of the VMH enhancer, transgenic mouse lines were generated with this construct (Ad4-enV). To identify the core sequence of the VMH enhancer, nucleotide substitution was performed by the PCR-based site-directed mutagenesis method.
Generation and Characterization of Transgenic Mice
Transgenic mice were generated as described previously (47). In brief, linearized DNA fragments were purified by electro-elution followed by phenol, phenol/chloroform extraction and precipitation with ethanol. The purified DNA fragments were injected into fertilized eggs of the mouse strain B6C3F1 prepared by an in vitro fertilization method. The manipulated eggs were then transferred into foster mothers. The transgene in founder animals was detected by PCR using tail or amniotic membrane DNA. The sequences of PCR primers were 5'-GCCGAAATCCCG AATCTCTATC-3' (nucleotide residues 21932214 of lacZ; GenBank accession no. J01636) and 5'-GATTCATTCCCCAGCGACCAG-3' (26512671). LacZ staining of the transgenic mice was performed as described previously (48). Briefly, fetuses were fixed in a solution containing 1% formaldehyde, 0.2% glutaraldehyde, 0.02% Nonidet P-40, and 1 mM MgCl2 at 4 C. Postnatal animals were perfused transcardially under deep pentobarbital anesthesia, first with PBS and then with the fixative above. Fixed whole fetuses at E10.5, E11.5, and E12.5 were incubated overnight in PBS containing 1 mg/ml X-gal, 5 mM K3 Fe(CN)6, 5 mM K4 Fe(CN)6, 2 mM MgCl2, and 0.2% Nonidet P-40 at 37 C. For detailed observation, the samples after lacZ staining were sliced using a vibratome (Micro Slicer DTK-1000, Dosaka Co. Ltd., Kyoto, Japan). The fixed fetuses at E14.5, E16.5, and E18.5, and adult brain were sliced by vibratome before lacZ staining.
Immunohistochemistry
Whole fetuses or isolated brains were fixed with 0.2% paraformaldehyde-PBS at 4 C. The fixation time varied according to the stage of the fetus: 30 min for E10.5, 2 h for E12.5 and E14.5, and overnight for E16.5 fetuses. For adult brains, perfusion fixation was followed by overnight fixation. After fixation, the buffer was substituted for 20% sucrose/PBS by rotating at 4 C overnight, and the samples were cryosectioned. The sectioned specimens were LacZ stained as reported previously (47). Immunohistochemical staining for Ad4BP/SF-1 was performed by a standard avidin-biotin complex method with a Histofine kit (Nichirei Co., Tokyo, Japan) using a rabbit antiserum to bovine Ad4BP/SF-1 (9, 49).
EMSAs
Preparation of nuclear extracts from the hypothalami of adult mice and EMSAs were performed as described previously (4). The following double-stranded oligonucleotides were used as probes or competitors: c1.3; 5'-GGGCTCAGAGGCAGGTAAGGAGGCTT-3' and 5'-GGAAGCCTCCTTACCTGCCTCTGAGC-3', HD2/3; 5'-GGGGACTTAATCGAAGCTTAATGGATTGACCTA-3' and 5'-GGTAGGTCAATCCATTAAGCTTCGATTAAGTCC-3', HD45; 5'-GGAAAGGATTATTCAAATATTCAATTAAACCG-3' and 5'-GGCGGTTTAATTGAATATTTGAATAATCCTTT-3', HD2/3m; 5'-GGGGACTGCCGCGAAGCTGCCGGGATTGACCTA-3' and 5'-GGTAGGTCAATCCCGGCAGCTTCGCGGCAGTCC-3', HD4/5m; 5'-GGAAAGGCGGCTTCAAATATTCACGGCAACCG-3' and 5'-GGCGGTTGCCGTGAATATTTGAAGCCGCCTTT-3'. The mutations introduced in HD2/3m and HD4/5m were identical with those used in the transgenic mouse assays.
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ACKNOWLEDGMENTS
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We thank Mrs. Tomoo Etoh, and Akira Miyakawa for their suggestions on the in vitro fertilization experiment. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and in part by The Cell Science Research Foundation.
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FOOTNOTES
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First Published Online June 30, 2005
Abbreviations: Ad4BP/SF-1, Ad4 binding protein/steroidogenic factor-1; E, embryonic day; poly (A), polyadenylation; SV40, simian virus 40; VMH, ventromedial hypothalamic nucleus.
Received for publication October 22, 2004.
Accepted for publication June 23, 2005.
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