CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression
© Postberg et al.; licensee Biomed Central. 2015
Received: 5 November 2014
Accepted: 22 December 2014
Published: 8 February 2015
An adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the underlying epigenetic signatures involved in its regulation.
NOS3 and STAT3 mRNA levels were elevated in HUAEC of patients who suffered from placental insufficiency. 5-hydroxymethylcytosine, H3K9ac and Histone 2A (H2A).Zac at the NOS3 transcription start site directly correlated with NOS3 mRNA levels. Concomitantly, we observed entangled histone acetylation patterns and NOS3 response upon hypoxic conditions in vitro. Knock-down of either NOS3 or STAT3 by RNAi provided evidence for a functional NOS3/STAT3 relationship. Moreover, we recognized massive turnover of Stat3 at a discrete binding site in the NOS3 promoter. Interestingly, induced hyperacetylation resulted in short-termed increase of NOS3 mRNA followed by deferred decrease indicating that NOS3 expression could become self-attenuated by co-expressed intronic 27 nt-ncRNA. Reporter assay results and phylogenetic analyses enabled us to propose a novel model for STAT3-3′-UTR targeting by this 27-nt-ncRNA.
An adverse intrauterine environment leads to adaptive changes of NOS3 expression. Apparently, a rapid NOS3 self-limiting response upon ectopic triggers co-exists with longer termed expression changes in response to placental insufficiency involving differential epigenetic signatures. Their persistence might contribute to impaired vascular endothelial response and consequently increase the risk of cardiovascular disease later in life.
KeywordsPlacental perfusion miRNA Nitric oxide Intrauterine growth retardation
Epidemiological evidence suggests that early environmental factors such as placental insufficiency correlate with increased disease risks later in life, e.g. cardiovascular disease . The phenotypic changes conveying these risks are necessarily due to deregulated expression of specific genes during the acute phase of the insult. Gene expression changes are known to be strongly associated with the plasticity of chromatin, whose structure can be influenced by CG dinucleotide (CpG) signalling and post-translational modifications (PTMs) of histones. Their combinatorial signatures control the spatiotemporal expression of genes in a potentially heritable way. In more detail, desoxyribonucleic acid (DNA) methylation targets cytosines (5-methylcytosine (5meC)) predominantly at isolated asymmetric CpG motifs (‘open sea’) or in the genomic context of so-called ‘CpG islands’ and adjacent regions (‘shores/shelves’) [2-4]. 5meC enrichment in promoters is frequently associated with transcriptional repression . Recently, hydroxymethylated cytosines (5-hydroxymethylcytosine (5hmeC)) were recognized as another functional DNA modification. Currently, the most widely accepted hypothesis is that 5hmeC is an intermediate state of active DNA demethylation conferred by members of the Tet protein family  and thus influencing gene expression [7,8]. Yet, another level of gene expression regulation is conveyed by PTMs of histones, which occur in a dynamic fashion through variable combinations at a given site. PTMs can influence chromatin compaction either directly or in conjunction with ‘reader’ proteins, thus regulating either activation or repression of genes .
The placenta represents the interface between the foetal and maternal organism. Its perfusion is therefore closely linked to foetal well-being and critically influences foetal development . One important factor influencing short- and long-term placental perfusion is hypoxia [11-13], representing the most adverse intrauterine milieu for the foetus. In response—particularly in chronic hypoxia —foetal circulation compensatory mechanisms involve nitric oxide (NO) synthesis by endothelial nitric oxide synthase (eNOS), which eventually contributes to blood vessel tone regulation. NOS3 mRNA synthesis, which gives rise to the eNOS protein, was recently found to be diminished in response to hypoxia in human umbilical vein endothelial cells (HUVEC) . In contrast, NOS3 mRNA was increased in human umbilical artery endothelial cells (HUAEC) . On the molecular level, NOS3 expression and eNOS activation are complex, mainly mediated by PI3K/Akt- or AC/PKA-signalling involving transcriptional, post-transcriptional and post-translational factors [17,18]. Attributes of current short-term regulation concepts involve rapid modulation of NOS3 mRNA stability or activation of the eNOS protein via serine phosphorylation . Adaptations to long-term stimuli (e.g. chronic hypoxia) apparently involve regulation on the transcriptional level and hence epigenetic plasticity . Recently, it has been shown in cell culture experiments that hypoxic repression of eNOS transcription in HUVEC is coupled with eviction of promoter histones . Further detailed insights into this aspect of NOS3/eNOS regulation are lacking to date, particularly in relation to its corresponding clinical significance.
