Characterization of the zebrafish cx36.7 gene promoter: Its regulation of cardiac-specific expression and skeletal muscle-specific repression

Gene 577 (2016) 265–274

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Research paper

Characterization of the zebrafish cx36.7 gene promoter: Its regulation of cardiac-specific expression and skeletal muscle-specific repression Hisako Miyagi, Kakon Nag 1, Naznin Sultana, Keijiro Munakata, Shigehisa Hirose, Nobuhiro Nakamura ⁎ Department of Biological Sciences, Tokyo Institute of Technology, 4259-B13 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

a r t i c l e

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Article history: Received 21 July 2015 Received in revised form 28 October 2015 Accepted 3 December 2015 Available online 9 December 2015 Keywords: Connexin GATA Heart Repressor Skeletal muscle Transgenic zebrafish

a b s t r a c t Zebrafish connexin 36.7 (cx36.7/ecx) has been identified as a key molecule in the early stages of heart development in this species. A defect in cx36.7 causes severe heart malformation due to the downregulation of nkx2.5 expression, a result which resembles congenital heart disease in humans. It has been shown that cx36.7 is expressed specifically in early developing heart cardiomyocytes. However, the regulatory mechanism for the cardiac-restricted expression of cx36.7 remains to be elucidated. In this study we isolated the 5′-flanking promoter region of the cx36.7 gene and characterized its promoter activity in zebrafish embryos. Deletion analysis showed that a 316-bp upstream region is essential for cardiac-restricted expression. This region contains four GATA elements, the proximal two of which are responsible for promoter activation in the embryonic heart and serve as binding sites for gata4. When gata4, gata5 and gata6 were simultaneously knocked down, the promoter activity was significantly decreased. Moreover, the deletion of the region between −316 and −133 bp led to EGFP expression in the embryonic trunk muscle. The distal two GATA and A/T-rich elements in this region act as repressors of promoter activity in skeletal muscle. These results suggest that cx36.7 expression is directed by cardiac promoter activation via the two proximal GATA elements as well as by skeletal muscle-specific promoter repression via the two distal GATA elements. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Heart development is a highly conserved, complex process in vertebrates, including cardiovascular progenitor specification and migration, the formation and rotation of the myocardial tubule, and tubular heart looping. Coordinated expression of transcription factors in the early developing heart regulates cardiomyocyte differentiation and morphogenesis. Genetic studies using Drosophila melanogaster have established the identity of certain transcription factors that regulate these cardiac processes (Tao and Schulz, 2007). Orthologous genes have been identified in vertebrates, such as the GATA-binding factor (GATA), NK2 transcription factor (NKX2), myocyte enhancer factor 2 (MEF2) and T-box (TBX) families. These gene products are expressed in early cardiac progenitor cells and cooperate with each other as well Abbreviations: Cx36.7, connexin 36.7; NKX2, NK2 transcription factor; MEF2, myocyte enhancer factor 2; CHD, congenital heart disease; hpf, hours post-fertilization; EGFP, enhanced green fluorescent protein; RLM-RACE, RNA-ligase mediated rapid amplification of cDNA ends; PCR, polymerase chain reaction; MO, morpholino antisense oligonucleotide; dpf, day(s) post-fertilization; NKE, Nkx2.5-binding element. ⁎ Corresponding author. E-mail addresses: [email protected] (H. Miyagi), [email protected] (K. Nag), [email protected] (N. Sultana), [email protected] (K. Munakata), [email protected] (S. Hirose), [email protected] (N. Nakamura). 1 Current address: Biomaterials Center for Regenerative Medical Engineering, Foundation for Advancement of International Science, 3-24-16 Kasuga, Tsukuba 3050821, Japan.

http://dx.doi.org/10.1016/j.gene.2015.12.013 0378-1119/© 2015 Elsevier B.V. All rights reserved.

as with other transcriptional regulators to promote heart development. Mutations in GATA4, NKX2-5 and TBX5, respectively, are associated with congenital heart disease (CHD) in humans (McCulley and Black, 2012). Over the last decade, the zebrafish (Danio rerio) has emerged as one of the ideal model organisms for studying heart development. Unlike mammals, zebrafish embryos do not completely depend on a functional cardiovascular system for survival, because their small size allows them to receive oxygen by passive diffusion (Pelster and Burggren, 1996). This feature enables embryos with cardiovascular defects to survive during embryogenesis. In addition, the rapid development and optically transparent nature of zebrafish embryos provide advantages for the in vivo imaging of cellular structure and dynamics in the course of heart development. Moreover, in the zebrafish it is relatively easy to link genotype to phenotype by forward and reverse genetic approaches (Bakkers, 2011). Sultana et al. (2008) previously reported the zebrafish mutant futka (ftk) that exhibits severe cardiac phenotypes, including thin, dilated heart chambers, an irregular and slow heartbeat, reverse blood flow and disorganized myofibril orientation. In addition, the expression of nkx2.5 is downregulated during early heart development, leading to the cardiac phenotype of the ftk mutants. A missense loss-of-function mutation in connexin 36.7 (cx36.7/ecx) is the cause of the cardiac phenotypes in ftk (Sultana et al., 2008). Connexin, a tetra-spanning transmembrane protein, assembles into a hexameric pore structure called a

