CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Intrauterine growth retardation; Nitric oxide; Placental perfusion; miRNA.

Figure 1

NOS3 mRNA correlates with placental perfusion indices and does not depend on NOS3 intronic 27 nt-ncRNA polymorphism. (A) NOS3 mRNA levels increased >15-fold in patients suffering from severe impairment of placental perfusion (group 3). (B) Protein levels of eNOS and eNOS(Ser1177ph) correlated with NOS3 mRNA levels. NOS3 mRNA levels were as follows: 12.9 (lane 1), 22.6 (lane 2), 157.7 (lane 3), 56.8 (lane 4), 74.7 (lane 5), 136.9 (lane 6). (C) No correlation between NOS3 mRNA levels and genotype at the 27 nt-ncRNA locus in intron 5 of the NOS3 gene was found. In human alleles, two tandem-repeated homologous motifs were found in typical allele-specific order (top right). The number of patients analysed for each group is stated (n).

Figure 2

5-hydroxymethylcytosine (5hmeC) levels adjacent to the NOS3 TSS correlate with impaired placental perfusion and upregulation of NOS3 mRNA. (A) 5meC (red line) within the NOS3 promoter exhibited a local depression in all patients with minimal variations. In contrast, the amounts of precipitated 5hmeC-enriched DNA (green line) showed a local peak, particularly at the TSS (P4) with significant variations between individuals. In the gene structure cartoon (below), the first 5 NOS3 exons are marked as red boxes and a black arrow indicates the TSS. CpG-enriched regions analysed by qPCR are shown as green boxes (P1 to P6). The blue box marks the location of the 27 nt-ncRNA in intron 5. (B) 5hmeC levels at the TSS (P4), but not at other CpG-rich loci, directly correlate with NOS3 mRNA levels.

Figure 3

Dynamic histone acetylation levels adjacent to the transcription start site of NOS3 correlate with impaired placental perfusion and differential NOS3 expression. (A-B) Histone acetylation patterns differed significantly between patients with high- (green line) and low- (red line) NOS3 mRNA levels. Interestingly, levels of H3K9ac (A) as well as H2A.Zac (B) were significantly higher surrounding the transcription start site (TSS). (C-D) The combined illustration of H3K9ac and H2A.Zac patterns—calculated as mean of both fold changes—demonstrated an even more pronounced effect of histone acetylation at the TSS on NOS3 mRNA levels (C) suggesting that both acetylation hallmarks have similar biological consequences. Moreover, the overall level of acetylation around the TSS directly correlated with NOS3 mRNA levels (D). Features of the gene structure cartoon are described in Figure 2. (E-F) H3K9me3 levels adjacent to the TSS were higher in patients with high-NOS3 mRNA levels when compared to patients with low-copy number (E)—except for one patient with low-NOS3 mRNA copy number who also had a high level of H3K9me3 (UC13). At the same time, patients with high-NOS3 mRNA copy number also showed an increase in H3K9me3/S10ph (F).

Figure 4

Histone acetylation changes underlie NOS3 expression in HUAEC in response to trichostatin A treatment and is followed by deferred self-attenuation. (A) Upon HDAC inhibition via trichostatin A (TSA) in HUAEC, there is a rapid, up to 55-fold increase prior to a steep and long-lasting decrease of NOS3 mRNA levels (A). A similar pattern is seen for STAT3 expression (A). (B) Treatment of HUAEC by a restricted TSA pulse leads to an initial boost of NOS3 expression followed by deferred, continuously decreasing amplitudes upon further TSA pulses. (C) Expression of STAT3 α not STAT3β is induced by TSA treatment. (D) Transfection of HUAEC with siRNA targeted to STAT3 or NOS3 mRNA leads to a significant reduction of NOS3 mRNA levels and vice versa. Thereby, the effect of STAT3 inhibition on NOS3 is much stronger than of NOS3 on STAT3.

Figure 5

Hypoxia induces STAT3 and H2A.Zac and H3K9ac turnover at the NOS3 promoter region. (A) Predicted STAT3 binding site in the NOS3 promoter region based on the STAT3 consensus motifs are shown. During normoxia in human umbilical artery endothelial cells (HUAEC), only one region 1554 bp upstream of the transcription start site (TSS) was enriched in immune precipitates using STAT3 specific antibodies. Enrichment significantly increased during hypoxia with additional sequences being amplified 1,260 and 620 bp upstream of the TSS. (B) NOS3 and STAT3, particularly STAT3α expression, is upregulated in HUAEC in response to hypoxia. (C) Enrichment of the promoter sequence 1554 upstream of the TSS in STAT3 immune precipitates is upregulated during hypoxia in HUAECs. (D) Hypoxia induces a substantial increase in H2A.Zac and H3K9ac turnover at the NOS3 promoter region in HUAECs.

Figure 6

27 nt-siRNAs target STAT3-3′UTR and correlates with NOS3 mRNA levels in healthy infants. We used both homologous, slightly different 27 nt-motifs as siRNA for the transfection of HUAEC (A-B). In both motifs, a significant reduction of NOS3 and STAT3 mRNA levels (A) and a tendency to lower eNOS and eNOS(Ser1177ph) protein levels (B) were observed (A) 48 h post-siRNA treatment. No differences in 27 nt-RNA concentrations were found between flow group 0 and 3 (C). Whereas, in infants with normal placental perfusion (flow group 0), 27 nt-RNA enrichment correlated with lower NOS3 mRNA levels (D), no such correlation was found in infants with severe placental insufficiency (flow group 3, E).

Figure 7

Targeting of STAT3-3′UTR by 27 nt-siRNAs is reminiscent of miR targeting. (A) The biogenesis of 27 nt-ncRNA is still enigmatic. This figure shows secondary structure simulations of the different tandem repeats exhibiting regular extended stem-loops. (B) The expression of luciferase containing the STAT3 wild-type (wt) 3′-UTR target motif is significantly reduced when compared with luciferase containing a mutated putative STAT3 3′-UTR recognition motif (mut). (C) Phylogenetic analyses show that the intronic 27 nt-ncRNA motif is conserved in numerous primate species within the NOS3 gene. Analyses of the corresponding putative STAT3 recognition motifs in those primates suggest a putatively conserved miR recognition mechanism in Catarrhini, whereas less support is given for Platyrrhini and Tarsidae. No evidence for the 27 nt-ncRNA motif was found in species other than primates.