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Human Molecular Genetics, 2003, Vol. 12, No. 4 423-433
© 2003 Oxford University Press

Differential binding of transcription factor E2F-2 to the endothelin-converting enzyme-1b promoter affects blood pressure regulation

Heiko Funke-Kaiser1,{dagger}, Florian Reichenberger2,{dagger}, Karla Köpke3, Stefan-Martin Herrmann1,4, Jacqueline Pfeifer1, Hans-Dieter Orzechowski1, Walter Zidek2, Martin Paul1 and Eva Brand2,*

1Institute of Clinical Pharmacology and Toxicology, Department of Clinical Pharmacology and 2Department of Internal Medicine, Division of Endocrinology and Nephrology, Benjamin Franklin Medical Center, Freie Universität Berlin, Berlin, Germany, 3Max-Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Germany and 4Institute of Pharmacology, Toxicology and Natural Products, Department of Natural Products and Clinical Pharmacology, University of Ulm, Ulm, Germany

Received November 17, 2002; Accepted December 14, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The endothelin-converting enzyme (ECE)-1 gene is a candidate for human blood pressure (BP) regulation and we report the identification of the new gene variants T-839G, C-338A, L75F, A677V and C+295T. Transient transfection of the reporter constructs containing the -338A allele showed an increase in promoter activity compared with the wild-type promoter. EMSA revealed the specific binding of E2F-2 to both ECE-1b promoter sequences, with the -338A allele being associated with an increased affinity to E2F-2 compared with -338C. The clinical relevance of this finding was analyzed in 704 hypertensive patients. In untreated hypertensive women, both the -338A and -839G alleles were significantly associated with ambulatory BP values. This study provides the first evidence of a link between the cell-cycle-associated E2F family and BP regulation via a component of the endothelin system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Arterial blood pressure (BP) is a complex quantitative trait influenced by genetic as well as environmental factors (1). High BP and hypertensive end-organ damage tend to cluster in families, suggesting that genetic markers can be identified that would allow the early detection of individuals at risk for the development of hypertension and its sequelae (2).

Endothelin-converting enzyme (ECE) is a key component in endothelin (ET) biosynthesis, leading to the generation of ET-1, a potent vasoconstricting peptide, and contributing to BP control. Two different ECE genes, ECE-1 (3) and ECE-2 (4), have been identified so far. In contrast to ECE-1, for which the vascular endothelium has been identified as a main site of constitutive expression in the context of a broad tissue expression (5), ECE-2 is mainly expressed in neuronal tissues (4). ECE-1 plays an essential role in vertebrate development as demonstrated by studies of ECE-1 knock-out mice, which show congenital intestinal aganglionosis, craniofacial and cardiovascular abnormalities (6). Furthermore, ECE-1 expression is altered in human cardiovascular diseases, including atherosclerosis (7), and ischemic cardiomyopathy (8). Human ECE-1 is expressed in four different isoforms (ECE-1a, ECE-1b, ECE-1c and ECE-1d) (9–12) generated by isoform-specific, alternative promoters (12–15). The gene is located on chromosome 1 (16), spanning over 120 kbp and consisting of 20 exons (11,12,14). The ECE-1b isoform is expressed in endothelial and vascular smooth muscle cells (12,13) and may, therefore, contribute to vascular ET generation and BP regulation, through variation in its constitutive expression level or pathophysiological upregulation mediated by binding of transcription factors to corresponding cis-elements. We, therefore, hypothesized that genetic variants within the ECE-1b gene promoter region, especially those affecting consensus sequences for transcription factor binding, may be associated with different BP phenotypes, such as ambulatory BP (ABP), which gives a better prediction of clinical outcome than clinic or casual BP measurements alone (17).

For this purpose, we scanned the ECE-1b promoter and the entire coding region for genetic polymorphisms, investigated possible associations with the different BP phenotypes in vivo in a Caucasian study population comprising hypertensive males and females (n=704) with and without antihypertensive medication, and tested the potential functionality of polymorphisms in vitro where appropriate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of the study population
The clinical characteristics of all participants, specified for gender, are shown in Table 1, including means and standard deviations of age, body mass index (BMI), onset of hypertension, systolic BP (SBP), diastolic BP (DBP), 24 h SBP and 24 h DBP measurements separately for the daytime (6 a.m.–10 p.m.) and night-time (10 p.m.–6 a.m.) period.