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2.2. RNA-ligase mediated rapid amplification of cDNA ends (RLM-RACE) To determine the transcription initiation site, 5′ RACE was performed on total RNA prepared from 16-hpf zebrafish embryos with First Choice RLM-RACE kit (Ambion, Austin, TX) according to the manufacturer's instructions. The sequences of the cx36.7-specific outer and inner primers were 5′-ctcctgctcgtcagtgtaga-3′ and 5′atcgaattcagcagcgtccattctgtcat-3′, respectively. 2.3. Construction of cx36.7 promoter–EGFP reporter plasmids

Fig. 1. Nucleotide sequence of the 5′-flanking region of the zebrafish cx36.7 gene. The transcription initiation site determined by RLM-RACE is indicated as +1 (arrow). The ATG translation start codon is in bold. The putative transcription factor-binding elements are boxed.

“connexon” (hemi-channel) in the plasma membrane, which often docks with another connexon on an adjacent cell to form a gap junction. In ftk mutants, a single amino-acid substitution (D12V) in cx36.7 causes a defect in trafficking to the cell surface, which is thought to be likely to prevent intercellular and/or intracellular signaling via cx36.7 (Sultana et al., 2008). The expression of cx36.7 mRNA is initially detectable in cardiac precursor cells in 50% epiboly embryos [5 hours post-fertilization (hpf)] and is almost absent at 48 hpf, at which time heart looping has been completed and functional valves are formed (Sultana et al., 2008). Therefore, cx36.7 is likely to mediate signaling so as to sustain the nkx2.5 expression that is essential for proper myofibril orientation and cardiac morphogenesis. Since inactivation of NKX2-5 is linked to CHD, ftk mutants have utility as a model for studying the molecular mechanisms of CHD pathogenesis as well as heart development. However, the regulatory mechanism underlying the cardiac-specific expression of cx36.7 remains unknown at present. In this study, to investigate the transcriptional regulation of cx36.7 gene expression, we characterized the cx36.7 promoter activity in zebrafish embryos by using an enhanced green fluorescent protein (EGFP)-reporter gene. The results show that cx36.7 expression is regulated by two types of GATA elements: one that promotes transactivation in the embryonic heart by binding to gata4, gata5 and gata6, while the other suppresses promoter activity in skeletal muscle.

2. Materials and methods 2.1. Zebrafish culture The wild-type zebrafish (a cross between the AB and TL lines) were provided by Dr. Atsushi Kawakami (Tokyo Institute of Technology, Yokohama, Japan). The zebrafish were maintained as previously described (Saito et al., 2010). The animal protocols and procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Institute of Technology.