We therefore undertook comprehensive analyses on the regulation of NOS3 expression in foetal HUAEC cells. This included NOS3 mRNA quantification, genotyping of an intronic putative non-coding RNA (ncRNA) locus suspicious for pathomechanistic relevance in cardiovascular diseases , monitoring of CpG signalling at the NOS3 promoter and quantification of PTMs relevant for chromatin structure regulation. We further tested a yet sparsely evidenced connection between Stat3 signalling and NOS3/eNOS regulation .
Impaired placental perfusion is associated with enriched NOS3 mRNA independent of tandem repeat polymorphisms of the NOS3 intronic 27 nt-ncRNA
Total ( n = 39)
Flow group 0 ( n = 10)
Flow group 1 ( n = 12)
Flow group 2 ( n = 10)
Flow group 3 ( n = 7)
Gestational age (SD)
31.9 ± 4.9
31.8 ± 4.8
31.6 ± 5.7
32.1 ± 4.9
31.2 ± 4.6
Birth weight in g (SD)
1,655 ± 917
1,746 ± 1,008
1,652 ± 810
1,636 ± 909
1,336 ± 657
Birth length in cm (SD)
41.4 ± 6.1
41.0 ± 7.1
40.7 ± 6.1
41.9 ± 5.5
40.7 ± 5.2
Maternal age (SD)
31.9 ± 4.9
31.8 ± 4.8
31.6 ± 5.7
31.2 ± 4.9
31.2 ± 4.6
21.9 ± 3.5
23.3 ± 2.8
22.9 ± 3.5
19.7 ± 3.0
18.8 ± 1.98
Apgar at 5 min
8.5 ± 1.2
8.5 ± 1.4
8.7 ± 1.3
8.4 ± 1.3
8.3 ± 1.1
pH at birth
7.34 ± 1.1
7.34 ± 1.2
7.33 ± 1.1
7.35 ± 1.4
7.32 ± 1.2
HELLP syndrome (%)
High-5-hydroxymethylcytosine levels adjacent to the NOS3 transcription start site (TSS) correlate with impaired placental perfusion and enriched NOS3 mRNA
Oligonucleotides used in this study
Sequence 5′ to 3′
27 nt-siRNA motif 1
27 nt-siRNA motif 2
Turnover of histone acetylation adjacent to the NOS3 TSS correlates with impaired placental perfusion and differential NOS3 expression
For transcriptional repression, 5meC presumably must be read out by 5meCpG-binding proteins, which entails assembly of repressive complexes. These complexes can contain histone deacetylase (HDAC) activity [5,25-27]. Vice versa, local chromatin conformation could also depend on 5hmeC signatures. To obtain comprehensive insight into the epigenetic regulation of NOS3 expression, we thus decided to analyse whether modifications of the chromatin signature might also be involved in NOS3 regulation. Out of seven patients’ samples belonging either to flow group 0 (n = 3) or flow group 3 (n = 4), we could purify sufficient amounts of chromatin for chromatin immunoprecipitation (ChIP) followed by qPCR analyses. We focused these analyses on histone modifications frequently associated with the promoters of either transcriptionally competent/active genes (histone 2A (H2A).Zac, H3K9ac) or repressed genes (H3K9me3). Further, we targeted histone 3 (H3) concomitantly modified at lysine-9 and serine-10 (H3K9me3/S10ph), since it has been reported that phosphorylation of serine-10 is sufficient to eject heterochromatin protein 1 (HP1) bound at H3K9me3  thus counteracting heterochromatin formation. This selection enabled us to analyse three switch positions at one discrete PTM target site, which could potentially be associated with biological consequences.