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  		Table 1. Clinical parameters of hypertensive subjects
 
Polymorphism detection and allele frequencies
Five bi-allelic ECE-1b variants were identified, two within the 5'-flanking region (T-839G, C-338A), and three within exonic regions (exon 3, L75F C/T; exon 17, A677V C/T; exon 19, C+295T). C-338A is located within a putative consensus site for E2F and GATA proteins. The minor allele frequencies at positions -839 and -338 were 8 and 26%, respectively. All three exonic variants were rare (<1%), two of which represented missense variants (L75F, A677V).

Linkage disequilibrium between ECE-1b variants
None of the ECE-1b gene polymorphisms showed significant deviation from Hardy–Weinberg expectation. Linkage disequilibrium was tested for all pairs of variants, but only C-338A and T-839G were in strong linkage disequilibrium (P<0.0001) with a correlation coefficient r=0.49.

Gender-specific effects of genotypes and haplotypes on BP phenotypes
Nine different haplotypes as combinations of the five variants were observed in the study population, three of which were rare (<1%). Three-hundred and thirty-three patients (51% of the study population) carried the prevalent haplotype where all frequent alleles were represented. The frequencies of haplotypes differing from the latter at position -338 only or both at -839 and -338 (heterozygous state) were 30 and 12%, respectively. The remaining haplotypes were rare. For simplicity, the following haplotypes were described only as combinations of the two common variants in the order T-839G and C-338A. The other three variants were rare so that altogether <1% of genotypes vary in these positions.

Both identified promoter polymorphisms were significantly associated with 24 h BP values in non-treated hypertensive females (Table 2). In women without antihypertensive medication, the -839G and -338A alleles were associated with higher daytime and night-time 24 h SBP as well as higher daytime and night-time 24 h DBP (P=0.012, P=0.021, P=0.022 and P=0.067, respectively for -839G; P=0.049, P=0.017, P=0.028 and P=0.035, respectively for -338A).


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  		Table 2. Effects of both promoter variants on 24 h blood pressure in hypertensive females without medication
 
Furthermore, females carrying haplotypes containing (1) -839T and -338C, (2) -839T and -338A (3) -839G and -338A showed significant associations with ABP phenotypes. The mean 24 h daytime and night-time BP values were highest among females carrying a haplotype containing both mutated ECE-1b promoter alleles, and were lowest for those carrying a haplotype containing the frequent variants. The haplotype which was mutated at position -338 had an intermediate effect on BP phenotypes. The differences of 24 h daytime BP and 24 h night-time BP values between females with genotype 11 on the one hand and genotypes 22, 23 or 33, which all contain at least one haplotype with both mutations, on the other hand, were statistically significant (24 h daytime: SBP, 144.5±13.4 versus 157.0±17.4 mmHg, P=0.008; DBP, 89.6±10.4 versus 97.5±12.0 mmHg, P=0.009; 24 h night-time: SBP, 128.7±15.7 versus 143.4±22.5 mmHg, P=0.008; DBP, 76.3±11.4 versus 83.7±13.2 mmHg, P=0.022; Table 3). In the entire study population, neither genotypes nor haplotypes were associated with assessed end-organ damage phenotypes.


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  		Table 3. Effects of ECE-1b haplotypes on 24 h-blood pressure in hypertensive females without medication
 
Subcloning and transient transfections of polymorphic promoter reporter constructs
To examine the putative functional significance of the newly identified polymorphisms in the human ECE-1b promoter, genomic PCR products of the four different promoter haplotypes were subcloned into the luciferase reporter plasmid pGL3basic, followed by transient transfection into the endothelial cell line EA.hy926. Compared with the ‘wild type’ promoter construct (-338C/-839T), those containing the -338A allele (-338A/-839T and -338A/-839G) showed increased promoter activities (130 and 136%, respectively; Fig. 1). This increase was statistically significant (P=0.02) with respect to the -338A/-839G haplotype, whereas the difference in promoter activity observed with construct -338A/-839T showed a high standard deviation, therefore statistical significance was not reached. A small increase in promoter activity compared with the wild-type was also observed for the -338C/-839G construct (117%; Fig. 1).



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  		Figure 1. Haplotype-specific ECE-1b promoter activity in endothelial cells. Promoter luciferase constructs driven by 1282 bp of the polymorphic ECE-1b promoter (relative to the putative translation initiation start codon) and representing the four promoter haplotypes [(1) C(-338)/T(-839) (wild-type); (2) C(-338)/G(-839); (3) A(-338)/T(-839); (4) A(-338)/G(-839)] were transfected into the endothelial cell line EA.hy926. The promoter activities are expressed as relative luciferase activity (RLA) with the activity of the wild-type (w.t.) construct set to 100%. A SV40 promoter driven pGL3 vector (SV40) served as standard for promoter activity. The transfection data represent the mean RLA values of five independent experiments (each n=4); P-values are related to the w.t. construct.

