D-mef2 is a target for Tinman activation during Drosophila heart development
1997; Springer Nature; Volume: 16; Issue: 3 Linguagem: Inglês
10.1093/emboj/16.3.515
ISSN1460-2075
AutoresKathleen Gajewski, Yongsok Kim, Young Mi Lee, Eric N. Olson, Robert A. Schulz,
Tópico(s)Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities
ResumoArticle1 February 1997free access D-mef2 is a target for Tinman activation during Drosophila heart development Kathleen Gajewski Kathleen Gajewski Department of Biochemistry and Molecular Biology, The University of Texas M.D.Anderson Cancer Center, Houston, TX, 77030 USA Search for more papers by this author Yongsok Kim Yongsok Kim Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Young Mi Lee Young Mi Lee Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Eric N. Olson Eric N. Olson Department of Molecular Biology and Oncology, Hamon Center for Basic Cancer Research, The University of Texas Southwestern Medical Center, Dallas, TX, 75235 USA Search for more papers by this author Robert A. Schulz Robert A. Schulz Department of Biochemistry and Molecular Biology, The University of Texas M.D.Anderson Cancer Center, Houston, TX, 77030 USA Search for more papers by this author Kathleen Gajewski Kathleen Gajewski Department of Biochemistry and Molecular Biology, The University of Texas M.D.Anderson Cancer Center, Houston, TX, 77030 USA Search for more papers by this author Yongsok Kim Yongsok Kim Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Young Mi Lee Young Mi Lee Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Eric N. Olson Eric N. Olson Department of Molecular Biology and Oncology, Hamon Center for Basic Cancer Research, The University of Texas Southwestern Medical Center, Dallas, TX, 75235 USA Search for more papers by this author Robert A. Schulz Robert A. Schulz Department of Biochemistry and Molecular Biology, The University of Texas M.D.Anderson Cancer Center, Houston, TX, 77030 USA Search for more papers by this author Author Information Kathleen Gajewski1, Yongsok Kim2, Young Mi Lee2, Eric N. Olson3 and Robert A. Schulz1 1Department of Biochemistry and Molecular Biology, The University of Texas M.D.Anderson Cancer Center, Houston, TX, 77030 USA 2Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892 USA 3Department of Molecular Biology and Oncology, Hamon Center for Basic Cancer Research, The University of Texas Southwestern Medical Center, Dallas, TX, 75235 USA The EMBO Journal (1997)16:515-522https://doi.org/10.1093/emboj/16.3.515 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The NK-type homeobox gene tinman and the MADS box gene D-mef2 encode transcription factors required for the development and differentiation of the Drosophila heart, and closely related genes regulate cardiogenesis in vertebrates. Genetic analyses indicate that tinman and D-mef2 act at early and late steps, respectively, in the cardiogenic lineage. However, it is unknown whether regulatory interactions exist between these developmental control genes. We show that D-mef2 expression in the developing Drosophila heart requires a novel upstream enhancer containing two Tinman binding sites, both of which are essential for enhancer function in cardiac muscle cells. Transcriptional activity of this cardiac enhancer is dependent on tinman function, and ectopic Tinman expression activates the enhancer outside the cardiac lineage. These results define the only known in vivo target for transcriptional activation by Tinman and demonstrate that D-mef2 lies directly downstream of tinman in the genetic cascade controlling heart formation in Drosophila. Introduction Recent studies suggest that the molecular pathways controlling heart formation are evolutionarily ancient and conserved in flies and vertebrates (reviewed in Scott, 1994; Bodmer, 1995; Olson and Srivastava, 1996). The heart is formed in these organisms from a subset of mesodermal precursor cells that become committed to a cardiogenic fate in response to inductive signals from adjacent cells. In Drosophila melanogaster, heart precursor cells originate from the dorsal-most region of the mesoderm (Bodmer et al., 1990; Azpiazu and Frasch, 1993; Rugendorff