Histone acetylation modifies NOS3 expression in HUAEC in response to trichostatin A treatment entailed by deferred self-attenuation
However, evidence for a direct connection between STAT3 signalling and NOS3 remains marginal to date. Since non-redundant biological relevance of two isoforms, Stat3α and Stat3β (encoded by two transcript variants), is controversially discussed [30,31], we decided to discriminate these isoforms. Interestingly, primarily the transcript-variant-encoding Stat3α is expressed in HUAEC cells. This variant was shown to be responsive to TSA treatment in our experiments (Figure 4C). To gain deeper insight into a putative interdependence of STAT3 and NOS3, we analysed whether RNA interference targeting either STAT3 or NOS3 in HUAEC might affect the expression of NOS3 or STAT3α/β (Figure 4D). Quantitative PCR analyses confirmed that both siRNAs led to significant reduction of their target mRNAs. Importantly, levels of STAT3α, but not STAT3β mRNA, were also significantly decreased when NOS3 was targeted by siRNA and vice versa (Figure 4C).
Site-specific STAT3 enrichment at the NOS3 promoter correlates with increased turnover of H2A.Zac and H3K9ac at the NOS3 TSS in response to hypoxia
Self-attenuation of NOS3 is a mediated expression of NOS3 intronic 27 nt-ncRNA
To gain further insights into the clinical relevance of this negative feedback mechanism, we measured 27 nt-ncRNA concentrations in biosamples from our patients. Surprisingly, at first sight, we did not find any differences between 27 nt-ncRNA concentrations when comparing patients with normal (flow group 0) and severely impaired placental perfusion (flow group 3) (Figure 6C). However, in patients without impairment of placental perfusion, enrichment of 27 nt-ncRNA correlated with lower NOS3 mRNA levels and vice versa (r 2 = 0.69, p = 0.07; Figure 6D). Importantly, such a correlation could not been found for patients with severe placental insufficiency (r 2 = 0.004, p = 0.91; Figure 6E).
NOS3-intronic 27 nt-ncRNA targets the STAT3-3′-UTR and acts reminiscently of 5′-dominant microRNAs
In this study, we showed that the foetal response upon alterations in placental perfusion involves differential expression of NOS3 in HUAEC. There was no influence of known genotype variations as described for other disorders in adult patients . Importantly, NOS3 expression correlated with perfusion indices in both the foetal and maternal circulation, thus for the first time linking human clinical data on placental perfusion to molecular changes in the foetus. These in vivo data are in line with a recent in vitro study demonstrating increased NOS3 activity in HUAEC in response to hypoxia .
We also demonstrated that the changes observed in response to placental insufficiency are associated with changes of the epigenetic signatures at the NOS3 gene locus. We noted that the 5meC level at the NOS3 promoter was low in all patients without being correlated with NOS3 expression. This corresponds well with the observation of widespread CpG hypomethylation within the NOS3 promoter in endothelial cells, and hypermethylation in non-endothelial cells . However, we found significant differences in the level of 5hmeC adjacent to the TSS correlating with placental and foetal perfusion indices and NOS3 mRNA levels suggesting a regulatory role for 5hmeC in NOS3 transcription. Mechanistically, the overlap of a local 5meC depression with a 5hmeC peak at the same site might be interpreted as rapid turnover of DNA methylation at this site .
Next, we analysed selected histone modifications in relation to NOS3 transcription. Similar to 5hmeC levels, we found increased levels of H3K9ac and H2A.Zac adjacent to the TSS in patients with high-NOS3 mRNA levels who suffered from intrauterine placental insufficiency. Interestingly, in patients with high-NOS3 mRNA levels, H3K9ac and H2A.Zac seemed to be interchangeable with patients having either highly increased turnover of H3K9ac or H2A.Zac or moderately increased acetylation at both sites. This corresponds well to the concept that histone acetylation adjacent to histone-DNA contact sites could directly compensate the positive charge of lysines leading to weaker interactions of the negatively charged DNA phosphate backbone which thus counteracts repressive chromatin configurations . Remarkably, significant changes of histone PTM levels were consistently found at two sites flanking the TSS, whereas the TSS itself behaved relatively inert. This pattern could indicate the existence of at least two well-positioned nucleosomes adjacent to a nucleosome-depleted region in direct proximity to the TSS in NOS3. This observation is also in line with data presented by Fish et al. demonstrating reduced levels of H3K9ac and H2A.Zac in HUVEC associated with decreased eNOS expression in vitro .
