Ets Motifs Are Necessary for Endothelial Cell–Specific Expression of a 723-bp Tie-2 Promoter/Enhancer in Hprt Targeted Transgenic Mice
Objective— Tie-2 is an endothelial cell–specific receptor tyrosine kinase that is involved in the remodeling of blood vessels and angiogenesis. Our goal was to characterize Tie-2 promoter function as a means of providing insight into the mechanisms of endothelial cell–specific gene regulation.
Methods and Results— When targeted to the Hprt locus of mice, a small Tie-2 promoter fragment (containing a 300-bp intronic enhancer coupled upstream to a 423-bp core promoter) (T-short) directed widespread endothelial cell expression in vivo. The T-short promoter contains 2 clusters of Ets sites, one in the first exon, the other in the intronic enhancer. In cultured endothelial cells, a combined mutation of the Ets motifs resulted in a significant reduction in promoter activity. Consistent with these results, the same Ets mutations resulted in a loss of detectable expression of the T-short promoter in all vascular beds with the notable exception of the brain.
Conclusions— These results suggest that the T-short promoter contains information for widespread expression in the vascular tree, Ets sites are necessary for in vivo promoter activity, and the shorter Tie-2 fragment may be useful as a tool to direct heterologous gene expression within the intact endothelium.
Endothelial cells play an important role in the development of the cardiovascular system as well as in a wide array of physiological and pathological processes. There is little understanding of the molecular mechanisms that underlie the differentiation of the endothelium. Moreover, there is a large gap in our knowledge of how endothelial cell phenotypes are generated and maintained in different vascular beds. One approach to address these questions is to study the molecular basis of endothelial cell–specific gene expression. The identification of transcriptional control elements and their interactions with DNA-binding proteins may provide important insight not only about the molecular switch that occurs during endothelial cell differentiation but also about the regulatory networks that couple the microenvironment to vascular bed–specific phenotypes. In addition, the elucidation of the molecular mechanisms of endothelial cell–specific gene regulation should provide a foundation for targeting biologically active genes to the intact endothelium.
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Several endothelial cell–specific promoters have been cloned and characterized, including von Willebrand factor (vWF),1 platelet-endothelial cell adhesion molecule-1,2 endothelial specific molecule-1,3 Flt-1,4 Flk-1,5 Tie-1, Tie-2,6,7 intercellular adhesion molecule (ICAM)-2,8 endothelial nitric oxide synthase (eNOS),9 E-selectin,10 and V̇E-cadherin.11 Although most of these studies have been carried out in cell culture systems, a growing list of endothelial cell–specific promoters has also been shown to direct expression in the endothelium of transgenic mice. One of the interesting themes emerging from the in vivo studies is the remarkably heterogeneous nature of endothelial cell gene regulation. Indeed, in standard transgenic mouse assays, virtually every endothelial cell–specific promoter tested to date, with the possible exception of the mouse Tie-2 and human ICAM-2 genes, has been shown to direct expression in distinct and restricted sites of the vascular tree.8,12–16 These findings support a model of modular gene regulation in which the expression of a single endothelial cell–specific gene within the endothelium is mediated by the sum of cell subtype-specific signaling pathways, each communicating with distinct regions of the promoter.
In a previous study of standard transgenic mice, a fragment of the Tie-2 promoter that contained 5′-flanking sequence, the first exon and 10-kb fragment of first intron, directed uniform expression throughout the vascular tree.17 One limitation of this construct is that it is sufficiently large as to hamper the design of novel targeting constructs. The elucidation of shorter promoter fragments with comparable expression profiles in vivo would greatly facilitate future gene-targeting strategies.
One way to control for the variable influence of the integration site is to insert a single copy of the transgene into a defined locus of the mouse genome. We have recently used this plug-in-socket approach to target a human eNOS, Flt-1, vWF, as well as murine Tie-2 promoter.18–20 We demonstrated that endothelial cell–specific genes retain tissue specificity at this locus, that the expression pattern is independent of transgene orientation, and that the different promoters contain information for expression in distinct subsets of endothelial cells.