 
E2F-2 binds to the polymorphic site at position -338in the human ECE-1b promoter
In silico sequence analysis for potential cis-elements indicated that the -338A allele is located within a consensus sequence for transcription factors of the E2F-family, whereas the -338C allele is part of a consensus for binding of GATA proteins (18). To examine potential protein–DNA interactions, an electrophoretic mobility shift assay (EMSA) was performed using allele-specific oligodeoxynucleotides (ODNs) (‘-338A’ and ‘-338C’, positions relative to the putative translation start site), respectively (Fig. 2). Using increasing amounts of unlabeled competitor ODN and nuclear proteins, we were able to identify two specific band shifts (S1 and S2), regardless of the ODN sequence (Fig. 2). A third, less intense band, unaffected by excess amount of competitor, was considered as non-specific (ns).



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  		Figure 2. Protein–DNA interactions of nuclear and cytosolic proteins of EA.hy926 cells with polymorphic consensus sequences at position -338 in the human ECE-1b promoter. EMSAs were performed with radiolabeled oligonucleotides containing the -338A (left) or -338C (right) polymorphism, respectively. Ø: without antibody or competitor. E2F-1,2,3 and GATA-2: binding reactions were performed using 6 µg of the respective antibody. Triangles indicate competition experiments in the presence of increasing amounts of unlabeled ODNs. All lanes represent nuclear proteins, with the exception of lanes C, where the binding reactions were performed with cytosolic protein extracts. ns, non-specific band shift; S1, S2, specific band shift; SS, super-shifted band shift.

 
To identify the transcription factors binding to the polymorphic consensus ODNs, we performed super-shift assays adding antibodies specific for E2F-1, E2F-2, E2F-3 and GATA-2, respectively. Addition of an antibody specific for the transcription factor E2F-2 induced a super-shifted band (SS) associated with complete disappearance of band S2 (Fig. 2), indicating that S2 (and not S1) represents the ‘super-shiftable’ band. Antibodies against other E2F proteins, E2F-1 and E2F-3, or against GATA-2 failed to induce any super-shifted (SS) band. The super-shift specific for E2F-2 was observed using -338A as well as -338C containing ODNs.

E2F-2 displays an increased affinity to the ECE-1 -338Aallele carrying promoter
Because the specific band shifts observed with -338A ODN were more intense than those obtained with -338C ODN (Fig. 2), we performed an additional EMSA experiment using increasing concentrations of anti-E2F-2 antibody in the binding reaction to analyze the affinity of E2F-2 with -338A and -338C alleles, respectively. As expected, decreasing antibody concentrations were associated with decreased intensities of the SS band and, conversely, with increased intensities of the corresponding ‘super-shiftable’ band shifts (S2) (Fig. 3A). Regardless of the antibody concentration used, SS and S2 bands were apparently more intense for -338A ODN.




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  		Figure 3. Analysis of differential E2F-2 binding to -338A and -338C. (A) EMSA binding reactions were performed using nuclear proteins of EA.hy926 cells, the indicated ODN (-338A or -338C) and different amounts of an anti-E2F-2 antibody (Ab). Abbreviations as in Figure 2. (B) Quantitative signal analysis of the EMSA shown in (A). Ordinate and abscissa represent the super-shift/super-shiftable shift ratio (SS/S2) and the antibody amounts, respectively. (C) Quantitative signal analysis showing the total E2F-2 binding (summation of SS and S2 intensities).