et al., 1994; Dunin Borkowski et al., 1995) and are formed in response to the Wingless (Wg) and Decapentaplegic (Dpp) growth factors (Staehling-Hampton et al., 1994; Frasch, 1995; Lawrence et al., 1995; Wu et al., 1995; Park et al., 1996). These cardiac precursors give rise to a bilaterally symmetric heart-like organ called the dorsal vessel, which is comprised of inner contractile cardial cells and outer pericardial cells (Rugendorff et al., 1994). NK-type homeobox genes (Kim and Nirenberg, 1989) have been shown to play important roles in cardiogenesis (reviewed in Harvey, 1996). Among this class of genes is tinman, which is required for the genesis of cardiac precursor cells and heart formation in Drosophila (Azpiazu and Frasch, 1993; Bodmer, 1993). Several tinman-related genes have been identified in vertebrates (Komuro and Izumo, 1993; Lints et al., 1993; Tonissen et al., 1994; Evans et al., 1995; Schultheiss et al., 1995; Buchberger et al., 1996; Lee et al., 1996), including Nkx-2.5 which is expressed throughout the development of the cardiac lineage (Lints et al., 1993) and is required for correct morphogenic and myogenic development of the mouse heart (Lyons et al., 1995). Although the cardiogenic functions of Tinman and Nkx-2.5 have been determined genetically, it is unknown how they exert their effects in the cardiac lineage as no direct target genes for these factors have been identified. Members of the myocyte enhancer factor-2 (MEF2) family of MADS box transcription factors have also been shown to play important roles in heart development in flies and vertebrates (Bour et al., 1995; Lilly et al., 1995; Ranganayakulu et al., 1995; reviewed in Olson et al., 1995; Q.Lin, J.Schwartz and E.N.Olson, submitted). The single mef2 gene in Drosophila (D-mef2) is expressed in the cardiac, somatic and visceral muscle lineages (Lilly et al., 1994; Nguyen et al., 1994; Taylor et al., 1995) where it is required for the differentiation of these different muscle types. In the cardiac lineage, D-MEF2 expression is detected initially in heart precursor cells and later is restricted to the cardial cells of the heart tube. In D-mef2 mutant embryos, heart precursors are specified properly based on the correct expression of tinman (Lilly et al., 1995) and Even-skipped (Bour et al., 1995), but cardial cells are unable to differentiate and fail to express contractile protein genes (Bour et al., 1995; Lilly et al., 1995; Ranganayakulu et al., 1995). Vertebrate species contain four mef2 genes which are also expressed in the early cardiogenic lineage where they are important for activating muscle structural genes (Chambers et al., 1994; Edmondson et al., 1994; Molkentin et al., 1996; Q.Lin, J.Schwartz and E.N.Olson, submitted). The phenotypes of tinman and D-mef2 mutant embryos suggest that tinman acts at an early step in the cardiogenic pathway to establish cardiac cell identity, whereas D-mef2 acts later to control the expression of cardiac structural genes. However, the potential hierarchical relationship between these two genes has not been investigated previously. To define further the roles of MEF2 factors in heart development, we have initiated an analysis of the cis-acting sequences that control the expression of D-mef2 in the cardiogenic lineage. Here we describe a novel cardiac enhancer that directs D-mef2 expression in the developing heart and we show that this enhancer is a direct target for transcriptional activation by Tinman. Results Tinman binding sites in the regulatory sequences flanking the D-mef2 gene Sequences controlling D-mef2 gene expression in embryonic cardiac, somatic and visceral muscle cell lineages have been mapped previously to a 13 kb interval between −12.4 and +0.6 relative to the start of the D-mef2 transcription unit (Lilly et al., 1995; Schulz et al., 1996). The 5.4 kb region II DNA (between −8.8 and −3.4 kb) contained enhancer sequences for all three cell types and was the only DNA that directed lacZ reporter gene expression in the developing heart (Figure 1A). To delimit further the D-mef2 heart enhancer element, this DNA was sequenced and scanned for potential Tinman binding sites which might be