For H3K9me3, which binds snuggly to heterochromatin protein 1 (HP1) and hence promotes heterochromatin formation and gene repression, we found generally low levels in most patients independent of NOS3 mRNA levels. This is in line with the status of a transcriptionally competent gene which is also reflected by the low-5meC levels at the NOS3 promoter observed in all patients. Occasionally and somewhat unexpectedly, we also observed H3K9me3 enrichment in association with high-NOS3 mRNA levels. However, further investigation revealed that concomitantly serine-10 phosphorylation occurred at the same site (H3K9me3/S10ph) in the affected individuals most likely representing a binary methylation/phosphorylation switch, which was reported to counteract HP1 binding and heterochromatin formation, thus disabling the repressive competence of H3K9me3 [28,43]. This status is also somewhat reminiscent of a poised gene associated with bivalent PTM, i.e. the existence of opposing histone modifications at the same nucleosome. Notably, in one patient’s sample exhibiting low-NOS3 mRNA amounts in combination with enriched H3K9me3, H3K9me3/S10ph was absent. Here, H3K9me3 is likely to be involved in the establishment of a repressive chromatin structure at the NOS3 promoter. Overall, the variances in histone acetylation and methylation patterns observed in this study clearly confirm that chromatin signatures involved in the regulation of a specific gene (such as NOS3) act in combination and are interdependent. In clinical specimens from individual patients, they thus need to be interpreted with caution always considering the greater context .
In the second part of this study, we aimed to provide deeper mechanistic insights into NOS3 gene regulation using an in vitro cell culture model. This was important, because a previous study by Rossig and co-workers reported a marked reduction in NOS3 expression upon treatment of endothelial cells with the HDAC inhibitor TSA for at least 12 h suggesting—in contrast to our in vivo findings—a repressive effect of histone acetylation on NOS3 expression . One possible explanation for this contradictory observation could be a negative feedback mechanism counteracting the increased NOS3 transcription enforced by histone acetylation possibly involving a 27 nt-ncRNA encoded in NOS3 intron 5 as proposed earlier . To evaluate this hypothesis, we performed TSA time course experiments in HUAEC also including STAT3 in these analyses, which has been previously linked to NOS3 regulation [23,45]. We could confirm that prolonged TSA treatment indeed resulted in a marked reduction in NOS3 mRNA copy number. However, short-termed treatment significantly increased the NOS3 mRNA copy number. Importantly, a parallel pattern was observed for STAT3 mRNA. Pulse-release experiments with TSA moreover showed that the deferred self-attenuation effect was sustainable. This observation is in agreement with a negative feedback loop entangled with NOS3 expression, which is possibly driven by a putatively co-processed 27 nt-ncRNA encoded in NOS3 intron 5  and involves targeting and cleavage of STAT3 mRNA . To explore this possible connection, we aimed to characterize the functional relationship between NOS3 and STAT3 expressions as well as Stat3 protein function. With respect to known Stat3-binding motifs , we identified several potential binding sites within the NOS3 promoter. Using the HUAEC hypoxia model for placental insufficiency , we showed that Stat3 selectively bound to one of the predicted sites (namely −1.554 bp upstream of the TSS). Also Stat3 turnover was significantly increased in the course of hypoxia, strongly suggesting a functional connection between Stat3 and NOS3 regulation. In addition, dynamics of NOS3 expression and histone acetylation patterns at the NOS3 TSS in response to hypoxia perfectly corresponded to data obtained from clinical samples.
Next, we explored the mechanism of self-attenuation by intronic 27 nt-ncRNA using its corresponding sequence for RNA interference in HUAEC cultures. As hypothesized, STAT3 and NOS3 mRNA levels decreased significantly in response to treatment with both 27 nt-ncRNA motifs found in the human genome, thus giving further support to the proposed concept of a negative feedback mechanism. Importantly, we were also able to demonstrate that 27 nt-ncRNA levels correlated with NOS3 mRNA levels in patients with normal placental flow whereas this relationship seemed to be dissolved in patients with severe placental insufficiency. This might be an indicator for a more permanent disruption of epigenetic regulatory mechanisms in these infants. If so, this possibly predisposes these patients for vascular disorders later in life.