 
Quantitative analysis of band intensities (expressed as density units) revealed that the ratio SS to S2, which reflects E2F-2 affinity and which is insensitive to variations in gel loading, was higher for -338A ODN compared to -338C ODN for all antibody concentrations tested (e.g. 0.73 versus 0.45 using 2 µg antibody; Fig. 3B). In addition, total E2F-2 binding (by summation of SS and S2 intensities) was higher for the -338A ODN, further indicating increased E2F-2 binding affinity of the -338A promoter allele (Fig. 3C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, the ECE-1b promoter haplotype -839G/-338A was significantly associated with higher daytime and night-time 24 h SBP as well as higher daytime and night-time 24 h DBP in non-treated hypertensive women. Additionally, the mean values of the 24 h daytime and night-time BP were highest among females carrying a haplotype containing both less frequent ECE-1b promoter alleles, and lowest for those carrying the frequent alleles. The haplotype with the less frequent allele at position -338 had an intermediate (e.g. codominant) effect on the assessed BP phenotypes. The exonic variants L75F, A677V and C+295T were not included in the present haplotype analysis since their less frequent alleles were too rare. It has already been observed that low-frequency alleles are more often represented among non-synonymous (missense variants such as L75F and A677V in the present study) than among synonymous polymorphisms (19), and with respect to protein properties they are more likely to be functional (20). Indeed, the single male patient carrying the ECE-1b L75F variant in our study had an early onset of hypertension at the age of 12 years and a positive family history in that his father also reported an early onset of hypertension. However, the functional role of this missense variant, e.g. the mechanism by which this might affect ECE-1 properties, explaining the link with essential hypertension, should be evaluated further in appropriate experimental studies.

The apparent gender-specific genotype effect observed in our present analysis remains conjectural. A possible gender-specific modulation of the ET system activation in hypertension has been reported in a rat model (21), and recent findings have indicated that estradiol inhibits ET-1 synthesis (22,23). Assuming a dose–effect curve between ET levels and BP, based on the fact that ET contributes to the maintenance of vascular tone in humans (24), and taking further into account that women exhibit lower plasma ET-1 levels than men (25), it is tempting to speculate that the ECE-1 promoter variants and, therefore, ECE-1 expression, might have a greater impact on BP phenotypes in women than in men, since women may be represented on the steeper part of a dose–effect curve.

However with respect to plasma ET-1 levels, it should be noted that ~80% of ET-1 is secreted at the abluminal side of the vessel (26). Hence, ET-1 is secreted from the endothelial cells towards the vascular wall, and acts predominantly on the underlying vascular smooth muscle cells, where its concentration may be several orders of magnitude higher than it is in plasma (26). Thus, ET acts more as an autocrine/paracrine system, rather than a systemic peptide, and therefore assessment of plasma ET-1 levels might not be the appropriate parameter (27).

Assuming that the ECE-1 promoter polymorphisms are also functional in the renal endothelium with respect to ET levels, these might influence renal cortical blood flow via the ETA receptor and in turn sodium reabsorption (28). Furthermore, the ETB receptor plays a crucial role in renal sodium handling by mediating an inhibiting effect on the epithelial Na+ channel (ENaC) (29,30) and ETB receptor expression was shown to be upregulated in human renal medulla by low salt diet (28). Therefore, the ECE-1b polymorphisms might also influence BP via vessel-independent, renal mechanisms. With respect to the gender-specific effects of the polymorphisms identified here, it might be possible for ETs to interfere with sex hormones regarding salt homeostasis. Nevertheless, renal sodium retention is not enhanced in the luteal phase of the normal ovulatory cycle (31,32). In this context, it is important to note that ECE-1 is expressed in endothelial and steroidogenic cells of the corpus luteum and that the expression level depends on the phase of the menstrual cycle (33).

In the context of genetic differences, African-American hypertensives have also been reported to have a higher ECE-1 activity compared with white hypertensive patients (34), indicating that genetic variability in the ET system, e.g. ECE-1 gene, could be linked with severity of essential hypertension. With respect to complex genetic diseases such as essential hypertension, genotype–phenotype relationships are complex as well. At the population level, studying only one or a few gene polymorphisms might obscure the identification of an existing genotype–phenotype relationship if pairs of polymorphisms under study display a significant linkage disequilibrium one with each other, and if they have opposing effects on the phenotype of interest (20). With respect to causality, it is most important to identify those alleles responsible for distinct genotype–phenotype relationships using in vitro models, and this is more important when linkage disequilibrium between polymorphisms is not exhaustive, rendering the study of ‘markers only’ insufficient.

We performed promoter assays in cultured human endothelial cells which were selected for two reasons. First, the vascular endothelium represents a major site of ECE-1 expression in humans in vivo (5). Second, according to a generally accepted model, ET-1 generated by endothelial cells acts on vasoconstricting ETA (and ETB) receptors expressed on underlying vascular smooth muscle cells (35). The functionality of the -338 variant seemed to be more pronounced compared with the variant -839, especially when considering the mean values of RLA, since alteration of the nucleotide at position -338 alone was associated with a greater alteration in promoter activity.

It should, however, be noted that the -839G allele also seems to influence promoter activity in transient transfections assays, but to a minor extent (117% of wild-type activity) and only in combination with the -338C polymorphism, since no statistical difference was observed comparing the constructs -338A/-839T and -338A/-839G.