Serial deletion fragments of the 5′ flanking region of the cx36.7 gene were amplified by polymerase chain reaction (PCR) from zebrafish genomic DNA with a common reverse primer (5′gaagtcgaccactgctcccagccatg-3′) and the following forward primers: 5′-gaactcgagtggtctaggtatttcagaaac-3′ for − 1600/− 1; 5′-gaactcgag ctataattttgagccatgcaa-3′ for − 500/− 1; 5′-gaactcgagatccatttagcagac ttttgcc-3′ for − 316/− 1; 5′-gaactcgaggtaatctgccgacgctatcag-3′ for − 133/− 1; and 5′-gaactcgagcctgctttctaaatgcactcg-3′ for − 110/− 1. The PCR products were then inserted into the SalI/XhoI sites of a pT2KXIGΔin vector (Kawakami and Shima, 1999), yielding Tol2 transposon plasmids containing the 5′-flanking region linked to an EGFP reporter. To obtain the 15-bp promoter construct, a pair of oligonucleotides (5′-gagggccctggctgggagcagtggtcgacag-3′ and 5′ctgtcgaccactgctcccagccagggccctc-3′) were annealed, digested with ApaI and SalI, and then inserted into the ApaI/SalI sites of a pT2KXIGΔin vector. Point mutations were introduced by PCRmediated site-directed mutagenesis, and their sequences are listed in Supplementary Table 1. The sequences of all of the constructs were verified by DNA sequencing. 2.4. Transient EGFP reporter assay and generation of stable transgenic zebrafish lines The mRNA encoding medaka fish Tol2 transposase was synthesized by in vitro transcription from a pCS2+ vector encoding Tol2 transposase (Kawakami and Shima, 1999) with the SP6 mMESSAGE mMACHINE kit (Ambion). The Tol2 transposase mRNA (250 ng) was then mixed with the Tol2 transposon constructs (250 ng) and phenol red dye (1 μl; Sigma-Aldrich, St. Louis, MO) in 10 μl of 1 × Danieau buffer [58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2 and 5 mM HEPES, pH 7.6]. The mixture (5 pl) was injected into the yolk of fertilized eggs at the one-cell stage. EGFP signals were observed by using the fluorescent microscopes Leica MZ16F (Leica, Wetzlar, Germany) and Zeiss Axio Zoom.V16 (Carl Zeiss, Jena, Germany). To generate stable transgenic zebrafish lines, the injected embryos were raised to adulthood and outcrossed with wild-type zebrafish. Their progeny were screened for heart-specific EGFP expression at 1 dpf. EGFP-positive progeny (F1 generation) were outcrossed with wild-type zebrafish to generate F2 progeny. The EGFP-positive F2 adult fish were incrossed to obtain homozygous transgenic lines. 2.5. Gene knockdown with morpholino antisense oligonucleotides (MOs) MOs that had previously been shown to target gata4, gata5, gata6 and nkx2.5 were synthesized by Gene Tools (Philomath, OR) (Holtzinger and Evans, 2005, Sultana et al., 2008, Tseng et al., 2011). Standard Control Oligo (5′-cctcttacctcagttacaatttata-3′; Gene Tools) was used as a negative control MO that targets a human beta-globin intron mutation. The MOs were dissolved in 1× Danieau buffer at a concentration of 2 ng/μl. Approximately 10 pg of the MOs, with 5 pl of phenol red dye (0.5% in D-PBS) as a tracer, was injected into the yolk of fertilized eggs at the one-cell stage. For rescue experiments, the pCS2+ vector encoding either gata4, gata5 or gata6 was linearized by NotI, and used as a template to synthesize capped and polyadenylated mRNA with the mMESSAGE mMACHINE SP6 transcription kit.

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Fig. 2. Deletion analysis of the cx36.7 promoter in 1-dpf zebrafish embryos. (A) Top: schematic representation of the 5′-flanking region of the cx36.7 gene, depicting the positions of the GATA (filled circle), A/T-rich (open triangle), E-box (filed triangle), NKE (filed reversed triangle) and Sp1 (open reversed triangle) elements. Bottom: schematic representation of the 5′ deleted promoter–EGFP chimeras that were inserted between the tol2 elements in the pT2KXIGΔin vector. The reporter constructs were injected into fertilized one-cell eggs. EGFP signals in transgenic zebrafish were analyzed at 1 dpf. The bar graphs represent the percentage of embryos with cardiac-specific EGFP expression. Data are expressed as the mean ± SEM of at least three independent experiments. Significant differences for the value of the 133-bp promoter construct (⁎⁎, p b 0.001) were found by one-way ANOVA with Tukey's post-hoc test. ns, no significant difference. (B–H) Representative images of 1-dpf embryos showing no EGFP expression (B and H) or cardiac-specific EGFP expression (C–G, arrows). Bar, 1 mm.

Partial open reading flames of zebrafish cx36.7 (nucleotides +1 to + 500), gata4 (+ 1 to + 500), gata5 (+ 1 to + 580), gata6 (+ 1 to +540) and nkx2.5 (+1 to +540) were inserted into pBlueScript SK− plasmid vectors (Stratagene, La Jolla, CA). Using the linearized plasmid vectors as templates, digoxigenin (DIG)-labeled antisense RNA probes were synthesized by in vitro transcription with T3 and T7 RNA polymerases (Stratagene) and DIG RNA labeling Mix (Roche, Mannheim, Germany). Whole mount in situ hybridization was performed as described (Esaki et al., 2009).

unlabeled complementary oligonucleotides. The mutant probes contained point mutations (tatc→gatc) in the GATA #1 and GATA#2 elements. The probes (10 pmol) were then incubated with 20 μl of streptavidin agarose beads (Pierce, Rockford, IL) in PBS. Zebrafish gata4 was in vitro translated by using the TNT T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI) with [35 S]methionine and [35S]cysteine (PerkinElmer, Boston, MA) according to the manufacturer's instructions. The labeled proteins (5 μl) were incubated with the probe-bound beads in 500 μl of PBS at 4 °C overnight. After washing four times with PBS, the beads and the labeled proteins (2 μl; 40% of the input) were incubated with Laemmli buffer (60 mM Tris–HCl, pH 6.8, 2% SDS, 8% glycerol, and 0.005% bromophenol blue), which was then subjected to by SDS-PAGE. The radioactive signals were analyzed by using FLA7000 (Fujifilm, Tokyo, Japan).

