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Expression of Fgf15 is regulated by both activator and repressor forms of Gli2 in vitro
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Biochemical and Biophysical Research Communications 369 (2008) 350–356 www.elsevier.com/locate/ybbrc
Expression of Fgf15 is regulated by both activator and repressor forms of Gli2 in vitro Munekazu Komada, Hirotomo Saitsu 1, Kohei Shiota, Makoto Ishibashi * Department of Anatomy and Developmental Biology, Kyoto University Graduate School of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan Received 30 January 2008 Available online 13 February 2008
Abstract Fibroblast growth factor 15 (Fgf15) is expressed in the medial region of diencephalon and midbrain by the seven-somite stage. In the previous studies, we showed that Sonic hedgehog signaling through Gli protein is required for Fgf15 expression in this region. The Fgf15 expression domain overlapped with that of Gli2 and the Gli-binding site (GliBs) is located in the 3.6-kb 50 -flanking enhancer/promoter region of the Fgf15 gene. In this study, we identified the two additional Gli-binding sites in row, called Gli-responsive elements (GliREs). Chromatin immunoprecipitation assay indicated that Gli2 directly binds to GliREs. The results from luciferase assays indicated that the Gli2 activator form binds to the GliBS and that the Gli2 repressor form binds to the GliREs. These findings suggest that the repressor form of Gli2 preferentially binds to the GliREs to control Fgf15 expression. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Fgf15; Gli2; Sonic hedgehog; Enhancer/promoter; Activator/repressor
The Hedgehog (Hh) family of signaling molecules mediates various morphogenesis processes during invertebrate and vertebrate development. In vertebrates, Sonic hedgehog (Shh), a member of the Hh family, has been shown to play important roles in cell fate determination, proliferation and survival in the developing central nervous system. In Drosophila, there is only one Hh protein and the cellular response to Hh signaling is mediated by the zinc finger type transcription factor Cubitus interruptus (Ci) [1]. Ci is regulated post-translationally by Hh signaling. In the absence of Hh signaling, Ci protein is cleaved into a truncated repressor form, CiR, which inhibits transcription of target genes. In the presence of Hh signaling, the proteolytic processing of Ci is suppressed to form an activator Ci, CiA [2]. While both CiA and CiR can bind to the sim*
Corresponding author. Fax: +81 75 751 7529. E-mail address: [email protected] (M. Ishibashi). 1 Present address: Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.015
ilar sequence (Gli-binding site, see below), previous works indicated that there is some preference between CiA and CiR for binding in different genes. For example, the Glibinging site of the dpp gene responds to both CiA and CiR [3]. In the case of Patched, only CiA binds to its Glibinding site [3]. The mechanisms of this preference remain elusive. In vertebrates, there are three Ci homologs, Gli1, Gli2 and Gli3 [4]. Unlikely fly Ci, mouse Gli1 contains only an activator domain and is not subject to proteolytic processing [5]. Therefore, Gli1 functions only as a transcriptional activator. On the other hand, mouse Gli2 and Gli3 contain repressor and activator domains on their N- and C-terminal, respectively, as fly Ci does [5]. Similarly to Ci, Gli3 can be proteolytically processed into a C-terminal truncated repressor, and this processing is inhibited by Hh signaling. Expression analysis of marker genes has shown that Gli3 functions mainly as a transcriptional repressor [6], though recent studies have also shown that Gli3 can act as a weak activator in vivo [7–9]. Most of Gli3 protein is cleaved to generate a Gli3-83 transcriptional repressor in
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the absence of Shh signaling. In the presence of Shh, Gli3 protein is thought to remain full-length (Gli3-190) and act as an activator [10–12]. Mouse Gli2 appears to exhibit distinct transcriptional activity from that of Gli3 while they share a high degree of sequence similarity. The C-terminal truncated forms act as dominant negatives [7]. Gli2 protein has been thought to be processed to form both an activator and a repressor. In vitro reporter assays have shown that Gli2 exhibits a stronger transcriptional activity than Gli3, albeit Gli2 is a weaker activator than Gli1 [5]. In mice, Gli2 is required for the most ventral neural progenitors in the developing spinal cord [13]. A definitive answer to the question of whether Gli2 functions only as a transcriptional activator or as both an activator and a repressor in vivo has not been achieved. Several lines of evidence suggest that Gli2 may normally function only as a transcriptional activator. For example, the loss-of-function mutation of mouse Gli2 resulted in loss of the most of ventral cell types in the developing neural tube [14,15]. Furthermore, Gli1 can rescue Gli2 mutant phenotypes when it is inserted into the Gli2 locus [13], but Gli3 cannot [10]. However, recent genetic analysis of Gli2 mutants on the Gli3 null background has also indicated that Gli2 could have repressor activity in addition to its activator function [11,16]. In zebrafish, Gli2 may act as a transcriptional activator or a repressor, depending on different genes in different tissues [17]. In mice, the vast majority of Gli2 protein is thought to be present in the fulllength activator form and only a small fraction might exist in the truncated repressor form [6]. An antiapoptotic factor BCL2 is regulated by Gli1 and Gli2 [18,19]. The human BCL2 promoter region has three Gli-binding sites [20] and one of these binding sites is critical for conferring Gli2-specific activation. Other two Glibinding sites are dispensable while they cooperate with the critical site in transactivation of BCL2 [19]. Little is known about how Gli2 transcriptional activity is regulated to activate target gene expression in response to Hh signaling. Fgf15 is a member of the Fibroblast growth factor (Fgf) family. Its expression in the diencephalon and midbrain is directly initiated by Shh signaling [21,22]. We previously demonstrated that the 3.6-kb Fgf15 enhancer/promoter region has a Gli-binding site (GliBS) at 1-kb upstream of the transcription start site. This site is required for Fgf15 expression in the medial/ventral diencephalon/midbrain in transgenic embryos and for activation in luciferase assay. Luciferase assay indicated that only Gli2 activates the Fgf15 enhancer/promoter through the GliBS. When the GliBS was mutated, activation was not completely abolished in luciferase assay. These results led us to the idea that there should be additional Gli2-specific responsive elements in the enhancer/promoter region. In this study, we identified two additional Gli-binding sites which are involved in regulation by Gli2. Luciferase assay demonstrated that Gli2 regulates Fgf15 expression