Therefore, only ODNs containing C-338A were used in subsequent gel shift experiments. Our EMSA experiments have clearly identified a polymorphic cis-acting element in the human ECE-1b promoter, which has the ability to bind E2F-2. Furthermore, we were able to show that the -338A allele, which was associated with higher BP phenotypes in women, is characterized by a higher affinity to E2F-2 compared with -338C. The ability of the -338C ODN to bind E2F-2 was unexpected, since in silico sequence analysis for potential cis-elements using TRANSFAC (18) identified a consensus sequence for GATA, but not E2F binding. We assume that ‘low affinity’ binding sites such as the ECE-1b promoter -338C allele are under-represented in databases such as TRANSFAC, which indicates that all predictions of putative transcription factor binding should be confirmed by appropriate experiments.

Furthermore, it is conceivable that, in another cellular context, the polymorphism at position -338 even changes the type of transcription factor, which binds to the promoter (E2F-2 versus GATA-2), in contrast to a quantitative alteration of the affinity to a certain transcription factor, which we observed in the endothelial cell line EA.hy926. Regarding cell-specific expression, GATA-1 to GATA-3 are mainly expressed in hematopoietic cells, whereas GATA-4 to GATA-6 are particularly expressed in the heart (36,37). In addition, GATA-2 was also isolated from aortic endothelial cells and the ET-1 gene promoter itself was the first described non-erythroid GATA-2 target (38). However, we were not able to detect GATA-2 in endothelial EA.hy926 cells by western blot analysis (data not shown), which explains why we did not observe a GATA-2 super-shift. The specific band shift S1 in our EMSA experiments is possibly caused by so-called differentiation regulated transcription factor (DRTF) proteins (DPs), which are known binding partners of E2F family members, or by binding of GATA proteins other than GATA-2 (39).

The E2F family of transcription factors comprises six members (E2F-1–E2F-6) and plays a crucial role in cell cycle control (39). Genes regulated by E2F proteins include cell cycle regulators (e.g. cyclin E and Cdc2) and enzymes for nucleotide biosynthesis (e.g. dihydrofolate reductase) (39). The effects of E2F proteins, with the exception of E2F-6, are regulated by interaction with three different pocket proteins (retinoblastoma protein, p107 and p130). E2F-2, together with E2F-1 and E2F-3, constitute the subclass of ‘activating E2Fs’, in contrast to E2F-4–E2F-6, which generally mediate repression (39). With regard to pathophysiology, E2F-1 was the first gene fulfilling the criteria of both an oncogene as well as a tumor-suppressor gene (40–42), and E2F-1-based gene therapy was used in animal models of glioblastoma multiforme (43,44). Our knowledge about specific functions of E2F proteins in endothelial cells is limited, but recently prevention of tumor necrosis factor-induced apoptosis of proliferating human endothelial cells by overexpression of E2F-1 has been reported (45). Proliferation of neointimal smooth muscle cells in an experimental model of vascular injury was suppressed by E2F transcription factor decoy, revealing an important role for this transcription factor in cell cycle regulation of vascular smooth muscle cells in vivo (46). In the PREVENT Study, ex vivo application of E2F decoy resulted in a decreased occlusion rate of venous bypass grafts at 12 months (47).

To the best of our knowledge, a link between essential hypertension and the E2F transcription factors has not yet been described. With regard to hypertension, it has been reported that angiotensin II can increase DNA synthesis and expression of cell-cycle-dependent kinase complex Cdk4-cyclin D in smooth muscle cells of hypertensive rats (48,49). Furthermore, cultured vascular smooth muscle cells from spontaneously hypertensive rats show a faster cell cycle progression than cells from normotensive controls (50,51).

With respect to future studies, it is important to note that, apart from ECE-1 expression in endothelial and vascular smooth muscle cells (5,12,13), ECE-1 immunoreactivity was additionally observed in the adrenal gland (5) and in several nuclei of the central nervous system (52). The potential functionality of the described polymorphisms in these tissues could also contribute to interindividual BP differences as well as other disease entities (53).

To conclude, our results suggest the implication of the ECE-1b C-338A polymorphism in the pathogenesis of elevated BP phenotypes in human essential hypertension, and support the hypothesis of differential, i.e. allele-dependent, binding affinities of the transcription factor E2F-2 to the ECE-1b promoter. This study provides the first evidence of a link between the cell-cycle associated E2F family and BP regulation via a component of the ET system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Study population
The study protocol was approved by the ethical committee of the Benjamin Franklin Medical Center at the Freie Universität Berlin. Written informed consent for genetic studies was obtained from all participants.

