The Expression of Endothelial Nitric-oxide Synthase Is Controlled by a Cell-specific Histone Code

Abstract

Expression of endothelial nitric-oxide synthase (eNOS) mRNA is highly restricted to the endothelial cell layer of medium to large sized arterial blood vessels. Here we assessed the chromatin environment of the eNOS gene in expressing and nonexpressing cell types. Within endothelial cells, but not a variety of nonendothelial cells, the nucleosomes that encompassed the eNOS core promoter and proximal downstream coding regions were highly enriched in acetylated histones H3 and H4 and methylated lysine 4 of histone H3. This differentially modified chromatin domain was selectively associated with functionally competent RNA polymerase II complexes. Endothelial cells were particularly enriched in acetylated histone H3 lysine 9, histone H4 lysine 12, and di-and tri-methylated lysine 4 of histone H3 at the core promoter. Histone modifications at this region, which we have previously demonstrated to exhibit cell-specific DNA methylation, were functionally relevant to eNOS expression. Inhibition of histone deacetylase activity by trichostatin A increased acetylation of histones H3 and H4 at the eNOS proximal promoter in nonexpressing cell types and led to increased steadystate eNOS mRNA transcript levels. H3 lysine 4 methylation was also essential for eNOS expression, since treatment of endothelial cells with methylthioadenosine, a known lysine 4 methylation inhibitor, decreased eNOS RNA levels, H3 lysine 4 methylation, and RNA polymerase II loading at the eNOS proximal promoter. Importantly, methylthioadenosine also prevented the trichostatin A-mediated increase in eNOS mRNA transcript levels in nonendothelial cells. Taken together, these findings provide strong evidence that the endothelial cell-specific expression of eNOS is controlled by cell-specific histone modifications.Endothelial nitric-oxide synthase (eNOS, 1 NOS3) is constitutively expressed in vascular endothelial cells, especially the endothelial layer of medium to large sized arterial blood vessels, where it is known to play a key role in vascular wall homeostasis and regulation of vasomotor tone. Studying the mechanisms regulating the constitutive transcription of eNOS in endothelial cells is essential to understand how these mechanisms may be perturbed in diseases characterized by a decrease in eNOS mRNA in the vascular endothelium. For example, constitutive expression of eNOS is compromised in the endothelial cells overlying advanced human atherosclerotic plaques (1-3).In general, the basis for endothelium-specific gene expression is not known. Whereas models involving DNA-binding transcription factors (e.g. AP-1, Ets family members, GATA-2, octamer proteins, or Sp1) (4 -7) have been invoked to explain the transcriptional control of a variety of endothelial genes, these models cannot fully account for the exquisite specificity of these endothelium-specific promoters, given that these transfactors are ubiquitously expressed. This can be contrasted with the control of muscle-specific or adipocyte-specific genes, which are controlled by "master regulators," i...

Histone H3 Lysine 4 Dimethylation Signals the Transcriptional Competence of the Adiponectin Promoter in Preadipocytes

“…To study the role of H3-K4 dimethylation on apM1 expression, we differentiated 3T3-L1 cells in the presence of MTA, a known inhibitor of AdoMet-dependent methyltransferases, that has previously been shown to inhibit H3-K4 methylation (36). Accordingly, by D5 of the differentiation process, we observed a marked decrease of global H3-K4 dimethylation in cells treated with the inhibitor.…”

“…Histone Modifications Spread through the apM1 Locus, Correlating with the Beginning of Gene Transcription-A host of previous studies have shown elevated levels of histone H3 hyperacetylation and K4 methylation localized to the 5Ј-proximal regions of transcriptionally active genes (2,7,36). The presence of the same modifications downstream of the transcription initiation site, on the other hand, is not as preeminent (12,37,38).…”

“…Cells-To check if the presence of H3-K4 dimethylation at the promoter regions of adipogenic genes is necessary for their later expression, we treated differentiating 3T3-L1 cells with MTA, an inhibitor of AdoMet-dependent methyltransferases that has been shown to inhibit histone H3-K4 methylation (36). We treated postconfluent 3T3-L1 fibroblasts 24 h before the stimuli of differentiation with 1 mM MTA to inhibit histone methylation prior to the start of adipogenesis.…”

Transcriptional regulation of endothelial nitric oxide synthase expression in uterine artery endothelial cells by c-Jun/AP-1

“…However, the NOS3 promoters of all species isolated so far, including sheep, also contain binding sites for multiple additional transcription factors participating in the complex regulation of NOS3 transcription in endothelial cells in a tissue/cell, and possibly species specific manner (Chan et al, 2004). Moreover, tissue and cell-specific transcriptional regulation of NOS3 expression is controlled by methylation (Chan et al, 2004) and histone acetylation (Fish et al, 2005;Gan et al, 2005) of the NOS3 promoter. In the present study, according to the sequence information of the 5′-flanking region of the sheep NOS3 gene, we hypothesized that the proximal consensus AP-1 (TGAGTCA) site positioned at the −682 to −676 upstream the ATG start codon in the sheep NOS3 gene plays an important role in the regulation of uterine artery endothelial NOS3 expression.…”

Epigenetic regulation of pulmonary arterial hypertension

“…Within endothelial cells, the eNOS core promoter is highly enriched in acetylated histone H3/K9 and H4/K12, and methylated H3/K4. 77 Furthermore, HDAC inhibitor trichostatin A may induce eNOS expression in non-endothelial cells, and small RNA may suppress eNOS expression by altering histone acetylation and DNA methylation in endothelial cells. 78,79 In our rat model for PPHN, the levels of eNOS mRNA and protein expression were significantly upregulated.…”