In the present study, we have extended the analysis of the Tie-2 promoter in the Hprt locus. A shorter fragment of the Tie-2 gene was engineered by linking the 300-bp intronic enhancer to the 5′ end of the core promoter. The promoter enhancer was coupled to the LacZ reporter gene, and the resulting construct was targeted to the Hprt locus of mice. Analysis of hemizygous male offspring revealed widespread expression in the vascular tree. Compared with the larger promoter, the short Tie-2 construct was more active in the heart and less active in the lung. Finally, we demonstrate that tandem Ets binding sites in the first exon and enhancer are indispensable for widespread expression of the Tie-2 promoter in the endothelium. Taken together, these results suggest that a short 723-bp fragment of the Tie-2 promoter may be used to direct expression of heterologous gene products in the vasculature. Moreover, the Hprt locus targeting strategy represents a novel tool for mapping endothelial cell–specific promoter elements in vivo.
Cell Cultures and Transfections
Mouse embryonic endothelial progenitor (MEEP) cells were generated and grown as previously described.21 Human umbilical vein endothelial cells (HUVECs) (Clonetics) and coronary artery endothelial cells (HCAECs) (Clonetics) were cultured in EGM-2 MV medium. Bovine aortic endothelial cells (BAECs) (Clonetics), human embryonic kidney (HEK)-293 cells (ATCC CRL-1573), COS-7 (ATCC CRL-1651), and mouse pancreas microvascular endothelial cells (MS1) (ATCC CRL-2279) were cultured in DMEM supplemented with 10% heat-inactivated FBS. Stable transfection of MEEP cells and transient transfection of HUVECs, HCAECs, BAECs, and MS1 cells were performed as described previously.22 β-galactosidase assays were performed with High-Sensitivity β-galactosidase Assay Kit (Stratagene) according to the manufacturer’s instruction. BK4-ES cells (kindly provided by Sarah Bronson, Penn State College of Medicine, Hershey, PA) were propagated and transfected with the Tie-2 promoter-containing Hprt-targeting vectors as previously described.19,20
Nuclear Extracts and DNase I Footprint Analysis
Nuclear extracts were prepared as previously described.23 A DNA fragment spanning the region between −118 and +317 of the murine Tie-2 gene was isolated and labeled with [α-32P] dCTP. For DNase I footprint, 9 fmol of the labeled probe and 40 μg of nuclear extracts were used as described previously.22,24
Generation and Analysis of Hprt-Targeted Transgenic Mice
Hprt-targeted ES cells containing the T-short-LacZ transgene were used to generate chimeric mice as previously described.19 Chimeric male were bred to C57BL/6 female mice to obtain agouti offspring. Female agouti offspring were then bred to wild-type males to generate hemizygous male mice.
Functional Analysis of the Tie-2 Promoter In Vitro
To determine the relative contribution of the 5′-flanking region and the 300-bp intronic core enhancer in directing endothelial cell expression, Tie-2 deletion constructs (Figure 1A) were stably transfected into MEEP cells. Consistent with previously published studies, the addition of the 1.7-kb intronic enhancer region to the 2100-bp Tie-2 promoter resulted in a 2-fold induction of expression (compare activity of T1-LacZ and T5-LacZ, Figure 1B).17 In a recent report, Fadel et al6 showed that the deletion of a region between −2000 and −153 bp of the Tie-2 gene had little impact on expression in endothelial cells, whereas the deletion of a region between −153 and −105 bp resulted in a significant increase in expression, suggesting that this latter region possesses net silencing activity. In keeping with these findings, a promoter containing the −105 to +318 region of the Tie-2 promoter (T′-short-LacZ) was 4.2-fold more active in stably transfected MEEP cells compared with T1-LacZ (Figure 1B). Finally, the addition of the 300-bp core intronic enhancer to the 5′ end of the short promoter in either orientation (T-short-LacZ and T-short-LacZ-rev) resulted a 7.7-fold induction of expression compared with T1-LacZ (Figure 1B). These in vitro results suggested that the T-short promoter might be used to direct high-level, widespread expression within the intact endothelium.