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through these sites. We designated them as Gli-responsive elements (GliREs). ChIP assay suggested that Gli2 directly binds to GliREs. Chimeric protein study indicated that the Gli2 activator form binds to the GliBS and the Gli2 repressor form binds to the GliREs. The results imply that the repressor form Gli2 binds to the GliREs to control the amount of Fgf15 expression. Materials and methods Plasmid construction and mutagenesis. The 3.6-kb Fgf15 enhancer/ promoter was inserted into the luciferase vector and Gli-binding site was mutated as described previously [22]. The 2-kb and 1.4-kb Fgf15 50 franking regions were also subcloned into pGL3-Basic reporter (Promega). The 8GliRE::luciferase reporter plasmids were generated by subcloning eight tandemly repeated copies of GliRE (50 GCCGTCGACGGTGCTGCCACCCGCATGCCTCGAGGCC-30 ) into the pGL3-d51 luciferase reporter. For luciferase assays shown in Fig. 2, the reporter plasmids were constructed by inserting the fragments of the Fgf15 enhancer/promoter into pGL3-d51-GliBS as indicated in the figure. pGL3-d51-GliBS luciferase reporter was constructed by inserting Gli-binding site of Fgf15 enhancer/promoter into pGL3-d51 luciferase reporter. No. 6 and 7 plasmids were constructed by inserting the PCR products (by KOD-Plus (TOYOBO, Japan)) into pGL3-d51-GliBS. Mutations were introduced into the GliREs by PCR-based site directed mutagenesis. To construct VP-Gli2ZF chimeric protein, the HindIII–BamHI DNA fragment that covers the entire five zinc finger domains of mouse Gli2 was fused to the C-terminus of the HSV VP16 activation domain. The resultant expression vector was designated as pVP-Gli2ZF. Luciferase assay. Luciferase assays were performed as described previously [22]. DNA transfection was performed with FuGene6 (Roche) according to the manufacturer’s instructions. C3H10T1/2 cells were transfected with the expression vector of Gli2 or pVP-Gil2ZF and the reporter plasmids. All luciferase assays were done in duplicates and repeated three times. Chromatin immunoprecipitation assay. ChIP assay was performed according to the manufacturer’s manual (LP Bio, Chromatin Immunoprecipitation Kit, KA-0137) using anti-FLAG antibody (Sigma, M2). C3H10T1/2 cells were transfected with FLAG-tagged mouse Gli2 (FLAGGli2) [23] and FLAG-tagged Hes1 (FLAG-Hes1) [24] expression vectors and the reporter plasmid.
Results Identification of two putative Gli-responsive elements in the 3.6-kb Fgf15 enhancer/promoter Previously, we reported that the 3.6-kb 50 -flanking enhancer/promoter of the Fgf15 gene is necessary and sufficient for induction of Fgf15 in the medial/ventral diencephalon/midbrain by transgenic analysis [22]. Luciferase assay showed that the 3.6-kb Fgf15 enhancer/promoter is activated by Gli2 transcription factor. A GliBS is located 1-kb upstream of the transcriptional start site. It is required for expression in the medial/ventral diencephalon/midbrain in the transgenic embryos and for activation in luciferase assay [22]. These results suggest that Gli2, but not Gli1, directly can interact with GliBS and transactivate the 3.6-kb Fgf15 enhancer/promoter. While mutation of this GliBS led to loss of expression in the medial/ventral
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diencephalon/midbrain in vivo, up-regulation of luciferase activity was not completely abolished, suggesting the presence of additional Gli-responsive elements. We performed luciferase assay to explore additional elements in the 3.6-kb Fgf15 enhancer/promoter required for expression of the Fgf15 gene and for interaction with GliBS and/or Gli2 transcription factor. First, to locate interactive elements with Gli2-mediated activity in the 3.6-kb Fgf15 enhancer/promoter region, we constructed a new deletion series of luciferase reporter vectors containing 2-kb (CN5) and 1.4-kb (CN3) 50 -flanking Fgf15 enhancer/promoter (Fig. 1). Secondly, we introduced base-substitution mutations that destroy the core GliBS (CN5-GliBSmt, CN3-GliBSmt, in Fig. 1). Gli2 transcription factor activated the 3.6-kb (CN7) and CN5 Fgf15 enhancer/promoter regions and the GliBS mutation resulted in significant decrease of the transcriptional activity (Fig. 1, [22]). In contrast, the GliBS mutation in the CN3 of the Fgf15 enhancer/promoter did not cause significant change of transcriptional activity by Gli2. These data suggest that the 620-bp sequence, between CN5 and CN3 of the Fgf15 enhancer/promoter includes interactive elements with the Gli2-mediated transcriptional activity. To identify essential regions interacting with Gli2 and/ or the GliBS of Fgf15 enhancer/promoter, seven luciferase reporter constructs containing deletion series of the 620-bp Fig. 2. Deletion analysis of the 50 -flanking 620-bp Fgf15 enhancer/ promoter region revealed the essential elements for the transcriptional activity of Fgf15 by Gli2. (A) Schematic representation of the EcoRV– KpnI 620-bp fragment of the Fgf15 enhancer/promoter and serial deletion constructs. These constructs include the GliBS of Fgf15 enhancer/ promoter and the chicken d-crystallin promoter. The 20-bp sequence around the SphI site that was required for transactivation by Gli2 is indicated by the open box. (B) The series of deletion constructs were cotransfected with the Gli2 expression vector. Luciferase activities were indicated as fold activation relative to the control. No. 1, 2, 4 and 7 constructs were activated by Gli2, respectively.