Patient selection and clinical investigation
Treated (n=377) and untreated (n=327) white hypertensive patients (n=704, 350 males, 354 females) from the outpatient hypertension clinic of the Benjamin Franklin Medical Center of the Freie Universität Berlin, Germany, were enrolled in the study. Resting BP was measured at each presentation by a trained nurse. Hypertension was defined as a SBP>=140 mmHg and/or a DBP >=90 mmHg on at least two separate occasions. Additionally, ABP measurements (90207, SpaceLabs Medical Inc.) were performed in all patients. Patients with white-coat hypertension, as evidenced by elevated clinic BP but 24 h-average ABP <135/85 mmHg, were excluded from the study. Secondary forms of hypertension were excluded and hypertensive end-organ damage was assessed by standard clinical, laboratory and imaging procedures. Age of onset was determined by history and direct contact with the family physician at the first time the patient was ever told by a physician that he or she was hypertensive. Patients who had never been treated previously or had stopped antihypertensive medication for any reason at least 4 weeks before presenting at our clinic were defined as untreated. Once the presence of hypertension was established, venous blood samples were drawn for DNA extraction, genetic screening and genotyping.

DNA extraction and identification of polymorphisms
Genomic DNA from all study subjects was extracted by standard techniques (54). From the known genomic structure of the human ECE-1b gene (15), 29 overlapping sets of oligonucleotides (sequences are available upon request) were designed to perform a total systematic scan of a 5575 bp region containing exons 1b to 19, intron 2, parts of intron 3, exon–intron boundaries and also comprising 1734 bp of the 5'-flanking region (upstream of the putative ECE-1b translation start site) in 95 patients (190 alleles) with hypertension.

All PCR amplifications were performed with 20 ng of DNA in a total volume of 25 µl containing 10 mM Tris–HCl (pH 9), 50 mM KCl, 1.5–3.0 mM MgCl2, 0.1% Triton-X100, 0.2 mg/ml BSA, 200 µM dNTPs, 25 pmol of each primer and 0.2 U Taq polymerase, followed by single-strand conformation polymorphism (SSCP) analysis as previously described (55). DNA samples from patients presenting mobility shifts as variant electrophoretic patterns were reamplified by PCR with unlabeled primers and subsequently sequenced using the chain-termination method (Sequenase Version 2.0 DNA Sequencing Kit, Amersham Life Science) and an automated sequencing device (ABI PRISM 377).

Genotyping
Genotyping of the entire study population for bi-allelic polymorphisms in the ECE-1b gene was performed by a hybridization technique using allele specific oligonucleotides (hybridization conditions and nucleotide sequences are available upon request) (56,57).

The ECE-1b T-839G genotypes were determined by PCR amplification of the relevant region, followed by restriction with AvaI (New England Biolabs, Frankfurt, Germany) (restriction conditions and nucleotide sequences are available upon request). Complete restriction of the PCR product (272 bp for the -839TT genotype) generated bands of 203 and 69 bp (-839GG genotype) in length.

Statistical analysis
Hardy–Weinberg equilibrium of the analyzed polymorphisms was tested in the study population by the likelihood-ratio-based G-test (58) or by {chi}2 analysis for the rare polymorphisms. Linkage disequilibrium between pairs of polymorphisms was tested by using the LINKDOS program described by Garnier-Gere and Dillmann (59). All data are presented as mean±SD. The non-parametric Kruskal–Wallis test was used to reveal differences in BP means between the status of a single ECE-1b variant, genotype or haplotype groups, respectively. Mann–Whitney U-test was used for the comparison of two groups. Categorical data were tested by contingency table analysis using {chi}2 statistics. P<=0.05 was considered statistically significant. Statistics were calculated with SPSSTM (version 10.0, SPSS Inc., Chicago, USA).

Haplotype analysis
Initially, haplotypes were constructed as combinations of the five newly identified variants in the order T-839G, C-338A, C+223T (exon 3), C+2030T (exon 17), and C+295T (exon 19). For further haplotype analysis, we included only those variants with a frequency >1% in our population. Therefore, haplotypes were constructed as combinations of two of five newly identified variants in the order T-839G, and C-338A. Genotypes and haplotypes are given as a sequence of these two variants as follows: with respect to genotypes, 1 codes for homozygous state (in our sample the frequent alleles, reference sequence); 2 for heterozygous state, and 3 for homozygous mutated state (rare alleles). With respect to haplotypes, 1 denotes the reference sequence at this position, and 2 denotes the mutated sequence. Each genotype was assigned one haplotype pair. For those individuals homozygous at both polymorphic sites or heterozygous at only one polymorphic site, haplotypes can be assigned unambiguously. Only for one of the nine genotypes (‘22’) a second haplotype pair is theoretically possible (haplotype pair ‘12’/‘21’), even if only the haplotype pair ‘11’/‘22’ is likely to exist (calculated by the program ithap-cgi) (60).