Since previous hypotheses on the 27 nt-ncRNA targeting mechanism did not seem to hold with regard to current models of miRNA targeting principles, we decided to reinvestigate the underlying mechanisms using an in vitro reporter assay as well as phylogenetic analyses. These experiments enabled us to propose a novel hypothesis of STAT3-3′-UTR targeting by the 27 nt-ncRNA, which is reminiscent of 5′-dominant miRNA targeting, including perfect anchor and seed matching, and therefore most probably fulfilling the requirements of target matching and for the assembly of a functional Argonaute-miRNA complex. Furthermore, the proposed mechanism seems to be conserved in a large number of primate species.
Overall, our study provides comprehensive evidence how an adverse intrauterine milieu directly influences human foetal gene expression by means of various levels of gene regulation in vascular endothelial cells, including CpG signalling, chromatin plasticity and non-coding regulatory RNA. Whether the observed changes are reversible or sustained remains an open problem to address. Due to ethical concerns, this question is currently impossible to answer in human individuals given the methodology available. However, epidemiological studies reporting correlation between epigenetic signatures at the NOS3 gene locus in HUAEC and obesity as well as bone mineral content suggest that a hypoxia-induced epigenetic memory state might persist . Such persistence would provide a pathophysiological explanation for the linkage between impaired foetal growth and later vascular abnormalities observed in numerous epidemiological studies . In theory, the epigenetic signature at the NOS3 locus at birth might define an individual baseline level of NOS3 transcription for further life. Alterations of this baseline might subsequently modify the bandwidth of variation in which an individual can respond to adverse events later in life. This in turn could explain the impaired endothelial vascular response observed in patients who were exposed to an adverse intrauterine milieu.
Written informed consent for human specimen was obtained from all legal guardians. Ethical approval for this study was obtained from the Witten/Herdecke University ethics committee. All work has been conducted according to the principles expressed in the Declaration of Helsinki.
The authors of foregoing studies use deviating annotation of the tandemly repeated 27 nt-ncRNA VNTR; namely, it is annotated being encoded within NOS3 intron 4. We refer to human NOS3 mRNA [RefSeq Gene NM_000603] encoded on chromosome 7q36, GenBank ID 4846, which spans a 23.54-kb region from base 150688144 to 150711687 [NC_000007.13]. Thereafter, NOS3 mRNA encoding the canonical transcript variant 1 derives from 27 introns, and the 27-bp tandem repeat is located within intron 5 between exons 5 and 6.
Patients were recruited over a 6-month period. Eligible infants obtained Doppler examination of placental and foetal circulation 12–24-h predelivery. GA estimation relied on ultrasound classification before week 14 of gestation. We aimed to depict the whole gestational age range and to include ten patients per flow group (see below). Epidemiological parameters, hospital course data and the outcome were collected. Foetuses with gestational diabetes were excluded since resistance and pulsatility indices differ from normal patients without correlation to foetal growth parameters in these patients [48,49].
Placental and foetal perfusion assessment
group 1: abnormal UA pulsatility index (PI) >2 standard deviations (SD) above mean, or absent UA end-diastolic flow, and normal MCA PI (mean ± 2 SD);
group 2: abnormal UA PI >2 SD above mean, or absent or reverse UA end-diastolic flow, and abnormal MCA PI (mean < 2 SD) and normal DV PI (mean ± 2 SD);
group 3: absent or reverse UA end-diastolic flow, and abnormal MCA PI (mean < 2 SD), and abnormal DV PI (mean > 2 SD, a-wave present or absent or reverse end-diastolic flow).