Fig. 1. Additional Gli-binding sites possibly exist in the 3.6-kb Fgf15 enhancer/promoter region. (A) Schematic representation of the Fgf15 enhancer/promoter luciferase reporter vectors. The restriction enzyme sites used for construction of deletion constructs are shown as indicated. (B) The Fgf15 enhancer/promoter with luciferase reporter was cotransfected into C3H10T1/2 cells with the Gli2 expression vector. Luciferase activities were indicated as fold activation relative to the control. Transactivation of CN7 (3.6-kb) and CN5 (2-kb) by Gli2 was significantly reduced by mutation of the GliBS. In contrast, there was no significant change when the GliBS of CN3 (1.4-kb) was mutated.
fragment were fused with GliBS (Fig. 2). As shown in Fig. 2, Gli2 up-regulated luciferase activity with the constructs No. 1, 2, 4, 7 and not with No. 3, 5 and 6. These results indicated that the 20-bp region flanking SphI site was required for up-regulation by Gli2. We identified two similar sequences to the GLI-binding consensus sequence [20] around the SphI site (Fig. 3A). These 9-base sequences had 3-base mismatch with human GLI-binding consensus sequences, and we designated these sequences as GliREs. The above results suggest that Gli2 binds to GliREs to regulate the transcriptional activity of Fgf15 enhancer/promoter. To confirm that these GliREs are involved in the transcriptional activity by Gli2, the luciferase reporter vector with eight copies of the first GliRE sequence was co-transfected with the Gli2 expression vector into the C3H10T1/2 cells. Then 60-fold up-regulation of the luciferase activity by Gli2 was observed (Fig. 3B).
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Fig. 3. Location of two putative Gli-binding sites upstream to GliBS, called GliREs. (A) Two putative GliREs were present around the SphI site within the 620-bp fragment. The core region of the putative GliREs contained three nucleotide-mismatch to the consensus Gli-binding sequence. Nucleotide substitution mutations were introduced into this Gli-binding sequence (GliREmt) to disrupt its binding to Gli2. (B) The luciferase reporter with eight times multimerized Gli-responsive element (8GliRE) or eight times multimerized Gli-binding site (8GliBS) from the 3.6-kb Fgf15 enhancer/promoter was cotransfected with the Gli2 expression vector. Gli2 up-regulated the 8GliRE reporter by 60-fold and 8GliBS reporter by 120-fold. (C) The luciferase reporter including wild type (CN7), the mutant of the first GliRE (CN7-1mt) or the mutant of both GliREs (CN7-1.2mt) was co-transfected with the Gli2 expression vector. In comparison with CN7, the transactivation by Gli2 was significantly increased with CN7-1mt and CN7-1.2mt. (D) The structure of the chimeric protein expression vector (pVP-Gli2ZF). cDNA encoding the VP16 activation domain (VP16-AD) and the Gli2 zinc finger domain (Gli2ZF) were inserted downstream of the SV40 promoter (P). (E) The Gli2 expression vector or pVP-Gli2ZF was co-transfected with CN7 and CN7-1.2mt reporter. Up-regulation of luciferase activity was significantly enhanced by pVP-Gli2ZF with the wild type enhancer/promoter (CN7), comparing with the Gli2 expression vector. With the mutant reporter (CN7-1.2mt), further up-regulation was observed when the reporter was co-transfected with the Gli2 expression vector (compare columns 3 and 1). This further up-regulation was cancelled with pVP-Gli2ZF (compare columns 3 and 4).
Next, we introduced base-substitution mutation at the 2nd–4th bases of the GliRE in the 3.6-kb fragment to obliterate binding capability. One mutant reporter (CN7-1mt) contains the mutation only in the first GliRE. The other
vector (CN7-1.2mt) carries mutation in both GliREs. Interestingly, these mutants showed a significant increase of transcriptional activity by Gli2 (Fig. 3C). The enhanced increase of luciferase activity by GliRE mutation indicates
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the presence of the repressor form of Gli2 (Gli2R) and its binding to the GliREs. Our data suggest that the GliREs interact preferentially with Gli2R in the context of 3.6-kb of the Fgf15 regulatory region.
demonstrates that Gli2 directly binds to the GliREs in the 360-bp Fgf15 enhancer/promoter region.