2.7. In vitro pull down assay

2.8. Quantification of EGFP signal intensity and statistical analysis

Probes were produced by annealing biotinylated oligonucleotides corresponding to nucleotides − 1 to − 73 and − 73 to − 133 with

EGFP signal intensity was quantified using the ZEN lite software (Carl Zeiss). Differences between groups were evaluated by one-way

Approximately 30 pg of the synthesized mRNA was injected into onecell stage fertilized eggs together with 10 pg of the MOs. 2.6. In situ hybridization

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(Fig. 2, A and G). In addition, the intensity of the cardiac EGFP signal was significantly reduced compared with that of embryos injected with the longer promoter constructs (Supplementary Fig. S2A). A further deletion up to −15 bp lacked promoter activity (Fig. 2, A and H). These results suggest that the 5′-flanking region between − 133 and −15 bp contains cis-acting regulatory elements that control the early cardiac-specific expression of cx36.7. 3.3. Identification of cardiac-specific regulatory elements

Fig. 3. Mutational analysis of the 316-bp promoter in 1-dpf zebrafish embryos. Schematic representation of the 316-bp cx36.7 promoter–EGFP chimera and its mutants with point mutations introduced into the GATA #1 (tatc → tgga), GATA #2 (tatc → tgga), GATA #3 (tatc → cggg), GATA #4 (tatc → gatc), A/T-rich (atatttttaa → atatttgcga), E-box (cacgtg → ccgggg) and NKE (tgaagtg → tgatgcg) elements. The reporter constructs were injected into fertilized one-cell eggs. EGFP signals in the injected zebrafish were analyzed at 1 dpf. The bar graphs represent the percentages of embryos with cardiac-specific EGFP expression. Data are expressed as the mean ± SEM of at least three independent experiments. Significant differences from the value of the 316-bp promoter construct ( ⁎⁎ , p b 0.001) were found by one-way ANOVA with Tukey's post-hoc test. ns, no significant difference.

ANOVA followed by Tukey's post hoc test or Student's t-test. The probability value p b 0.05 was considered as statistically significant. 3. Results 3.1. Identification of the zebrafish cx36.7 promoter region A search of the NCBI and Ensembl databases revealed that the zebrafish cx36.7 gene contains a single exon. The transcription initiation site was determined by RLM-RACE, which amplifies only 5′ capped mRNA. The transcription initiation site (numbered as +1) was 118 bp upstream of the translation start codon (Fig. 1, arrow). Based on the genomic sequence information obtained from the Zebrafish Information Network (ZFIN; www.zfin.org), a set of primers was designed to amplify a 1.6-kbp 5′-flanking region of the cx36.7 gene by PCR from the zebrafish genome. To confirm the ability of the obtained PCR fragment to drive cardiac-specific expression in vivo, it was fused to the open reading frame of an EGFP reporter gene in the Tol2 transposon vector (pT2KXIGΔin). The reporter construct was injected into fertilized eggs at the 1-cell stage with the transposase mRNA, which allows the promoter–EGFP chimera to be integrated into the genome (Supplementary Fig. S1). At 1 day post-fertilization (dpf), EGFP expression was detected specifically in the heart of approximately 86% of the injected zebrafish embryos (Fig. 2, A and C). When a promoter-less EGFP construct was injected, no EGFP expression was seen in any of the embryos examined (Fig. 2, A and B). These results indicate that the 1.6-kb 5′flanking region acts as a promoter that drives EGFP expression, mimicking early cardiac expression of endogenous cx36.7. 3.2. Deletion analysis of the cx36.7 promoter in zebrafish embryos To narrow down the region required for cardiac-specific expression, we performed deletion analysis of the cx36.7 promoter. Progressive 5′ deletions of the cx36.7 promoter were constructed and their relative ability to induce EGFP expression was analyzed in zebrafish embryos at 1 dpf. Cardiac-specific EGFP expression was not significantly affected by deletions of up to − 133 bp (Fig. 2, A and D–F). Further deletions from −133 onward resulted in a progressive decrease in promoter activity. Cardiac EGFP expression was observed in approximately 45% of the 1-dpf embryos injected with the deletion construct up to −110 bp