Detailed definitions of (compound) genotypes and haplotypes are as follows: genotype ‘11’ indicates that both chromosomes carry the T nucleotide at position -839 and the C nucleotide at position -338 (homozygous wild-type state at both positions). Genotype ‘22’ indicates that one chromosome at position -839 carries the T nucleotide and the other carries the G nucleotide. The same applies to position -338 regarding the C and A nucleotides (heterozygous states at both positions). Genotype ‘23’ indicates a heterozygous state at position -839 and that both chromosomes at position -338 carry the A nucleotide (homozygous mutated state). Genotype ‘33’ indicates that both chromosomes at position -839 carry the G nucleotide and both chromosomes at position -338 carry the A nucleotide (homozygous mutated states at both positions).

Haplotype ‘11’ indicates that both variants at positions -839 and -338 are identical to the reference sequence. Haplotype ‘12’ indicates, that the sequence carries the T nucleotide at position -839 as in the reference sequence and the A nucleotide at position -338, i.e. mutated at position -338. Haplotype ‘21’ indicates, that the sequence carries the G nucleotide at position -839, i.e. mutated at position -839, and the C nucleotide at position -338 as in the reference sequence. Haplotype ‘22’ indicates that both variants at positions -839 and -338 differ from the reference sequence, i.e. both are mutated.

Each genotype is therefore defined by a pair of haplotypes: genotype ‘11’, haplotype ‘11’xhaplotype ‘11’; genotype ‘22’, haplotype ‘11’xhaplotype ‘22’ or haplotype ‘12’xhaplotype ‘21’; genotype ‘23’, haplotype ‘12’xhaplotype ‘22’; genotype ‘33’, haplotype ‘22’xhaplotype ‘22’.

Promoter–reporter gene constructs
Based on the sequence of the human ECE-1b promoter [GI 4972242, GI 20537487 and Orzechowski et al. (13)], we performed a genomic PCR (sense-primer: 5'-TGCCACCAGGCCCAGCTG-3'; antisense-primer: 5'-GCTGTGCCCCAGACGCCT-3') using four different genomic templates representing the following allele combinations: (1) C(-338), T(-839) (wild-type); (2) A(-338), T(-839); (3) C(-338), G(-839); (4) A(-338), G(-839) (positions relative to the putative translation initiation start codon). The resulting PCR products (1282 bp of the 5'-regulatory region directly upstream of the putative translation initiation start codon) were subcloned into the luciferase reporter vector pGL3basic (Promega, Madison, USA) and the identity of the inserts was confirmed by sequencing.

Cell culture conditions
The human endothelial cell line EA.hy926 (a generous gift from Dr C.-J. Edgell) was cultured in Dulbecco's modified Eagle medium supplemented with 10% FCS, 1 mM sodium pyruvate, non-essential amino acids (1x), HAT-supplement (1x), 100 U/ml penicillin and 100 µg/ml streptomycin (all Biochrom).

Transient transfection experiments
EA.hy926 cells were transfected in 12-well plates using 1.5 µl Fugene-6 (Roche Molecular Biochemicals, Mannheim, Germany) and 0.25 µg firefly reporter plasmid per well. For standardization, 0.17 µg pRL-null vector (Promega), which encodes renilla luciferase, was cotransfected. Cells were harvested 48 h after beginning of the transfection procedure using Passive Lysis Buffer (Promega). Firefly luciferase and renilla luciferase activities were measured in a Lumat LB 9501 (Berthold Technologies, Bad Wildbad, Germany) using the Dual-Luciferase Reporter Assay System (Promega). Relative luciferase activity (RLA) is defined as the mean value of the firefly-luciferase/renilla-luciferase ratios of each construct related to the promoterless reporter plasmid pGL3 basic—[firefly-luciferase (construct)/renilla-luciferase (pRL-null vector)]/[firefly-luciferase (pGL3 basic)/renilla luciferase (pRL-null vector)]. The transfection data represent the mean RLA values of five independent experiments (each n=4) related to the wild-type promoter construct, which was set to 100%, with standard deviations indicated in the figures. With respect to statistical analysis of transient transfection experiments, a two-tailed t-test was performed and statistical significance was assumed at P<0.05.