Discussion Gli2 may be processed to form Gli2R which interact with the GliREs in the Fgf15 enhancer/promoter To confirm whether Gli2 functions as a transcriptional repressor via the GliREs, we engineered a chimeric protein of the Gli2 DNA-binding domain (zinc finger domain [ZF]) and the transcriptional activator domain of HSV VP16 (Fig. 3D, [25]). This chimeric protein should act exclusively as an activator through Gli2-binding sequences. The pVP16-Gli2ZF expression vector was co-transfected with CN7 or the double GliRE mutant (CN7-1.2mt). As shown in Fig. 3E, CN7 was more extensively up-regulated by VP16-Gli2ZF than Gli2 itself (compare columns 2 and 1 in Fig. 3E), suggesting that some ratio of Gli2 protein acts as repressors. Increased up-regulation of the CN7-1.2mt by Gli2 was almost completely cancelled by VP16-Gli2ZF (compare columns 3 and 4 in Fig. 3E), and the level of up-regulation was almost same as that with the combination of CN7 and Gli2 (compare columns 4 and 1 in Fig. 3E). These results suggest that the repressor form of Gli2 suppresses up-regulation of Fgf15 by the activator form of Gli2 to some extent and that this suppression was mediated by GliREs. Gli2 directly binds to the GliREs in the Fgf15 enhancer/ promoter To demonstrate that Gli2 directly binds to the GliREs in the Fgf15 enhancer/promoter region, we performed ChIP assay with FLAG-Gli2 and the 360-bp Fgf15 enhancer/ promoter DNA fragment. This DNA fragment has two GliREs and does not include GliBS (Fig. 4A). FLAGHes1 expression vector was used as a negative control because there is no Hes1-binding site in the 360-bp fragment. As shown in Fig. 4B, the DNA fragment including GliREs was amplified from the precipitates with FLAGGli2. No band was detected with FLAG-Hes1. This result
Our previous study indicated that the Fgf15 expression in the medial/ventral diencephalon/midbrain is directly initiated by Shh signaling, most likely through Gli2. We found a Gli-binding consensus sequence (GliBS) 1-kb upstream of the transcriptional start site, it was essential for expression in the medial/ventral diencephalon/midbrain in the transgenic embryos and for activation in luciferase assay. However, mutation of the GliBS did not completely abolish the increase of luciferase activity [22]. We assumed that there should be additional Gli-responsive elements. Sequence analysis revealed two putative Glibinding sites. Our data suggest that these two sites, GliREs, directly interact with Gli2 and may cooperate with the critical GliBS in regulation of transcriptional activity by Gli2. Similarly, the human BCL2 cis-regulatory region has three Gli-binding sites and only one of them is critical for Gli2specific activation of the BCL2 promoter [19]. Other two sites are dispensable, but still cooperative with the critical site [19]. In cultured cells, truncation of the activation domain in the C-terminal half of Gli2 results in a transcriptional repressor [5]. Although several studies have suggested that Gli2 functions only as a transcriptional activator [13–15], it has been reported that both the full-length activator form, Gli2-185, and the repressor form, Gli2-78, exist in vivo [6]. Furthermore, genetic analysis of Gli2 mutants on a Gli3 null background has also indicated that Gli2 could compensate repressor activity of Gli3 [11,16]. In zebrafish, Gli2 may act as a transcriptional activator or a repressor, depending on different target genes in different tissues [17]. Since Gli2 is expressed uniformly in the neural tube, it is reasonable to assume that the ratio of activator and a repressor forms determines amount of target gene expression. This ratio makes a gradient, depending on the strength of Shh signaling. Our results in this study further support that Gli2 might have the transcriptional bipotentiality, both an activator
Fig. 4. Gli2 directly interacts with these Gli-responsive elements in Fgf15 enhancer/promoter region. (A) The structure of the reporter plasmid. The BamHI–KpnI fragment of Fgf15 enhancer/promoter included two GliREs. The left and right primers used for PCR was indicated. (B) ChIP assay was performed on the reporter plasmid with FLAG-Gli2 or FLAG-Hes1 expression vector. FLAG-Hes1 was used as a negative control.
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and a repressor. Mutation of the GliREs in the 3.6-kb Fgf15 enhancer/promoter (CN7-1mt, CN7-1.2mt) resulted in significant increase of the luciferase activity (Fig. 3C). The data indicate that the repressor form Gli2 may interact with GliREs and attenuate the transcription of Fgf15. VP16-Gli2ZF, the strong transcriptional activator, further up-regulated the luciferase activity with the wild type 3.6kb Fgf15 enhancer/promoter. Disruption of the GliREs led to loss of this further up-regulation by VP16-Gli2ZF (Fig. 3E). These results suggest that VP16-Gli2ZF might interact directly to the GliREs and act as a transcriptional activator in vitro. We propose that Gli2 is present in both the full-length activator form and the processed repressor form [5,6] and regulates transcriptional levels by binding to GliBS and GliREs, depending on the context of Glibinding sites. The results are different from the case of the BCL2 enhancer [19]. This conclusion seems to be rather paradoxical because the residual activation of the Fgf15 3.6-kb enhancer/promoter with GliBSmt implies the binding of the Gli2 activator to the GliREs (Fig. 1). Also, combination of the GliREcontaining short fragment and GliBS showed up-regulation through the GliREs (Fig. 2). Moreover, the 8GliRE construct was substantially activated by Gli2 (Fig. 3B). These results suggest that the activator form of Gli2 can also bind to the GliREs and activate the enhancer through these sites in the absence of the GliBS or with the short fragment of the regulatory region. However, in the Fgf15 3.6-kb enhancer/promoter with the intact GliBS, mutation of the GliREs led to further up-regulation by Gli2, suggesting that the repressor form of Gli2 preferentially binds to these sites in this context. In addition, with the wild type Fgf15 3.6-kb enhancer, the VP16-Gli2ZF chimeric protein up-regulates the enhancer more than naive Gli2 itself. This effect of the VP16-Gli2ZF was completely reversed by mutation of the GliREs (Fig. 3E). These data suggest that, in more physiological conditions, the GliREs interact preferentially with the repressor form of Gli2 to regulate the expression of Fgf15.
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[13] [14]
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Acknowledgments We thank Dr. Sasaki for Gli2 and FLAG-Gli2 expression vector, Dr. Kageyama for FLAG-Hes1 expression vector and Dr. Kamachi for pd51lucII, and Dr. Aspland and Murre for pGL3-Fgf15 promoter. This study was supported by The Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant No. 15689004, 16015264, 18590168) and Kato Memorial Bioscience Foundation.