HUAEC isolation and cell culture
Immediately after delivery, umbilical cords were transported on ice from the maternity ward to the laboratory. HUAEC were separated as described previously . RNA was extracted using TRIzol (Life Technologies) and quantified at 260 nm. RNA integrity was assessed by agarose gel electrophoresis. For cDNA synthesis, we used 500 ng RNA per sample using the QuantiTect Reverse Transcription kit (Qiagen). DNA was isolated by phenol:chloroform:isoamylic alcohol extraction. For in vitro experiments, HUAEC (Promocell) were cultivated upon manufacturer’s recommendations. For HDAC inhibition, HUAEC were treated with 1-μM TSA. Hypoxia experiments were performed using a hypoxia incubator chamber (STEMCELL Technologies, Grenoble, France) exposing cells to a ppO2 of 0.1 bar corresponding to an oxygen fraction of 10% for 24 h. Control experiments were performed under normoxic conditions (ppO2 = 0.21 bar).
Gene expression analyses
Gene expression analyses were performed using qPCRanalyses on a Rotor-Gene 6000 (Qiagen). For PCR reactions, QuantiTect SYBR Green qPCR Master Mix (Qiagen) containing Hot Start Taq DNA polymerase and SYBR Green was used. Primers were used as listed in Table 2. The expression of genes of interest was normalized to at least three out of five housekeeping genes (BACT, GAPDH, PECAM1, RPL19, VIL1). Following TSA experiments, the mitochondrial ATP6 gene was used for normalization, since interference of this drug with the chromatin state of nuclear housekeeping genes was expected. PCR conditions were as follows: 95°C for 15 min, 40× [95°C for 15 s, 60°C for 30 s]. Melting of PCR product was done using a gradient from 55°C to 95°C rising in 0.5°C increments. For relative comparative quantification of gene expression fold changes, we utilized the ΔΔCt method  using at least three housekeeping genes for normalization.
Polymorphism and zygosity of the NOS3 intron 5 27 nt-ncRNA VNTR was determined by PCR or, in some cases, nested PCR (Table 2). PCR fragments were cloned into the pGEM-T easy vector (Promega) prior to Sanger sequencing (GATC Biotech). Homozygosity was evaluated by at least five replicates.
Primary antibodies used in this study were as follows: 1. Mouse anti-5meC (Diagenode mAb33D3C15200081), 2. Rat anti-5hmeC (Diagenode mAb633HMC-020), 3. Rabbit anti-H2A.Zac (Diagenode pAb-173-050), 4. Rabbit anti-H3K9ac (Active Motif pAb#39137), 5. Rabbit anti-H3K9me3 (Active Motif pAb#39161), Rabbit anti-H3K9me3S10ph (Diagenode pAbCS-128-100), 5. Rabbit anti-eNOS (Cell Signalling Technologies pAb#9572), 6. Rabbit anti-eNOS Ser1177ph (Cell Signalling Technologies mAbC9C3 #9570), 7. Rabbit anti-H3K4me3 (Diagenode pAbCSP-030-050), 8. Rabbit anti-α-Tubulin (Sigma Aldrich mAbDM1A T9026) and 9. Rabbit anti-Stat3 (D3Z2G, Cell Signalling Technologies mAb#12640).
(Hydroxy-)methylated DNA immunoprecipitation and qPCR analyses
Abbreviations: AE (amplification efficiency); Ct (cycle threshold values obtained from exponential phase of the PCR reaction); the dilution factor (DF) 10 corresponds to 10% ‘input’ sample—thus, the resulting compensatory factor in our experiments was 3.32.