Sequence motif searches with TRANSFAC predicted that the 316-bp promoter region contains several consensus sites for the binding of transcription factors, including the A/T-rich, GATA, E-box (CANNTG), Nkx2.5-binding (NKE; TNAAGTG), and Sp1-binding element (GGCT GGG) sites (Fig. 1). There is neither a TATA box nor a CAAT box. To determine if these sites are involved in cardiac-specific expression, we performed mutational analysis of the 316-bp promoter region. When point mutations were introduced into either one of two proximal GATA elements (GATA #1 and #2), the number of 1-dpf embryos with cardiac EGFP expression was significantly decreased, by approximately 40% and 35%, respectively (Fig. 3). Combining these mutations had an additive effect, with a further decrease of up to 18.8% (Fig. 3). In contrast, introduction of point mutations into either or both of the two distal GATA elements (GATA #3 and #4) had no effect on the promoter activity, although the embryos with cardiac EGFP expression appeared to be slightly but not significantly decreased (Fig. 3). Likewise, mutations introduced into the A/T-rich, E-box, or NKE elements had no or less effect on cardiac-specific EGFP expression at 1 dpf (Fig. 3). Quantification analysis showed that the intensity of cardiac EGFP signal was significantly reduced by point mutations into the GATA #1 and #2 elements (Supplementary Fig. S2B). Similar effects were observed in embryos injected with the 133-bp promoter construct (Supplementary Fig. S2C). These results suggest that the two proximal GATA elements act synergistically to activate promoter activity in the early developing heart. 3.4. Gata4, gata5 and gata6 transactivate the cx36.7 promoter in the early developing heart In the zebrafish, the three GATA-binding transcription factors gata4, gata5 and gata6 are predominantly expressed in the embryonic heart and play an essential role in myocardial gene expression and heart development (Serbedzija et al., 1998, Reiter et al., 1999, Reiter et al., 2001, Peterkin et al., 2003, Trinh et al., 2005). In situ hybridization experiments showed that the mRNA expression patterns of cx36.7 and the three gata genes are detected in the heart regions at 16 and 24 hpf (Fig. 4). We therefore sought to determine whether these gata proteins are involved in the transactivation of the cx36.7 promoter by analyzing the effects of their knockdown on EGFP expression. One-cell-stage fertilized eggs were injected with the 316-bp promoter construct along with control MOs, or MOs specific to gata4, gata5 and gata6, respectively. At 1 dpf, 77.3% of the embryos injected along with the control MOs exhibited cardiac-specific EGFP expression (Fig. 5). MO-mediated knockdown of gata4, gata5, and gata6 resulted in a significant decrease in the number of embryos exhibiting cardiac EGFP expression to 39.3%, 38.0%, and 34.7%, respectively (Fig. 5). These effects were rescued by coinjection of mRNA encoding gata4, gata5 or gata6, confirming MO specificity (Fig. 5, Rescue). Since the three gata proteins are known to be functionally redundant (Holtzinger and Evans, 2007, Peterkin et al., 2007), we performed double- and triple-knockdown experiments. As expected, a further reduction in promoter activity (2.3% to 10.3%) was observed by double gata knockdown (Fig. 5). The maximum inhibitory effect (1.3%) was reached by triple gata knockdown (Fig. 5), but we cannot exclude the possibility that this effect resulted from a loss of heart development in the morphants. We also observed that injection of nkx2.5-specific MOs did not significantly affect cardiac-specific EGFP

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Fig. 4. The expression of cx36.7, gata4, gata5, gata6 and nkx2.5 in zebrafish embryos. Whole mount in situ hybridization of zebrafish embryos at 16 hpf (top) and 24 hpf (bottom) was performed with antisense RNA probes specific to cx36.7, gata4, gata5, gata6 and nkx2.5. The cx36.7 expression was detected in the developing heart regions in which gata4, gata5, gata6 and nkx2.5 are expressed. Arrows, hearts; arrowheads, gut.

expression (Fig. 5; 80.7%), although nkx2.5 expression was detected along with cx36.7 in the heart regions (Fig. 4). In addition, in vitro pull down experiments revealed that gata4 directly binds to the GATA #1 and #2 elements (Fig. 6) Collectively these results suggest that gata4, gata5 and gata6 are responsible for cardiac-specific cx36.7 promoter activity as a result of binding to the proximal GATA elements. 3.5. Identification of the repressor elements that suppress promoter activity in skeletal muscle When the observations in the promoter deletion experiments were extended to the later developmental stages at 2 dpf and 3 dpf, we still detected cardiac-specific EGFP expression in embryos injected with the 316 bp (Fig. 7A, arrow and Fig. 8, WT) as well as longer promoter constructs (data not shown). Interestingly, 25.3% of the 2-dpf embryos injected with the 133-bp promoter construct exhibited EGFP expression in the trunk muscles as well as the heart (Fig. 7, B, arrowheads and C), and the number of such embryos increased at 3 dpf (Fig. 10, WT). Trunk muscle EGFP expression was also observed in a stable zebrafish