Comparison of T5 and T-Short Embryos
To test this latter hypothesis, the short Tie-2 construct, containing the 300-bp intronic enhancer sequence upstream of the −105 to +318 Tie-2 promoter and LacZ reporter gene (T-short-LacZ), was targeted to the Hprt locus of mice. Chimeric mice were bred to obtain germ-line transmission. The resulting line, termed T-short, contrasts with the previously described T5 line, which harbors a Tie-2 transgene containing 2100-bp 5′-flanking region of the murine promoter coupled to the LacZ cDNA and the 1.7-kb intronic enhancer.19 Six-week-old T5 and T-short hemizygous males were mated with C57BL/6 wild-type females. Embryos were removed and processed for LacZ staining at day E10.5. As shown in Figures 2A and 2B, whole-mount embryos from both T5 and T-short lines revealed widespread endothelial cell–specific staining of the vasculature. However, the T-short transgene seemed to be expressed at higher levels, particularly in the dorsal aorta, intersomitic vessels, and caudal veins. Moreover, there was additional expression in the sprouting blood vessels of the brain. In the yolk sac, the T-short promoter directed expression in both the larger and smaller blood vessels, whereas T5 directed expression only to the larger vessels (compare Figures 2C and 2D).
Comparison of T5 and T-Short Adult Mice
In whole-mount LacZ stains of adult organs, the level and pattern of reporter gene activity differed between the T5 and T-short lines. For example, there was increased β-galactosidase activity in the hearts of T-short mice compared with T5 mice (Figure 3A). In contrast, there was less LacZ staining in the lungs of T-short mice compared with the T5 mice (Figure 3B). Reporter gene activity in the brain, chest wall, and skeletal muscle was similar in the 2 lines of mice (Figures 3C through 3G).
In tissue sections of the T-short and T5 mice, β-galactosidase activity was detected in distinct populations of endothelial cells (please see http://atvb.ahajournals.org; Figures I and II). In the heart, the T-short transgene was expressed in a subset of arteries, veins, and capillaries, whereas the T5 transgene was expressed at far lower levels. In contrast, expression of the T-short promoter in the lung was much lower compared with that of the T5 promoter. The T-short and T5 transgenes directed comparable levels of reporter gene expression in a subset of large and small blood vessels of the kidney, spleen, and brain.
To quantify the differences in LacZ expression between the T-short and T5 mice, RNase protection assays were carried out with total RNA and a LacZ-specific riboprobe. LacZ expression in the T-short heart was 3.4-fold higher compared with age- and sex-matched T5 hearts (please see http://atvb.ahajournals.org; Figure IIIA). In contrast, LacZ expression in the T-short lung was 3.9-fold lower compared with T5 lungs. As predicted from the whole-mount and tissue-section staining observations, there were no significant differences in LacZ expression levels in the spleen, brain, kidney, and skeletal muscle from T-short and T5 mice (please see http://atvb.ahajournals.org; Figure IIIB).
Hprt locus targeting controls for copy number and integration site; however, this strategy introduces a new variable, namely, genetic background. It is formally possible that differences in LacZ expression between lines reflect mixed genetic background. To address this possibility, β-galactosidase activity was analyzed in 6 or more hemizygous mice from each line. The level and pattern of transgene expression was constant between animals (not shown). In addition, analysis of F1 agouti offspring of the chimeras, which are genetically identical across lines, revealed similar expression patterns compared with later generations (not shown). Taken together, these findings argue against a significant effect of genetic background on transgene expression.
Ets Motifs in the T-Short Were Sufficient for the Promoter Activity in Endothelial Cells
The above results suggest that T-short promoter construct contains information for widespread expression in the intact endothelium. The T-short construct contains several consensus transcription factor binding sites, including Ets and GATA motifs (Figure 4A). To determine whether the Ets or GATA motifs play a role in mediating Tie-2 promoter activity in vitro, we introduced point mutations into these sites and assayed promoter function in transient transfection assays. A point mutation of the 6 Ets motifs in the first exon (Exon-Ets-mut-luc) resulted in a significant reduction in promoter activity (>2-fold) in all endothelial cells tested (Figure 4B). In contrast, a point mutation of the Ets sites in the core enhancer of the first intron (Enhancer-Ets-mut-luc) had no effect on expression levels. However, a combined point mutation of Ets elements in the first exon and intronic enhancer (Double-Ets-mut-luc) resulted in more profound reduction of the activity compared with the point mutation of the first exon sites alone, particularly in BAECs and HCAECs (Figure 4B). A point mutation of the 2 GATA motifs in the first exon (Exon-GATA-mut-luc) resulted in a 58% and 26% reduction of promoter activity in MS1 and HUVECs, respectively, but had no significant effect in BAECs and HCAECs (Figure 4B). A deletion of the GATA motif in the core enhancer did not alter expression in endothelial cells (not shown).