Chromatin purification and ChIP assays
Chromatin was purified from HUAEC isolated from seven individuals belonging to the low- or high-level NOS3 expression groups. Cells were fixed in PBS/1% formaldehyde for 10 min at room temperature, washed with PBS and incubated with glycine stop solution, prior to additional washing with PBS. Cells and nuclei were then homogenized in ice-cold ChIP buffer (50 mM NaCl; 50 mM Tris–HCl, pH 7.5; 0.1 mM PMSF; 5 mM EDTA; 0.1% SDS) using a Qiagen TissueRuptor device. Following centrifugation for 10 min at 13.000 rpm in a microcentrifuge at 4°C, the supernatant containing the soluble chromatin fraction was collected, and the chromatin concentration was measured at 260 nm using a NanoPhotometer (Implen). Portions of 25-μg (0.1 ng/μl) chromatin were sheared by ultrasonic treatment using a Bioruptor UCD-200 (Diagenode) and 25× [30 s ON/30 s OFF] at position ‘high’. Chromatin fragment size was controlled on agarose gels, and one of the chromatin aliquots was saved as input. For ChIP 25 μg sheared chromatin was incubated with the respective antibody in a rotator for 16 h at 4°C in a total volume of 250 μl ChIP incubation buffer. Subsequently 25 μl protein G magnetic beads (Active Motif) were added and incubated for 4 h at 4°C rotating. Protein G magnetic beads were separated using a magnetic rack and washed repeatedly. To elute DNA fragments enriched by immunoprecipitation, immunocomplexes were incubated with elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, (pH 8.1)) for 30 min at 65°C on a shaker. Eluted immunocomplexes were treated with proteinase K. Quantitative PCR analyses were performed using a Rotorgene 6000 (Qiagen). The relative amounts of specifically immunoprecipitated DNA were estimated as ‘percent of input’ and quantified using individual standard curves for each amplicon. Primer pairs were used as described in Table 2.
RNA interference and reporter assay
For HUAEC transfection, oligonucleotides corresponding to two different 27 nt-ncRNA motifs were used. For transfection into HeLa cells, siRNAs targeted to STAT3 or NOS3 were used. HiPerFect transfection reagent (Qiagen) was used. Effects of 27 nt-ncRNA or siRNAs targeting STAT3 or NOS3 on the expression of NOS3 or STAT3 were assessed in HUAEC 72 h post-transfection by qPCR as described above. For other experiments, we cloned a 1.160-bp DraI/NheI-fragment from the STAT3-3′-UTR containing either the wild-type or a mutated miRNA targeting site into the pmirGLO Dual-Luciferase miRNA target expression vector (Promega). These constructs were transfected into HeLa using Lipofectamine 2000 (Life Technologies) 48 h prior to transfection of siRNAs targeting the [luciferase]-STAT3-3′-UTR. Here, the levels of luciferase mRNAs were measured by qPCR 24 h post-transfection.
MiR library generation and qPCR.
From selected specimens, we prepared microRNA libraries. Therefore, each sample was tagged with multiplex sequencing barcodes. Total RNA was separated by polyacrylamide gel electrophoresis. Gel fragments corresponding to 15 to 35 nt RNA molecules were cut and RNA was eluted. The obtained small RNA fraction was directly used for the construction of libraries in four steps. Step 1: Ligation of DNA oligonucleotides to the 3′-end of the RNA; Step 2: Ligation of RNA or, respectively, chimeric RNA/DNA oligonucleotides to the 5′-end of RNAs; Step 3: cDNA library synthesis by reverse transcriptase; Step 4: qPCR analyses of the cDNA libraries using pairs of Illumina (San Diego, California, US) index primers, which corresponded to the adapter oligonucleotides used, and a 27 nt-ncRNA-specific primer (5′-tagacctgctgcrggggtgag-3′) were performed as described above.
Mean expression levels were calculated at least from triplicate real-time PCR measurements. In all figures, data are presented as median ± interquartile range (IQR), minimum and maximum, and values for p < 0.05 were considered statistically significant unless depicted otherwise. Significance testing was performed using a one-way ANOVA test for values with Gaussian distribution and Mann–Whitney U test for values without Gaussian distribution. The Kolmogorov-Smirnov test was utilized to rule out non-Gaussian distribution. All analyses were performed using GraphPad version 5.01 (La Jolla, CA USA).
Heterochromatin protein 1
Human umbilical artery endothelial cell
Human umbilical vein endothelial cell
Inducible nitric oxide synthase
Methylated DNA immunoprecipitation
Quantitative real-time polymerase chain reaction
Testis-specific histone 2B
Transcription start site
Variable number tandem repeat
We thank the children and their parents for their participation in this study and Claudia Förster for excellent technical assistance and facilitating daily lab routine.
Sources of funding
This study was funded by HELIOS Research Center GmbH, Friedrichstraße 136, 10117 Berlin, Germany (ID 009694). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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