line carrying the 133-bp promoter construct [Tg(− 133cx36.7:EGFP)] but not the 500-bp promoter construct [Tg(−500cx36.7:EGFP)] (Fig. 7, E and F). We therefore hypothesized that the region between − 316 and −133 bp acts as a suppressor of promoter activity in skeletal muscle. To identify the region responsible for this suppression, we introduced point mutations into the distal GATA (GATA #3 and #4) and A/ T-rich elements of the 316-bp promoter, and assessed their effects on EGFP expression in skeletal muscle. Mutations in either or both of the distal GATA elements led to an apparent increase in EGFP expression in the trunk muscles from 2 dpf (Fig. 8, #3, #4, and #3 + 4 and Fig. 9, D, E, and F), which is consistent with the result obtained with the 133bp promoter construct (Fig. 10, WT). When the A/T-rich element was mutated, EGFP expression in skeletal muscle was clearly observed at 3 dpf, albeit to a lesser extent compared with the distal GATA mutations (Fig. 8, A/T and Fig. 9G). Mutations in both the distal GATA and A/T-rich elements had a similar effect of the distal GATA mutations (Fig. 8, the most rightward graphs and Fig. 9H), suggesting that the GATA elements have a strong effect on promoter inactivation. In contrast with these observations, trunk muscle EGFP expression was not significantly induced

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in the first GATA element (GATA #1) had no effect and exhibited an EGFP expression pattern similar to the embryos injected with the wild-type promoter construct (Fig. 10, A and B). In contrast, mutations in the second GATA element (GATA #2) resulted in a significant decrease in trunk muscle EGFP expression (Fig. 10, A and C). A similar inhibitory effect was observed when both of the two GATA elements were mutated (Fig. 10, A and D). These results suggest that the GATA #2 element plays an important role in activation of the 133-bp promoter in skeletal muscle. Next, to determine if gata4, gata5 and gata6 are involved in this promoter activation, we examined the effect of triple knockdown on EGFP expression in the trunk muscle of transgenic embryos with the 133-bp promoter construct containing the GATA #1 mutation. The ratio of embryos showing trunk muscle EGFP expression was comparable in the gata knockdown and control groups at 2 dpf (Fig. 11). We could not perform the quantification analysis at 3 dpf because many of the gata morphants had died. These results suggest that the promoter activation in skeletal muscle is likely to be independent of gata4, gata5 and gata6. 4. Discussion

Fig. 5. Effects of knockdown of cardiac gata and nkx2.5 on cx36.7 promoter activity. Fertilized one-cell eggs were injected with the 316-bp promoter construct along with negative control MOs, or MOs specific to gata4, gata5, gata6 and nkx2.5. EGFP signals in the injected zebrafish were analyzed at 1 dpf. For rescue experiments, mRNA encoding either gata4, gata5 or gata6 was co-injected with the promoter construct and the corresponding MOs. The bar graphs represent the percentages of embryos with cardiac-specific EGFP expression. Data are expressed as the mean ± SEM of at least three independent experiments. Significant differences from the value of control-MO-injected embryos (⁎⁎, p b 0.001) were found by one-way ANOVA with Tukey's post-hoc test. ns, no significant difference.

by the introduction of point mutations into the proximal GATA elements (GATA #1 and #2) (Fig. 8, #1, #2, and #1 + 2 and Fig. 9, A, B and C). These results suggest that the two distal GATA and A/T-rich elements serve as suppressors of cx36.7 expression in embryonic trunk muscle. 3.6. Skeletal muscle EGFP expression is dependent on the second proximal GATA element To investigate the promoter activation mechanism in skeletal muscle, we analyzed EGFP expression in embryos injected with the 133bp promoter constructs having proximal GATA mutations. Mutations

Fig. 6. Binding of gata4 to the proximal GATA elements. In vitro translated [35S]-labeled zebrafish gata4 (lane 1; 40% of the input) was incubated with streptavidin-agarose beads conjugated to biotinylated double-stranded DNA probes corresponding to the regions −1/−73 (lane 2) and −73/−133 (lane 4), or their mutants containing point mutations in the GATA #1 (lane 3) and #2 (lane 5) elements, respectively. For a negative control experiment, [35S]-labeled gata4 (lane 6; 40% of the input) was incubated with nonimmobilized streptavidin-agarose beads (lane 7). After washing beads, the bound materials were analyzed by SDS-PAGE followed by autoradiography to detect gata4.