involved in regulating cardial cell expression. Bacterially produced Tinman binds to the sequence T(C/T)AAGTG (Y.Kim, unpublished data) and two identical, convergently oriented CTCAAGTGG sequences that match the Tinman binding consensus were found between −5.8 and −5.5 kb upstream of D-mef2 in a region we refer to as IIA237 (Figure 1B). Figure 1.Location of a minimal D-mef2 heart enhancer. (A) Schematic of the D-mef2 gene and adjacent 5′-flanking sequence. DNA II is a 5.4 kb region previously shown to contain enhancer elements for cardiac, somatic and visceral muscle cell expression. DNA IIA237 is a 237 bp subregion containing an enhancer element for cardial cell expression. (B) Sequence of the IIA237 DNA. Two consensus Tinman binding sites (Tin 1 and Tin 2) are shown in bold. Download figure Download PowerPoint To test whether Tinman can bind to these sites, a DNase I protection assay was performed using a glutathione S-transferase (GST)–Tinman fusion protein and IIA237 DNA labeled specifically on either strand. The fusion protein protected both sites on both strands of the IIA237 DNA (Figure 2). These sequences have, therefore, been designated Tin 1 and Tin 2 for Tinman binding sites 1 and 2. Figure 2.Tinman binds in vitro to the D-mef2 IIA237 heart enhancer. DNase I protection assay performed with either a 32P-labeled (+) or (−) strand of the IIA237 DNA and increasing amounts of a glutathione S-transferase–Tinman fusion protein (GST:NK-4). Lanes: G+A, chemical sequencing reaction of the IIA237 DNA; NONE, reaction without fusion protein added; GST:NK-4, reactions with increasing amounts of fusion protein (50, 100 and 200 ng); BSA, reaction with bovine serum albumin control protein added. Tin1 and Tin2 boxes indicate the two sequences protected from DNase I digestion. Download figure Download PowerPoint IIA237 DNA functions as a D-mef2 heart enhancer regulated by the tinman gene To test the IIA237 region for heart enhancer function, the DNA was cloned upstream of a minimal Hsp43 promoter driving the lacZ gene, and germline transformant lines harboring this construct were established. Heart precursor cells first appear at stage 11, and IIA237 transformant embryos expressed the β-galactosidase marker in these progenitors by stage 12 (Figure 3A). In stage 13 embryos, β-galactosidase was detected in heart precursors located along the length of the dorsal-most part of the mesoderm (Figure 3B) and, in late stage embryos, staining was observed in the inner cardial cells of the newly formed dorsal vessel (Figure 3C). Thus, the 237 bp DNA was sufficient for directing reporter gene expression in the cardiac cell lineage and contained a heart enhancer element. Embryos from a total of six IIA237–lacZ transformant lines were assayed, and five showed expression in heart precursor and cardial cells. However, β-galactosidase expression was consistently lower in these embryos compared with the levels observed in the 5.4 kb DNA II–lacZ transformants, suggesting that an additional sequence(s) in the region II DNA is required for the proper quantitative expression of D-mef2 in the heart. Figure 3.IIA237 DNA functions as a D-mef2 heart enhancer. D-mef2–Hsp43–lacZ fusion gene expression in transformant embryos. (A) Stage 12, (B) stage 13, (C) stage 17 embryos. Arrows point to β–galactosidase expression in progenitor or differentiated heart cells. Download figure Download PowerPoint To verify that the reporter gene was being expressed in progenitor cells of the heart, we generated a fly strain that was homozygous for the D-mef2–lacZ fusion gene in a tin346/TM3 genetic background. By intermating these flies, we could monitor the expression of the lacZ gene in tinman null embryos that fail to specify cardial cells. β-Galactosidase was not detected in dorsal regions of the mutant embryos, consistent with the conclusion that the 237 bp D-mef2 enhancer is expressed in heart precursor cells (Figure 4A and C). In comparison, the D-mef2–lacZ fusion gene was expressed in the forming and mature heart of tin346 heterozygous embryos (Figure 4B and D). Figure 4.Regulation of the D-mef2 heart enhancer by tinman. D-mef2–Hsp43–lacZ fusion gene expression in stage 