The results of the functional promoter analysis raised the possibility that the Ets consensus sites in the first exon of the Tie-2 gene bind specifically to DNA proteins from endothelial cells. To test this hypothesis, we performed DNase I footprint analysis in nuclear extracts derived from nonendothelial cells (HEK-293 and COS-7) and endothelial cells (BAECs and MS1). As shown in Figure 5, incubation of the 32P-labeled Tie-2 promoter probe with nuclear extracts from MS1 and BAECs, but not from HEK-293 and COS-7 cells, resulted in DNase protection of the Ets motifs in the first exon. Consistent with the transfection results, the GATA region was protected by nuclear extracts derived from MS1 endothelial cells but not BAECs (Figure 5). In DNase I footprint assays of the core enhancer region, the Ets regions were protected in both endothelial and nonendothelial cells (not shown). In electrophoretic mobility shift assays, endothelial-derived nuclear protein specifically bound to a 32P-labeled oligonucleotide probe containing the Ets motif from the first exon of Tie-2 but not to a similar probe containing a point mutation of the Ets sequence (please see http://atvb.ahajournals.org; Figures IVA and IVB).
Comparison of T-Short and T-Short-mEts Mice
The above findings suggested that the Ets motifs in the first exon might be important for Tie-2 expression in vivo. To test this hypothesis, Hprt-targeted mice were generated with a T-short promoter containing point mutations of the Ets sites in the first exon and intronic enhancer (T-short-mEts). Whole-mount LacZ stains of embryos from T-short-mEts lines revealed a complete loss of reporter gene expression (Figure 6A). These findings were consistent in embryos derived from multiple male studs. In whole mounts of organs from 6-week-old adult hemizygous T-short-mEts males, the X-Gal reaction product was present at low levels in the brain (Figure 6B) but was undetectable in the heart (Figure 6C), lung (Figure 6D), diaphragm (Figure 6E), and skeletal muscle (Figure 6F). These findings were independent of age (between 4 and 9 weeks old) and were similar between littermates.
We have used an Hprt locus targeting strategy to study Tie-2 gene regulation in the endothelium. Using this approach, we previously reported that a Tie-2 promoter containing 2100-bp 5′-flanking sequence directs endothelial cell–specific expression in a subset of blood vessels in the developing embryo and in the brain and kidney of the adult mouse, whereas the inclusion of a 1.7-kb enhancing element, derived from intron 1 of the Tie-2 gene, directed more widespread expression in both the embryonic and adult vasculature.19 In the present study, we have extended these observations by showing that a smaller fragment of the promoter in which the 300-bp first intronic core enhancer is linked to the 5′ end of the promoter is sufficient for mediating widespread expression in the endothelium.
In standard transgenic assays of the Tie-2 promoter, a 10-kb fragment from the 5′ half of the first intron (which includes the 1.7-kb enhancer) was shown to direct uniform, integration site–independent expression in the endothelium of embryos and adult mice.17 A construct in which the 1.7-kb intronic enhancer was coupled to the Tie-2 promoter (or to a heterologous minimal TK promoter) displayed increased ectopic activity during embryonic development and was associated with a higher rate of nonexpression. Finally, a similar construct containing the 300-bp core enhancer resulted in incremental integration site-dependent effects.17
The T-short promoter fragment used in the present study has not been described in standard transgenic mouse assays. Based on the above considerations, it seems likely that the T-short promoter, which contains only the 300-bp core enhancer from the first intron, would be vulnerable to the unpredictable effects of random integration. However, in the absence of such data, it is not possible to definitively infer an advantage of Hprt targeting over standard transgenic mice for this particular promoter fragment. Finally, based on the results of standard transgenesis, it is conceivable that DNA sequences within the 5′ half of the first intron (outside the enhancer) would promote more uniform expression within the endothelium of Hprt-targeted mice.