In this study, in order to clarify the regulatory mechanism underlying the cardiac-specific expression of cx36.7, we performed in vivo EGFP reporter assays of the cx36.7 promoter in zebrafish embryos and determined the cis- and trans-acting factors that regulate the promoter activity. Deletion analysis showed that the 133-bp upstream region is sufficient for full promoter activity in the embryonic heart (Fig. 2 and Supplementary Fig. S2A). The activation of the cardiac promoter is mediated by the synergistic activity of the two GATA elements (GATA #1 and #2) to which gata4 binds (Figs. 3 and 6). Knockdown analysis revealed that gata4, gata5 and gata6 are required for this promoter activation (Fig. 5). In vertebrates, there are six GATA transcription factors (GATA1 through GATA6) that are classified into two subfamilies based on their expression patterns. GATA1, GATA2 and GATA3 are expressed in hematopoietic cells and are essential for T-lymphocyte, erythroid and megakaryocyte differentiation (Orkin, 1995). GATA4, GATA5 and GATA6 are required in the differentiation of the heart as well as endoderm-derived tissues, such as the gut and gonads (Laverriere et al., 1994), and the three forms share DNA binding sequences [(a/ t)gata(a/g)] (Molkentin The, 2000). The overlapping expression patterns and similar activation mechanisms are attributed to their redundant or overlapping biological function. The GATA-binding motifs are found in the promoter and enhancer regions of many cardiac-specific genes, such as cardiac troponin C (Ip et al., 1994), the myosin light chain I (Rotter et al., 1991), Na+/Ca2+ exchanger NCX1 (Koban et al., 2001) and A- and B-type natriuretic peptides (ANP and BNP) (Grepin et al., 1994). These are important for promoter activity, which is controlled by the cardiac GATA factors (McGrew et al., 1996, Di Lisi et al., 1998, Cheng et al., 1999, Peterkin et al., 2005). Similarly, our results suggest that the early cardiac expression of cx36.7 is regulated by gata4, gata5 and gata6, probably by binding to the two proximal GATA elements. Besides the GATA elements, the cx36.7 promoter contains several potential binding motifs for cardiomyogenic transcription factors, such as mef2 (A/T-rich), nkx2.5 (NKE) and basic helix-loop-helix (bHLH) factors (E-box). However, mutational analysis indicated that these motifs are unlikely to contribute to cardiac-specific promoter activation (Fig. 3 and Supplementary Fig. S2B). Unexpectedly, transgenic embryos with the 133-bp promoter construct exhibited EGFP expression in the skeletal muscle of the embryonic trunk (Figs. 7 and 10). This EGFP expression was dependent on the GATA #2 element but independent of gata4, gata5 and gata6 (Figs. 10 and 11). To the best of our knowledge, there is no report on the expression of the six GATA factors in zebrafish skeletal muscle. Indeed, in situ hybridization failed to detect the expression of gata4, gata5 and gata6 in the embryonic trunk muscle (data not shown). However, several studies have reported the presence of the GATA2 protein and GATA3

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Fig. 7. EGFP expression in transgenic embryos using the 316- or 133-bp promoter constructs. (A and B) One-cell stage fertilized eggs were injected with the 316- (A) or 133-bp (B and C) promoter constructs. At 2 dpf, EGFP expression was observed. Typical images of embryos with EGFP expression in the heart (arrows) and trunk muscle (arrowheads) are shown. (C) A high magnification image of the trunk region of the embryo in B. (D) An image of a control embryo without injection is shown. Zebrafish embryos exhibit high autofluorescence in the yolk sac (asterisk). (E and F) EGFP expression of 2-dpf stable zebrafish lines carrying the 500- (E) or 133-bp (F) promoter constructs. EGFP signal in the heart and trunk muscle is indicated by arrows and arrowheads, respectively. (G) An image of a control non-transgenic embryo is shown. Bars, 500 μm (A, B, and D–G) and 200 μm (C).

and GATA4 transcripts in rat skeletal muscle cells (Sakuma et al., 2003, Di Lisi et al., 2007, Downie et al., 2008). It is possible that the activation of cx36.7 promoter in the trunk muscle may be mediated by undetectable levels of the GATA factors or by different factors that bind to the GATA #2 element. We also found that the cx36.7 promoter activity is strongly repressed in the trunk muscle by the region between − 316 and − 133 bp (Fig. 7). The repressor activity is dependent on the GATA #3 and #4 elements, and partially on the A/T-rich element

Fig. 8. Mutational analysis of the 316-bp promoter. Fertilized one-cell eggs were injected with the 316-bp promoter construct (WT), or its mutants with point mutations in the GATA (#1–#4) and A/T-rich (A/T) elements, as described in the Fig. 3 legend. EGFP signals in the embryos were analyzed at 1, 2 and 3 dpf. The stacked bar charts represent the percentages of the following four groups of embryos: no EGFP expression (blue), cardiac-specific EGFP expression (green), EGFP expression in the heart and trunk muscle (red), and dead embryos (white). Data are expressed as the mean ± SEM of three independent experiments.