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Biochemical and Biophysical Research Communications 369 (2008) 350–356 www.elsevier.com/locate/ybbrc
Expression of Fgf15 is regulated by both activator and repressor forms of Gli2 in vitro Munekazu Komada, Hirotomo Saitsu 1, Kohei Shiota, Makoto Ishibashi * Department of Anatomy and Developmental Biology, Kyoto University Graduate School of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan Received 30 January 2008 Available online 13 February 2008
Abstract Fibroblast growth factor 15 (Fgf15) is expressed in the medial region of diencephalon and midbrain by the seven-somite stage. In the previous studies, we showed that Sonic hedgehog signaling through Gli protein is required for Fgf15 expression in this region. The Fgf15 expression domain overlapped with that of Gli2 and the Gli-binding site (GliBs) is located in the 3.6-kb 50 -flanking enhancer/promoter region of the Fgf15 gene. In this study, we identified the two additional Gli-binding sites in row, called Gli-responsive elements (GliREs). Chromatin immunoprecipitation assay indicated that Gli2 directly binds to GliREs. The results from luciferase assays indicated that the Gli2 activator form binds to the GliBS and that the Gli2 repressor form binds to the GliREs. These findings suggest that the repressor form of Gli2 preferentially binds to the GliREs to control Fgf15 expression. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Fgf15; Gli2; Sonic hedgehog; Enhancer/promoter; Activator/repressor
The Hedgehog (Hh) family of signaling molecules mediates various morphogenesis processes during invertebrate and vertebrate development. In vertebrates, Sonic hedgehog (Shh), a member of the Hh family, has been shown to play important roles in cell fate determination, proliferation and survival in the developing central nervous system. In Drosophila, there is only one Hh protein and the cellular response to Hh signaling is mediated by the zinc finger type transcription factor Cubitus interruptus (Ci) [1]. Ci is regulated post-translationally by Hh signaling. In the absence of Hh signaling, Ci protein is cleaved into a truncated repressor form, CiR, which inhibits transcription of target genes. In the presence of Hh signaling, the proteolytic processing of Ci is suppressed to form an activator Ci, CiA [2]. While both CiA and CiR can bind to the sim*
Corresponding author. Fax: +81 75 751 7529. E-mail address: [email protected] (M. Ishibashi). 1 Present address: Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.015
ilar sequence (Gli-binding site, see below), previous works indicated that there is some preference between CiA and CiR for binding in different genes. For example, the Glibinging site of the dpp gene responds to both CiA and CiR [3]. In the case of Patched, only CiA binds to its Glibinding site [3]. The mechanisms of this preference remain elusive. In vertebrates, there are three Ci homologs, Gli1, Gli2 and Gli3 [4]. Unlikely fly Ci, mouse Gli1 contains only an activator domain and is not subject to proteolytic processing [5]. Therefore, Gli1 functions only as a transcriptional activator. On the other hand, mouse Gli2 and Gli3 contain repressor and activator domains on their N- and C-terminal, respectively, as fly Ci does [5]. Similarly to Ci, Gli3 can be proteolytically processed into a C-terminal truncated repressor, and this processing is inhibited by Hh signaling. Expression analysis of marker genes has shown that Gli3 functions mainly as a transcriptional repressor [6], though recent studies have also shown that Gli3 can act as a weak activator in vivo [7–9]. Most of Gli3 protein is cleaved to generate a Gli3-83 transcriptional repressor in
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the absence of Shh signaling. In the presence of Shh, Gli3 protein is thought to remain full-length (Gli3-190) and act as an activator [10–12]. Mouse Gli2 appears to exhibit distinct transcriptional activity from that of Gli3 while they share a high degree of sequence similarity. The C-terminal truncated forms act as dominant negatives [7]. Gli2 protein has been thought to be processed to form both an activator and a repressor. In vitro reporter assays have shown that Gli2 exhibits a stronger transcriptional activity than Gli3, albeit Gli2 is a weaker activator than Gli1 [5]. In mice, Gli2 is required for the most ventral neural progenitors in the developing spinal cord [13]. A definitive answer to the question of whether Gli2 functions only as a transcriptional activator or as both an activator and a repressor in vivo has not been achieved. Several lines of evidence suggest that Gli2 may normally function only as a transcriptional activator. For example, the loss-of-function mutation of mouse Gli2 resulted in loss of the most of ventral cell types in the developing neural tube [14,15]. Furthermore, Gli1 can rescue Gli2 mutant phenotypes when it is inserted into the Gli2 locus [13], but Gli3 cannot [10]. However, recent genetic analysis of Gli2 mutants on the Gli3 null background has also indicated that Gli2 could have repressor activity in addition to its activator function [11,16]. In zebrafish, Gli2 may act as a transcriptional activator or a repressor, depending on different genes in different tissues [17]. In mice, the vast majority of Gli2 protein is thought to be present in the fulllength activator form and only a small fraction might exist in the truncated repressor form [6]. An antiapoptotic factor BCL2 is regulated by Gli1 and Gli2 [18,19]. The human BCL2 promoter region has three Gli-binding sites [20] and one of these binding sites is critical for conferring Gli2-specific activation. Other two Glibinding sites are dispensable while they cooperate with the critical site in transactivation of BCL2 [19]. Little is known about how Gli2 transcriptional activity is regulated to activate target gene expression in response to Hh signaling. Fgf15 is a member of the Fibroblast growth factor (Fgf) family. Its expression in the diencephalon and midbrain is directly initiated by Shh signaling [21,22]. We previously demonstrated that the 3.6-kb Fgf15 enhancer/promoter region has a Gli-binding site (GliBS) at 1-kb upstream of the transcription start site. This site is required for Fgf15 expression in the medial/ventral diencephalon/midbrain in transgenic embryos and for activation in luciferase assay. Luciferase assay indicated that only Gli2 activates the Fgf15 enhancer/promoter through the GliBS. When the GliBS was mutated, activation was not completely abolished in luciferase assay. These results led us to the idea that there should be additional Gli2-specific responsive elements in the enhancer/promoter region. In this study, we identified two additional Gli-binding sites which are involved in regulation by Gli2. Luciferase assay demonstrated that Gli2 regulates Fgf15 expression