13 (A) and stage 17 (C) tin346 homozygous mutant embryos, stage 14 (B) and stage 17 (D) tin346/TM3,ftz-lacZ heterozygous embryos, and stage 12 heat-shocked (E) and non-heat-shocked (F) embryos. Open arrows indicate the absence of β-galactosidase staining in heart precursor cells (A), cardial cells (C) or the ventral region of the extended germ band in heat-shocked Tinman embryos (E). Solid arrows point out β-galactosidase activity in cardial cells of the forming (B and F) or mature heart (D) and in expanded domains of heat-shocked Tinman embryos (E). Download figure Download PowerPoint To address further the relationship of tinman gene function to D-mef2 enhancer expression, we introduced the D-mef2–lacZ reporter into the hsTin9A strain which contains an inducible tinman gene under the control of a heat shock promoter. Ectopic expression of Tinman in heat-shocked embryos resulted in an expanded activation of the enhancer and expression of the β-galactosidase marker, especially in cells of the anterior procephalic region and the dorsal mesoderm of the extended germ band (Figure 4E). Relatively little induced β-galactosidase expression was detected in ventrally positioned cells of the heat-shocked embryos. Overall, these results indicate that Tinman can activate the expression of the D-mef2 heart enhancer, but only certain domains of the germ band extended embryo are permissive for transcriptional activation by Tinman. As a control, non heat-shocked embryos showed the normal pattern of β-galactosidase expression in the heart precursor cells (Figure 4F). Both Tinman binding sites are required for D-mef2 heart enhancer activity in transformant embryos The functional importance of the Tinman binding sites in the D-mef2 cardiac enhancer was assessed through the generation of a series of constructs that contained deletions or nucleotide changes in the Tin 1 and Tin 2 sequences (Figure 5). Transformant lines carrying the mutant D-mef2–lacZ fusion genes were established and embryos were stained for β-galactosidase. In the IIA166 construct, both ends of the enhancer including the Tin 1 and Tin 2 sites were deleted. This DNA failed to express in cardial cells. Likewise, deletion of only the 27 bp 5′ region (IIA210) or the 44 bp 3′ region (IIA193) inactivated the enhancer. These results suggested that either both Tinman binding sites were required, or other elements located in the 5′ and 3′ sequences flanking the Tin sites must be present for the functional integrity of the D-mef2 heart enhancer. To discriminate between these possibilities, we created three variant enhancers containing mutations in each Tinman binding site alone or in combination. The mutations in these sites are known to abolish Tinman binding to DNA (Y.Kim, unpublished data). None of the three mutant enhancers were able to direct lacZ expression in heart precursor or cardial cells in transformant embryos, indicating that both Tinman binding sites must be present in the IIA237 DNA for the activity of the D-mef2 enhancer. Together, these results demonstrate that Tinman is a direct activator of D-mef2 transcription in the developing heart of Drosophila and that transcriptional activation by Tinman requires binding to two sites in the cardiac enhancer. Figure 5.Effect of the mutagenesis of Tinman binding sites on D-mef2 enhancer activity in transformant embryos. The names of the different D-mef2–Chab constructs are listed on the left. Composition indicates the starting 237 bp IIA237 DNA and its deleted or mutated derivatives. Tinman binding sites are indicated by the numbers 1 and 2, and an X designates a mutated Tin site. These sites were changed from CTCAAGTGG to CTgtAGacG in the mutant constructs. Cardial cell and heart enhancer function of the tested DNAs and the number of lines assayed for each construct are shown on the right. Download figure Download PowerPoint Discussion Expression patterns and functions of tinman and D-mef2 The results of this and previous studies (Lilly et al., 1995; Schulz et al., 1996) reveal that D-mef2 transcription in the early mesoderm and in developing muscle cell lineages is regulated by a complex set of independent control regions located