We have recently used Hprt targeting to introduce other endothelial cell–specific promoters into the germ line of mice.20 Interestingly, every promoter studied to date has been found to direct expression in different subsets of endothelial cells in vivo. For example, a 1000-bp fragment of the human Flt-1 promoter was expressed predominantly on the arterial side of the circulation. In contrast, an Hprt-targeted vWF promoter fragment contained information for expression in the endothelial lining of brain blood vessels, as well as heart and skeletal muscle capillaries. Together with the present findings, these data provide strong support for a model of vascular bed–specific gene regulation, in which distinct DNA modules direct expression in unique sites of the vascular tree. An interesting question is the degree to which these modules are regulated by signals residing in the extracellular environment or by genetically predetermined transcriptional programs.
Our findings additionally suggest that DNA promoter sequences may be artificially manipulated as a means of fine-tuning expression within the vascular tree. As an extension of these results, we propose that promoter-enhancer elements from different endothelial cell–specific genes may be coupled to one another to target desired levels of biologically active genes to specific sites of the cardiovascular system. This hypothesis is readily testable using Hprt locus targeting. For example, it should be possible to determine whether intronic enhancers will boost overall expression of a heterologous endothelial cell promoter, 2 vascular bed–specific modules may be combined to induce additive or synergistic expression in the endothelium, and an environmentally responsive cis-regulatory element, such as the hypoxia response element, may be linked to an endothelial cell–specific promoter to confer temporal control under pathophysiological conditions.
The Tie-2 promoter contains several potential DNA-binding elements. For example, the core enhancer in the first intron contains putative binging sites for Ets, GATA, and octomeric transcription factors.17 The first exon also contains a cluster of 6 putative Ets motifs and 2 consensus GATA site motifs. In a previous study, 2 of these Ets motifs in particular were shown to play an important role in mediating expression of Tie-2 in cultured endothelial cells.7 In the present study, we have demonstrated that the GATA site in the first intron is not necessary for full promoter activity, whereas the 2 GATA motifs in the first exon are important for basal expression in some but not all types of endothelial cells. These findings suggest that GATA DNA-protein interactions may be important for cell subtype–specific expression of the Tie-2 gene. A mutation of the Ets cluster in the first exon, but not the first intronic enhancer, resulted in a significant reduction of promoter activity in all types of endothelial cells tested. Consistent with these results, DNase I footprint assays revealed endothelial cell–specific protection of the Ets cluster in the first exon, and mobility shift experiments confirmed the importance of the Ets consensus motif in mediating DNA binding. The observation that a double mutation of the Ets sites in the first exon and first intron resulted in additional reduction in expression suggest that these latter sites play a role in modulating promoter activity, at least under in vitro conditions.
Consensus Ets binding motifs have been identified in the promoters of several other endothelial cell–specific genes, including Flt-1,25 Tie-1,26 ICAM-2,8 and V̇E-cadherin.11 In many cases, these sites have been shown to be important for basal activity of the promoter in cultured endothelial cells. An important question is whether Ets DNA-protein interactions are essential for mediating expression of endothelial cell–specific genes under in vivo conditions. In transient transgenic mouse assays, a mutation in the Ets-binding site in the core intronic enhancer of Tie-2 was reported to result in reduced expression in the endothelium.17 Similarly, a mutation of the immediate upstream tandem Ets motif of the Tie-1 gene was shown to abolish most of the promoter activity, with weak staining in 1 of 5 transgenic embryos.26 Although these latter results implied a critical role for the Ets motifs in directing expression in the endothelium, they were limited by the unpredictable effect of random integration.
In the present study, we have used single-locus targeting to confirm the importance of the Ets sites in mediating expression of the Tie-2 gene. Specifically, we have shown that the mutation of the Ets motifs in the first exon and the intronic enhancer resulted in a loss of reporter gene expression in all organs except the brain. The determination of the relative contribution of the Ets motifs in mediating basal expression of Tie-2 will depend on additional targeting studies in which the various Ets motifs are mutated singly or in combination.
An important goal in vascular biology is to develop strategies for targeting biologically active genes to the endothelium. In this study, we have identified and characterized a short promoter fragment from the Tie-2 gene that directs widespread expression of LacZ in the vascular tree. Given the advantages of single locus targeting, we believe that the system described in this report will be useful for directing reproducible expression of other heterologous genes to the endothelium of mice.
This work was supported by National Institute of Health grants HL 60585-03, HL 63609-02, and HL 65216-02.
- Received February 27, 2003.
- Accepted July 5, 2003.
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