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through these sites. We designated them as Gli-responsive elements (GliREs). ChIP assay suggested that Gli2 directly binds to GliREs. Chimeric protein study indicated that the Gli2 activator form binds to the GliBS and the Gli2 repressor form binds to the GliREs. The results imply that the repressor form Gli2 binds to the GliREs to control the amount of Fgf15 expression. Materials and methods Plasmid construction and mutagenesis. The 3.6-kb Fgf15 enhancer/ promoter was inserted into the luciferase vector and Gli-binding site was mutated as described previously [22]. The 2-kb and 1.4-kb Fgf15 50 franking regions were also subcloned into pGL3-Basic reporter (Promega). The 8GliRE::luciferase reporter plasmids were generated by subcloning eight tandemly repeated copies of GliRE (50 GCCGTCGACGGTGCTGCCACCCGCATGCCTCGAGGCC-30 ) into the pGL3-d51 luciferase reporter. For luciferase assays shown in Fig. 2, the reporter plasmids were constructed by inserting the fragments of the Fgf15 enhancer/promoter into pGL3-d51-GliBS as indicated in the figure. pGL3-d51-GliBS luciferase reporter was constructed by inserting Gli-binding site of Fgf15 enhancer/promoter into pGL3-d51 luciferase reporter. No. 6 and 7 plasmids were constructed by inserting the PCR products (by KOD-Plus (TOYOBO, Japan)) into pGL3-d51-GliBS. Mutations were introduced into the GliREs by PCR-based site directed mutagenesis. To construct VP-Gli2ZF chimeric protein, the HindIII–BamHI DNA fragment that covers the entire five zinc finger domains of mouse Gli2 was fused to the C-terminus of the HSV VP16 activation domain. The resultant expression vector was designated as pVP-Gli2ZF. Luciferase assay. Luciferase assays were performed as described previously [22]. DNA transfection was performed with FuGene6 (Roche) according to the manufacturer’s instructions. C3H10T1/2 cells were transfected with the expression vector of Gli2 or pVP-Gil2ZF and the reporter plasmids. All luciferase assays were done in duplicates and repeated three times. Chromatin immunoprecipitation assay. ChIP assay was performed according to the manufacturer’s manual (LP Bio, Chromatin Immunoprecipitation Kit, KA-0137) using anti-FLAG antibody (Sigma, M2). C3H10T1/2 cells were transfected with FLAG-tagged mouse Gli2 (FLAGGli2) [23] and FLAG-tagged Hes1 (FLAG-Hes1) [24] expression vectors and the reporter plasmid.
Results Identification of two putative Gli-responsive elements in the 3.6-kb Fgf15 enhancer/promoter Previously, we reported that the 3.6-kb 50 -flanking enhancer/promoter of the Fgf15 gene is necessary and sufficient for induction of Fgf15 in the medial/ventral diencephalon/midbrain by transgenic analysis [22]. Luciferase assay showed that the 3.6-kb Fgf15 enhancer/promoter is activated by Gli2 transcription factor. A GliBS is located 1-kb upstream of the transcriptional start site. It is required for expression in the medial/ventral diencephalon/midbrain in the transgenic embryos and for activation in luciferase assay [22]. These results suggest that Gli2, but not Gli1, directly can interact with GliBS and transactivate the 3.6-kb Fgf15 enhancer/promoter. While mutation of this GliBS led to loss of expression in the medial/ventral
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diencephalon/midbrain in vivo, up-regulation of luciferase activity was not completely abolished, suggesting the presence of additional Gli-responsive elements. We performed luciferase assay to explore additional elements in the 3.6-kb Fgf15 enhancer/promoter required for expression of the Fgf15 gene and for interaction with GliBS and/or Gli2 transcription factor. First, to locate interactive elements with Gli2-mediated activity in the 3.6-kb Fgf15 enhancer/promoter region, we constructed a new deletion series of luciferase reporter vectors containing 2-kb (CN5) and 1.4-kb (CN3) 50 -flanking Fgf15 enhancer/promoter (Fig. 1). Secondly, we introduced base-substitution mutations that destroy the core GliBS (CN5-GliBSmt, CN3-GliBSmt, in Fig. 1). Gli2 transcription factor activated the 3.6-kb (CN7) and CN5 Fgf15 enhancer/promoter regions and the GliBS mutation resulted in significant decrease of the transcriptional activity (Fig. 1, [22]). In contrast, the GliBS mutation in the CN3 of the Fgf15 enhancer/promoter did not cause significant change of transcriptional activity by Gli2. These data suggest that the 620-bp sequence, between CN5 and CN3 of the Fgf15 enhancer/promoter includes interactive elements with the Gli2-mediated transcriptional activity. To identify essential regions interacting with Gli2 and/ or the GliBS of Fgf15 enhancer/promoter, seven luciferase reporter constructs containing deletion series of the 620-bp Fig. 2. Deletion analysis of the 50 -flanking 620-bp Fgf15 enhancer/ promoter region revealed the essential elements for the transcriptional activity of Fgf15 by Gli2. (A) Schematic representation of the EcoRV– KpnI 620-bp fragment of the Fgf15 enhancer/promoter and serial deletion constructs. These constructs include the GliBS of Fgf15 enhancer/ promoter and the chicken d-crystallin promoter. The 20-bp sequence around the SphI site that was required for transactivation by Gli2 is indicated by the open box. (B) The series of deletion constructs were cotransfected with the Gli2 expression vector. Luciferase activities were indicated as fold activation relative to the control. No. 1, 2, 4 and 7 constructs were activated by Gli2, respectively.