upstream of the gene. In the early Drosophila embryo, the expression pattern of D-mef2 is very similar to that of tinman. Both genes are expressed initially throughout the mesoderm within the ventral furrow and in the germ band extended embryo. D-mef2 transcripts accumulate normally in these cells in tinman mutant embryos (Lilly et al., 1994; Nguyen et al., 1994) and tinman is expressed normally in D-mef2 mutant embryos (Lilly et al., 1995), suggesting that the two genes are activated independently in the early mesoderm. The most likely regulator of tinman and D-mef2 is the basic helix–loop–helix transcription factor Twist, as neither gene is expressed in twist mutant embryos (Bodmer et al., 1990; Lilly et al., 1994; Nguyen et al., 1994; Taylor et al., 1995) and ectopic expression of Twist expands the expression pattern of both genes (Taylor et al., 1995). Later, as the mesoderm differentiates, tinman expression becomes regulated by Dpp and is restricted to dorsal mesodermal cells located adjacent to Dpp-expressing ectodermal cells (St Johnston and Gelbart, 1987; Bodmer, 1990; Azpiazu and Frasch, 1993; Frasch, 1995). The function of tinman in the dorsal mesoderm is to determine the formation of heart precursor cells and to activate the expression of bagpipe, another NK-type homeobox gene required for specification of visceral muscle precursor cells (Azpiazu and Frasch, 1993; Bodmer, 1993). During this refinement of tinman gene expression, D-MEF2 is expressed in cardiac, somatic and visceral muscle cell precursors and later is expressed in the differentiated heart, body wall, and gut musculature (Bour et al., 1995; Lilly et al., 1995). Despite the expression of D-mef2 throughout the course of mesoderm formation and differentiation, gene function is required only for the differentiation of heart, somatic and visceral muscle cells (Bour et al., 1995; Lilly et al., 1995; Ranganayakulu et al., 1995). Thus, genetic studies have demonstrated an early function for tinman, and a late function for D-mef2, in the development of specific derivatives of the mesoderm. Tinman regulation of D-mef2 in the cardial cell lineage D-MEF2 is expressed in all of the contractile cardial cells of the heart tube where one of its functions is to activate the transcription of the three myosin subunit genes (Bour et al., 1995; Lilly et al., 1995; Ranganayakulu et al., 1995). The IIA237 region upstream of D-mef2 functions as a transcriptional enhancer in most, but not all of these D-MEF2-positive cells as enhancer activity was not observed in short, segmentally repeated sections of the heart tube (Figures 3C and 4D). However, this result is consistent with the regulation of the IIA237 heart enhancer by Tinman, as tinman is expressed in this pattern, i.e. in most but not all of the cardial cells of the mature heart (Bodmer, 1993). Thus, D-mef2 requires Tinman and at least one other regulatory input for its expression in the complete complement of cardial cells. The requirement for two Tinman binding sites for the activity of the D-mef2 heart enhancer suggests some manner of cooperativity between Tinman proteins bound to these sites, perhaps mediated by the interaction of Tinman molecules with each other. Other NK-type homeodomain proteins can also bind this consensus sequence. For example, the NK-2 protein, which is the product of the ventral nervous system defective (vnd) locus of Drosophila (Jimenez et al., 1995), has a consensus binding site of T(T/C)AAGT(G/A)G (Tsao et al., 1994) which matches the Tinman sites in the D-mef2 cardiac enhancer. vnd is expressed in cells of the developing nervous system and midgut of the embryo (Jimenez et al., 1995; Mellerick and Nirenberg, 1995) and it is conceivable that NK-2 could bind to the IIA237 DNA in these cells. However, since the IIA237 enhancer is not expressed in CNS or midgut cells, it seems likely that additional factors must work with the NK proteins to regulate target genes in the appropriate cell types. Thus, the selective activation of the D-mef2 enhancer by Tinman may require a co-activator protein that is expressed in the cardial cells. Because the IIA237 region contains all the information necessary