Protein extraction
Proteins were isolated according to Schreiber et al. (61) with the following modifications: cells were harvested in PBS and resuspended in hyptonic buffer [10 mM HEPES–KOH (pH 7.9), 10 mM KCl, 1 mM DTT, 0.1 mM EGTA, 1x Complete (Roche Molecular Biochemicals)]. After addition of 37.5 µl/ml 10% IGEPAL CA-630 (Sigma, Taufkirchen, Germany) and vortexing, cytoplasmic proteins were recovered from the supernatant following centrifugation (10 000g, 10 min, 4°C). The nuclear pellet was resuspended in hypertonic buffer (20 mM HEPES–KOH (pH 7.9), 400 mM NaCl, 1 mM EGTA, 1 mM DTT, 1x Complete, 25% glycerol) and incubated for 1.5 h at 4°C under continuous shaking. Following centrifugation (13 000g, 4°C, 30 min), the supernatant containing nuclear proteins was recovered and protein concentrations were determined using the DC Protein Assay (Bio-Rad, Munich, Germany).

EMSA
Double-stranded (ds) ODNs (‘-338A’, 5'-GCTCTGGGCCAAATCGAGGGCACCT-3'; ‘-338C’, 5'-GCTCTGGGCCACATCGAGGGCACCT-3') were generated by reannealing of the corresponding complementary single-stranded ODNs. One picomole of dsODN was labeled using 2.5 µl [{gamma}-32P]ATP (10 µCi/µl, Amersham Pharmacia Biotech, Little Chalfont, UK) and 1.25 µl T4 polynucleotide kinase (10 U/µl, Promega) in a total volume of 25 µl for 1 h at 37°C, followed by purification using MicroSpin G-25 columns (Amersham Pharmacia Biotech, Freiburg, Germany). Binding reactions [12 µg protein, 60 000 cpm dsODN, 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM DTT, 300 mM KCl, 1 mM MgCl2, 5% glycerol and 2 µg poly[d(I-C)] (Roche Molecular Biochemicals) in a total volume of 25 µl] were performed overnight at 4°C. For competition analysis increasing amounts of unlabeled dsODN (1 or 50 pmol per binding reaction, respectively) were added. For super-shift analysis, proteins were preincubated with indicated antibody concentrations for 1 h at room temperature (RT) using the following antibodies: E2F-1 (sc-193X), E2F-2 (sc-633X), E2F-3 (sc-878X) and GATA-2 (sc-267X) (all Santa Cruz, Heidelberg, Germany). Binding reactions were resolved in a 4% polyacrylamide gel buffered with 1x Tris–glycine at RT. After electrophoresis (10 mA) gels were exposed to imaging plates (Fujifilm BAS-MP 2040S, Raytest Isotopenmessgeräte, Straubenhardt, Germany) and analyzed using an image plate reader (Fujifilm BAS-1500, Raytest Isotopenmessgeräte). Quantitative signal analysis was performed using the software TINA 2.09 g (Raytest Isotopenmessgeräte).

Sequence analysis
Promoter sequences were analyzed for potential cis-elements using the database TRANSFAC (version 3.5, Gesellschaft für biotechnologische Forschung, Braunschweig, Germany; mirrored at www.motif.genome.ad.jp).


   ACKNOWLEDGEMENTS
 
We wish to thank Brigitte Egbers and Katrin Kossatz-Eskandani for excellent technical assistance, and Alexander Thomas for his help in functional studies. This work has been supported by grants from the Bundesministerium for Education, Science and Technology (BMBF) to Martin Paul in the context of the Clinical Pharmacology Network Berlin-Brandenburg and the National Genome Research Network (NGFN) ‘Cardiovascular Disease’. Stefan-Martin Herrmann and Martin Paul are participants in the grant of the Deutsche Forschungsgemeinschaft: Graduierten-Kolleg 754 (supported Jacqueline Pfeifer), Myokardiale Genexpression und Funktion, Myokardhypertrophie. Karla Köpke was supported by the Berlin Center for Genome Based Bioinformatics (BMBF grant no. 031209B).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Internal Medicine, Division of Endocrinology and Nephrology, Benjamin Franklin Medical Center, Freie Universität Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. Tel: +49 3084453579; Fax: +49 3084454235; Email: ebrand{at}zedat.fu-berlin.de Back

{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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