Fig. 1. Additional Gli-binding sites possibly exist in the 3.6-kb Fgf15 enhancer/promoter region. (A) Schematic representation of the Fgf15 enhancer/promoter luciferase reporter vectors. The restriction enzyme sites used for construction of deletion constructs are shown as indicated. (B) The Fgf15 enhancer/promoter with luciferase reporter was cotransfected into C3H10T1/2 cells with the Gli2 expression vector. Luciferase activities were indicated as fold activation relative to the control. Transactivation of CN7 (3.6-kb) and CN5 (2-kb) by Gli2 was significantly reduced by mutation of the GliBS. In contrast, there was no significant change when the GliBS of CN3 (1.4-kb) was mutated.
fragment were fused with GliBS (Fig. 2). As shown in Fig. 2, Gli2 up-regulated luciferase activity with the constructs No. 1, 2, 4, 7 and not with No. 3, 5 and 6. These results indicated that the 20-bp region flanking SphI site was required for up-regulation by Gli2. We identified two similar sequences to the GLI-binding consensus sequence [20] around the SphI site (Fig. 3A). These 9-base sequences had 3-base mismatch with human GLI-binding consensus sequences, and we designated these sequences as GliREs. The above results suggest that Gli2 binds to GliREs to regulate the transcriptional activity of Fgf15 enhancer/promoter. To confirm that these GliREs are involved in the transcriptional activity by Gli2, the luciferase reporter vector with eight copies of the first GliRE sequence was co-transfected with the Gli2 expression vector into the C3H10T1/2 cells. Then 60-fold up-regulation of the luciferase activity by Gli2 was observed (Fig. 3B).
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Fig. 3. Location of two putative Gli-binding sites upstream to GliBS, called GliREs. (A) Two putative GliREs were present around the SphI site within the 620-bp fragment. The core region of the putative GliREs contained three nucleotide-mismatch to the consensus Gli-binding sequence. Nucleotide substitution mutations were introduced into this Gli-binding sequence (GliREmt) to disrupt its binding to Gli2. (B) The luciferase reporter with eight times multimerized Gli-responsive element (8GliRE) or eight times multimerized Gli-binding site (8GliBS) from the 3.6-kb Fgf15 enhancer/promoter was cotransfected with the Gli2 expression vector. Gli2 up-regulated the 8GliRE reporter by 60-fold and 8GliBS reporter by 120-fold. (C) The luciferase reporter including wild type (CN7), the mutant of the first GliRE (CN7-1mt) or the mutant of both GliREs (CN7-1.2mt) was co-transfected with the Gli2 expression vector. In comparison with CN7, the transactivation by Gli2 was significantly increased with CN7-1mt and CN7-1.2mt. (D) The structure of the chimeric protein expression vector (pVP-Gli2ZF). cDNA encoding the VP16 activation domain (VP16-AD) and the Gli2 zinc finger domain (Gli2ZF) were inserted downstream of the SV40 promoter (P). (E) The Gli2 expression vector or pVP-Gli2ZF was co-transfected with CN7 and CN7-1.2mt reporter. Up-regulation of luciferase activity was significantly enhanced by pVP-Gli2ZF with the wild type enhancer/promoter (CN7), comparing with the Gli2 expression vector. With the mutant reporter (CN7-1.2mt), further up-regulation was observed when the reporter was co-transfected with the Gli2 expression vector (compare columns 3 and 1). This further up-regulation was cancelled with pVP-Gli2ZF (compare columns 3 and 4).
Next, we introduced base-substitution mutation at the 2nd–4th bases of the GliRE in the 3.6-kb fragment to obliterate binding capability. One mutant reporter (CN7-1mt) contains the mutation only in the first GliRE. The other
vector (CN7-1.2mt) carries mutation in both GliREs. Interestingly, these mutants showed a significant increase of transcriptional activity by Gli2 (Fig. 3C). The enhanced increase of luciferase activity by GliRE mutation indicates
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the presence of the repressor form of Gli2 (Gli2R) and its binding to the GliREs. Our data suggest that the GliREs interact preferentially with Gli2R in the context of 3.6-kb of the Fgf15 regulatory region.
demonstrates that Gli2 directly binds to the GliREs in the 360-bp Fgf15 enhancer/promoter region.