(Figs. 8 and 9), but the trans-acting mechanism was not determined in this study. Recently, GATA factors have been shown to act as a negative regulator of tissue/cell-specific gene expression in mammals. For example, the expression of mouse erythropoietin in renal tubular epithelial cells is repressed by the GATA element which is a target for GATA2 and GATA3, thereby establishing the inducible expression in response to hypoxia (Obara et al., 2008). GATA2 is expressed in preadipocytes and functions as a gatekeeper for adipogenesis through the suppression of the promoter activity of peroxisome proliferator-activated receptor γ (PPARγ) (Tong et al., 2000). GATA1 is essential for hematopoiesis through the regulation of hematopoietic gene expression, which includes gene repression (Grass et al., 2003, Rylski et al., 2003, Welch et al., 2004, Munugalavadla et al., 2005, Rodriguez et al., 2005, Jing et al., 2008, Chou et al., 2009, Tripic et al., 2009). In this process, GATA1 interacts with its co-factor Friend of GATA-1 (FOG-1), which recruits the co-repressor complexes CtBP (C-terminal binding protein) and NuRD (nucleosome remodeling and deacetylase), thereby enabling chromatin remodeling and transcriptional activation/repression (Bresnick et al., 2005). FOG-2 regulates GATA4-mediated transcription in the developing and adult heart (Lu et al., 1999, Svensson et al., 1999). Less is known about the impact of GATA elements on gene repression in skeletal muscle. Di Lisi et al. (2007) reported that the promoter activity of cardiac troponin I is repressed in rodent skeletal muscle cells by the GATA elements through the binding of unidentified trans-acting factor(s) other than GATA2, GATA3 and GATA4. Our results suggest that the skeletal muscle-specific gene repression that occurs via GATA elements may be evolutionally conserved so as to ensure cardiacrestricted gene expression. In summary, this study shows that cx36.7 is a downstream target gene of the cardiac GATA factors, and its early cardiac-restricted expression is mediated by GATA-element-mediated transcriptional activation and repression (Fig. 12). The findings provide new evidence to help understand the mechanism for the regulation of gene expression in myocytes. Further studies are required to clarify the mechanism

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Fig. 9. EGFP expression in transgenic embryos with the mutant 316-bp promoter constructs. Fertilized one-cell eggs were injected with the 316-bp promoter constructs having point mutations in the indicated GATA and A/T-rich elements. Typical images of the trunk region of the 2-dpf (A–F) or 3-dpf (G and H) embryos are shown. The arrowheads indicate EGFP signals in the trunk muscle. The asterisks indicate yolk sac autofluorescence. Bars, 200 μm.

underlying skeletal muscle-specific transcriptional regulation, such as identification of the trans-acting factors that bind to the activator and repressor GATA elements of the cx36.7 promoter.

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.12.013.

Fig. 10. Mutational analysis of the 133-bp promoter. (A) Fertilized one-cell eggs were injected with the 133-bp promoter construct (WT) or its mutants with point mutations in either one or both of the GATA #1 and #2 elements. EGFP signals in the embryos were analyzed at 1, 2 and 3 dpf. The stacked bar charts represent the percentages of the following four groups of embryos: no EGFP expression (blue), cardiac-specific EGFP expression (green), EGFP expression in the heart and trunk muscle (red), and dead embryos (white). Data are expressed as the mean ± SEM of three independent experiments. (B–D) Representative images of the trunk region of 2-dpf embryos injected with the 133-bp promoter constructs having mutations in GATA #1 (B), GATA #2 (C), and both GATA #1 and GATA #2 (D). The asterisks indicate yolk sac autofluorescence. Bars, 0.2 mm.

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Fig. 12. Proposed model of the regulation of the cx36.7 gene expression. In the embryonic heart, cx36.7 transcription is up-regulated by gata4, gata5 and gata6 through binding to the GATA #1 and GATA #2 elements. In the embryonic skeletal muscle, the GATA #2 element also has the ability to activate the cx36.7 promoter. However, this activation is constitutively suppressed by the GATA #3, GATA #4 and A/T-rich elements, thereby enabling the cardiac-restricted expression of cx36.7.

Fig. 11. Effect of cardiac gata knockdown on promoter activation in skeletal muscle. (A and B) The 133-bp promoter construct with the GATA #1 mutation was co-injected into fertilized one-cell eggs along with control MOs (A) or gata4, gata5 and gata6-specific MOs (B). EGFP signals in the embryos were analyzed at 2 dpf. The arrow and arrowheads indicate EGFP signals in the heart and trunk muscle, respectively. The asterisks indicate yolk sac autofluorescence. Bars, 0.2 mm. (C) The bar graph represents the percentages of the embryos with EGFP expression in the trunk muscle. Data are expressed as the mean ± SEM of three independent experiments. No significant difference (ns) from the value of control-MOinjected embryos was found using Student's t-test.

Disclosure of conflict of interest statement All authors declare that there is no conflict of interest.

Acknowledgments We thank Dr. Mikiko Tanaka for access to the microscope, Yoko Yamamoto and Nana Shinohara for DNA sequencing, Yumiko Tanaka and Ippei Kasajima for technical assistance, Noriko Isoyama for maintaining zebrafish, and Yuriko Ishii for secretarial assistance. This work was supported by Kurata Memorial Hitachi Science and Technology Foundation (NN), Takeda Science Foundation (NN), and JSPS KAKENHI Grant Numbers 24570209 (NN) and 2402392 (NS and NN). Pacific Edit reviewed the manuscript prior to submission.

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