for transcriptional activation by Tinman, it will be of particular interest to screen for other potential regulatory elements within this enhancer to identify the transcription factors that may cooperate with Tinman to control cardial cell gene expression. Regulatory network controlling cardiac gene expression in Drosophila Our analysis of D-mef2 expression and function in the heart, coupled with the findings of others, suggests a genetic hierarchy controlling gene expression during Drosophila cardiogenesis (Figure 6). Wg is required for the specification of cardiac progenitors (Wu et al., 1995), while Dpp produced in the ectoderm determines the expression domain of tinman in the dorsal mesoderm (Staehling-Hampton et al., 1994; Frasch, 1995). Tinman is a direct activator of D-mef2 in heart precursor cells and this regulation probably requires a Tinman co-activator protein, since Tinman expression in the dorsal mesoderm is much broader than D-mef2, and ectopic Tinman expression can activate the D-mef2 cardiac enhancer in some but not all regions of the embryo. Additional factor(s) are required for the expression of D-mef2 in the full complement of cardial cells because the Tinman-dependent enhancer is active in only a subset of cells of the dorsal vessel. Late-expressed contractile and structural protein genes can be divided into D-mef2-dependent and D-mef2-independent subgroups. The cardial cell transcription of the three myosin subunit genes requires D-mef2 function (Ranganayakulu et al., 1995) while the βTub60D and TmI genes are expressed normally in the heart of D-mef2 mutant embryos (Bour et al., 1995; Ranganayakulu et al., 1995; Lin et al., 1996). Whether these latter genes are activated directly by Tinman or require other as yet unidentified factor(s) for their cardial cell expression remains to be determined. Figure 6.Schematic of a regulatory hierarchy controlling cardiac gene expression during Drosophila heart development. Download figure Download PowerPoint Conservation of genes and mechanisms controlling heart formation The NK-type homeobox genes Nkx-2.3, Nkx-2.5 and Nkx-2.7 are closely related to tinman and their expression during cardiac development in chick, fish, frog and mouse embryos suggests that these genes may perform functions related to those of tinman (Komuro and Izumo, 1993; Lints et al., 1993; Tonissen et al., 1994; Evans et al., 1995; Schultheiss et al., 1995; Buchberger et al., 1996; Lee et al., 1996). Moreover, the preferred DNA binding site for Nkx-2.5 (Chen and Schwartz, 1995; Durocher et al., 1996) is identical to the Tinman sites in the D-mef2 cardiac enhancer, and inactivation of the Nkx-2.5 gene in mice results in abnormal heart development (Lyons et al., 1995). Likewise, the four vertebrate mef2 genes are expressed in the developing heart (Chambers et al., 1994; Edmondson et al., 1994; Molkentin et al., 1996) like their Drosophila homolog, and recent studies indicate that mef2C is required for proper heart development in mice (Q.Lin, J.Schwartz and E.N.Olson, submitted). In light of our results on Tinman regulation of D-mef2 in cardial cells, it will be interesting to determine whether the Nkx-2.5 genes activate mef2 gene expression during vertebrate cardiac morphogenesis. The coincident expression of the Nkx-2.5 and mef2C genes in the primordial heart of the mouse embryo is consistent with this type of regulatory hierarchy (Lints et al., 1993; Edmondson et al., 1994; Molkentin et al., 1996). Parallel studies of such genetic interactions in flies and vertebrates should provide important insights into evolutionarily conserved mechanisms controlling heart development and cardiac gene expression. Materials and methods DNA sequence determination DNAs were sequenced using the Sanger dideoxy method combined with automated fluorescence detection (Spurgeon et al., 1995). The 5.4 kb D-mef2 genomic DNA II (Schulz et al., 1996) was sequenced using a series of internal primers. Overlapping sequences were linked together and scanned for potential Tinman binding sites using the GCG DNA sequence analysis program. Wild-type and mutated D-mef2 DNAs to be tested for enhancer function in the CaSpeR-Hsp43-AUG-βgal (Chab) transformation vector were sequenced using a primer from the Hsp43 promoter region. Primers were obtained from Genosys Biotechnologies (Woodlands, TX). DNase I protection assay Footprinting assays were performed as described (Hoey et al., 1988). The IIA237 D-mef2 heart enhancer in pCRII (Invitrogen, San Diego, CA) was digested with either HindIII (minus-strand labeling) or XbaI (plus-strand labeling) and digested DNAs were treated with alkaline phosphatase. Dephosphorylated DNAs were redigested with either XbaI (minus-strand labeling) or HindIII (plus-strand labeling), eluted from a gel, and 32P-end labeled with T4 kinase. Binding reactions were then performed with a GST–NK-4 fusion protein and 12.5 fmol of labeled DNA in 10 μl of binding buffer containing 25 mM HEPES (pH 7.5), 3 mM MgCl2, 1 mM EDTA, 0.5% NP-40, 10% glycerol, 1 μg of poly[d(I–C)]. After incubation for 15 min at room temperature, CaCl2 (final 0.5 mM) and DNase I (0.05 U) were added to the binding reactions for DNase I digestion. Reactions were terminated after 1 min incubation and analyzed on an 8% denaturing polyacrylamide gel. D-mef2–Chab enhancer test constructs The D-mef2 IIA237, IIA166, IIA210 and IIA193 DNAs were generated by PCR amplification using the DNA II template, the terminal 20 nucleotide 5′ and 3′ primers, and dNTP solutions, LA PCR buffer and LA Taq enzyme provided in the TaKaRa LA PCR kit (PanVera, Madison, WI). PCR products were cloned into the pCRII vector using the TA cloning kit from Invitrogen (San Diego, CA). To generate the D-mef2 IIA237mTin1, IIA237mTin2 and IIA237mTin1/2 mutant DNAs, a PCR-based method was also used. For IIA237mTin1, two separate DNA fragments (A1 and A2) were amplified by PCR with specific primers (A1 DNA: SP6 and mT2R; A2 DNA: mT25 and T7) and IIA237 template DNA. The A1 and A2 DNAs were mixed, denatured and reannealed. DNAs were gap-filled and reamplified by PCR using 30 amplification cycles (94°C, 1 min; 55°C, 1 min; 72°C, 1 min) with SP6 and T7 primers. The amplified DNA fragments were then cloned into pCRII. For the IIA237mTin2 construct, the same method was employed using different primers (A3 DNA: SP6 and mT1R; A4 DNA: mT15 and T7). For the IIA237mTin1/2 DNA, an A5 DNA was generated by PCR using the mT15 and T7 primers and IIA237mTin1 template DNA. The A5 and A3 DNAs were then mixed, reamplified by PCR and subcloned. Primers used were as follows: SP6, 5′-ATTTAGGTGACACTATA-3′; mT1R, 5′-GCAGCTAAAGGATGCGCCgtCTacAGACCGGGCTCGCTATC-3′; mT15, 5′-GATAGCGAGCCCGGTCTgtAGacGGCGCATCCTTTAGCTGC-3′; mT2R, 5′-AATGGAGGCACCCAAGCgtCTacAGCCCCTTATCCGTGTC-3′; mT25, 5′-GACACGGATAAGGGGCTgtAGacGCTTGGGTGCCTCCATT-3′; T7, 5′-TAATACGACTCACTATAGGG-3′. Lower case letters correspond to the introduced base changes. The wild-type or mutant D-mef2 sequences were isolated from the pCRII vector and cloned into Chab. Drosophila germline transformation The D-mef2–Chab DNAs were stably introduced into the Drosophila genome by P element-mediated germline transformation (Rubin and Spradling, 1982). The y w67c23 strain was used for embryo injections and transformed flies were identified by w+ eye color selection. A minimum of four lines were established for each construct. The insertion of the IIA237 D-mef2–Chab DNA on chromosome II in the IIA237-9 line was determined using standard linkage analysis. IIA237-9 flies were crossed with tin346/TM3,ftz-lacZ flies to generate a IIA237-9/IIA237-9;tin346/TM3,ftz-lacZ stock. Upon intermating of these flies, the expression of the IIA237–lacZ DNA was assayed in tin346 heterozygous or homozygous embryos, with the former identified by the expression of the marker linked to the balancer chromosome. tin346 has been classified as a null allele of the tinman gene (Azpiazu and Frasch, 1993). IIA237-9 flies were also crossed to hsTin9A/TM3,ftz-lacZ flies to generate embryos that could be heat treated to induce ectopic expression of the Tinman protein. The heat shock regimen was as follows. Embryos were collected for 2 h at 25°C on yeasted grape-agar plates and then aged for 4 h at 25°C. Embryos were heat-shocked for 30 min by placing the plates in a w
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