Discussion Gli2 may be processed to form Gli2R which interact with the GliREs in the Fgf15 enhancer/promoter To confirm whether Gli2 functions as a transcriptional repressor via the GliREs, we engineered a chimeric protein of the Gli2 DNA-binding domain (zinc finger domain [ZF]) and the transcriptional activator domain of HSV VP16 (Fig. 3D, [25]). This chimeric protein should act exclusively as an activator through Gli2-binding sequences. The pVP16-Gli2ZF expression vector was co-transfected with CN7 or the double GliRE mutant (CN7-1.2mt). As shown in Fig. 3E, CN7 was more extensively up-regulated by VP16-Gli2ZF than Gli2 itself (compare columns 2 and 1 in Fig. 3E), suggesting that some ratio of Gli2 protein acts as repressors. Increased up-regulation of the CN7-1.2mt by Gli2 was almost completely cancelled by VP16-Gli2ZF (compare columns 3 and 4 in Fig. 3E), and the level of up-regulation was almost same as that with the combination of CN7 and Gli2 (compare columns 4 and 1 in Fig. 3E). These results suggest that the repressor form of Gli2 suppresses up-regulation of Fgf15 by the activator form of Gli2 to some extent and that this suppression was mediated by GliREs. Gli2 directly binds to the GliREs in the Fgf15 enhancer/ promoter To demonstrate that Gli2 directly binds to the GliREs in the Fgf15 enhancer/promoter region, we performed ChIP assay with FLAG-Gli2 and the 360-bp Fgf15 enhancer/ promoter DNA fragment. This DNA fragment has two GliREs and does not include GliBS (Fig. 4A). FLAGHes1 expression vector was used as a negative control because there is no Hes1-binding site in the 360-bp fragment. As shown in Fig. 4B, the DNA fragment including GliREs was amplified from the precipitates with FLAGGli2. No band was detected with FLAG-Hes1. This result
Our previous study indicated that the Fgf15 expression in the medial/ventral diencephalon/midbrain is directly initiated by Shh signaling, most likely through Gli2. We found a Gli-binding consensus sequence (GliBS) 1-kb upstream of the transcriptional start site, it was essential for expression in the medial/ventral diencephalon/midbrain in the transgenic embryos and for activation in luciferase assay. However, mutation of the GliBS did not completely abolish the increase of luciferase activity [22]. We assumed that there should be additional Gli-responsive elements. Sequence analysis revealed two putative Glibinding sites. Our data suggest that these two sites, GliREs, directly interact with Gli2 and may cooperate with the critical GliBS in regulation of transcriptional activity by Gli2. Similarly, the human BCL2 cis-regulatory region has three Gli-binding sites and only one of them is critical for Gli2specific activation of the BCL2 promoter [19]. Other two sites are dispensable, but still cooperative with the critical site [19]. In cultured cells, truncation of the activation domain in the C-terminal half of Gli2 results in a transcriptional repressor [5]. Although several studies have suggested that Gli2 functions only as a transcriptional activator [13–15], it has been reported that both the full-length activator form, Gli2-185, and the repressor form, Gli2-78, exist in vivo [6]. Furthermore, genetic analysis of Gli2 mutants on a Gli3 null background has also indicated that Gli2 could compensate repressor activity of Gli3 [11,16]. In zebrafish, Gli2 may act as a transcriptional activator or a repressor, depending on different target genes in different tissues [17]. Since Gli2 is expressed uniformly in the neural tube, it is reasonable to assume that the ratio of activator and a repressor forms determines amount of target gene expression. This ratio makes a gradient, depending on the strength of Shh signaling. Our results in this study further support that Gli2 might have the transcriptional bipotentiality, both an activator
Fig. 4. Gli2 directly interacts with these Gli-responsive elements in Fgf15 enhancer/promoter region. (A) The structure of the reporter plasmid. The BamHI–KpnI fragment of Fgf15 enhancer/promoter included two GliREs. The left and right primers used for PCR was indicated. (B) ChIP assay was performed on the reporter plasmid with FLAG-Gli2 or FLAG-Hes1 expression vector. FLAG-Hes1 was used as a negative control.
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and a repressor. Mutation of the GliREs in the 3.6-kb Fgf15 enhancer/promoter (CN7-1mt, CN7-1.2mt) resulted in significant increase of the luciferase activity (Fig. 3C). The data indicate that the repressor form Gli2 may interact with GliREs and attenuate the transcription of Fgf15. VP16-Gli2ZF, the strong transcriptional activator, further up-regulated the luciferase activity with the wild type 3.6kb Fgf15 enhancer/promoter. Disruption of the GliREs led to loss of this further up-regulation by VP16-Gli2ZF (Fig. 3E). These results suggest that VP16-Gli2ZF might interact directly to the GliREs and act as a transcriptional activator in vitro. We propose that Gli2 is present in both the full-length activator form and the processed repressor form [5,6] and regulates transcriptional levels by binding to GliBS and GliREs, depending on the context of Glibinding sites. The results are different from the case of the BCL2 enhancer [19]. This conclusion seems to be rather paradoxical because the residual activation of the Fgf15 3.6-kb enhancer/promoter with GliBSmt implies the binding of the Gli2 activator to the GliREs (Fig. 1). Also, combination of the GliREcontaining short fragment and GliBS showed up-regulation through the GliREs (Fig. 2). Moreover, the 8GliRE construct was substantially activated by Gli2 (Fig. 3B). These results suggest that the activator form of Gli2 can also bind to the GliREs and activate the enhancer through these sites in the absence of the GliBS or with the short fragment of the regulatory region. However, in the Fgf15 3.6-kb enhancer/promoter with the intact GliBS, mutation of the GliREs led to further up-regulation by Gli2, suggesting that the repressor form of Gli2 preferentially binds to these sites in this context. In addition, with the wild type Fgf15 3.6-kb enhancer, the VP16-Gli2ZF chimeric protein up-regulates the enhancer more than naive Gli2 itself. This effect of the VP16-Gli2ZF was completely reversed by mutation of the GliREs (Fig. 3E). These data suggest that, in more physiological conditions, the GliREs interact preferentially with the repressor form of Gli2 to regulate the expression of Fgf15.
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[13] [14]
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Acknowledgments We thank Dr. Sasaki for Gli2 and FLAG-Gli2 expression vector, Dr. Kageyama for FLAG-Hes1 expression vector and Dr. Kamachi for pd51lucII, and Dr. Aspland and Murre for pGL3-Fgf15 promoter. This study was supported by The Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant No. 15689004, 16015264, 18590168) and Kato Memorial Bioscience Foundation.
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