Role of histone deacetylation in cell-specific expression of endothelial nitric-oxide synthase

Abstract

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of endothelial nitric-oxide synthase (eNOS) are not fully understood. In this study we investigated whether histone deacetylation was involved in repression of eNOS expression in non-endothelial cells. Induction of eNOS expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate was observed in all four different types of non-endothelial cells examined. Chromatin immunoprecipitation assays showed that the induction of eNOS expression by TSA was accompanied by a remarkable increase of acetylation of histone H3 associated with the eNOS 5'-flanking region in the non-endothelial cells. Moreover, DNA methylation-mediated repression of eNOS promoter activity was partially reversed by TSA treatment, and combined treatment of TSA and 5-aza-2'-deoxycytidine (AzadC) synergistically induced eNOS expression in non-endothelial cells. The proximal Sp1 site is critical for basal activity of eNOS promoter. The induction of eNOS by inhibition of HDACs in non-endothelial cells, however, appeared not mediated by the changes in Sp1 DNA binding activity. We further showed that Sp1 bound to the endogenous eNOS promoter and associated with HDAC1 in non-endothelial HeLa cells. Combined TSA and AzadC treatment increased Sp1 binding to the endogenous eNOS promoter but decreased the association between HDAC1 and Sp1 in HeLa cells. Our data suggest that HDAC1 plays a critical role in eNOS repression, and the proximal Sp1 site may serve a key target for HDCA1-mediated eNOS repression in non-endothelial cells.

Fig. 1. Induction of eNOS mRNA expression in non-endothelial cells by HDACs inhibitor TSA

A, induction of eNOS mRNA by TSA in four non-endothelial cell types. The cells were treated with 0.2 μg/ml TSA or Me₂SO in control cells for 24 h. The eNOS mRNA level was quantitated by real-time reverse transcription-PCR and normalized to the housekeeping gene β-actin. B, TSA dose-dependent induction of the eNOS mRNA in HeLa cells. The eNOS mRNA was quantitated as in Panel A. All data are presented as the mean ± S.D. of three separated experiments. CTR, control. HCSMC, human coronary artery smooth muscle cells.

Fig. 2. Comparison of basal activity of eNOS proximal promoter in endothelial and non-endothelial cells and activation of the eNOS promoter by the HDACs inhibitor TSA in non-endothelial

A, basal activity of the eNOS promoter in non-endothelial and endothelial cells. The eNOS promoter/reporter constructs were transfected into the cells, and luciferase activity was measured and normalized as the percentage of the pGL3 control vector. B, eNOS promoter was activated by TSA in HeLa. Luciferase activity was measured 24 h after the cells treated with or without TSA. All data are presented as the mean ± S.D. of three separated experiments. CTR, control.

Fig. 3. Effect of TSA on acetylation of histone H3 associated with eNOS 5′-flanking region

A, schematic graph of the eNOS promoter 5′-flanking region and the location of four pairs of primers designated as A, B, C, and D used for PCR amplification in the ChIP assay. TSS, transcription start site. kb, kilobases. B, accumulation of acetylation of histone H3 associated with eNOS in non-endothelial cells by TSA treatment. ChIP assays were performed with anti-acetylated histone H3 antibody for the cells treated with or without TSA. Primers for the eNOS 5′-flanking region, as indicated in panel A, were used to amplify the DNA isolated from the ChIP assay. The data shown are representative of three separated experiments. IP, immunoprecipitated; HUVEC, human umbilical vein endothelia cells.

Fig. 4. Effects of TSA on DNA methylation mediated-repression of eNOS promoter

In vitro methylated and mock-methylated eNOS promoter/reporter constructs were transiently transfected into HeLa cells followed by treatment with or without TSA. Luciferase activity was measured 24 h after the treatment. The data shown are the mean ± S.D. of three separated experiments.

Fig. 5. Synergistic induction of eNOS mRNA expression by TSA and AzadC

HeLa cells were treated with 7 μm AzadC for 8 days, 1 μg/ml TSA for 24 h, or AzadC for 7 days combined with TSA (1 μg/ml) for another 24 h. The eNOS mRNA level was quantitated as in Fig. 1. The data shown are the mean ± S.D. of three separated experiments.

Fig. 6. DNA methylation status of the eNOS proximal promoter after treated with AzadC or TSA

A, bisulfite genomic sequencing results of subcloned PCR products of HeLa cells treated with AzadC (7 μm) for 8 days. B, bisulfite genomic sequencing results of subcloned PCR products of HeLa cells treated with TSA (1 μg/ml) for 24 h. The x axis of each graph indicates the CpG doublet position in the proximal promoter region of eNOS with respect to the start site of transcription. CTR, control. Each column represents the percentage of the methylated CpG of total 10–15 individual clones. p < 0.05 (*) and p < 0.01 (**) are by the χ² analyses.

Fig. 7. The role of the Sp1 in TAS induced eNOS expression in non-endothelial cells

A, DNA binding activity of Sp1 in non-endothelial cells after TSA treatment. EMSA was performed by incubating DIG-labeled Sp1 probe with the nuclear extracts from HeLa cells treated with or without TSA. The competitor was unlabeled probe with a 100-fold molar excess. B, role of the proximal Sp1 site for basal activity of eNOS promoter. The Sp1 site mutant (MT) and wild type (WT) eNOS promoter/reporter constructs were transfected into HeLa cells, and luciferase activity was measured and normalized as the percentage of the wild type. C, Sp1 binding ability between Sp1 site mutant and wild type with DAPA assay. Biotinylated oligonucleotides were incubated with nuclear extracts of HeLa cells and pulled down by streptavidin-agarose and subjected to Western blot. WT, wild type Sp1 probe; MT, mutant Sp1 probe (the mutation is the same as in the mutant Sp1 site eNOS promoter/reporter construct in panel B). D, responsiveness of the proximal Sp1 site to TSA treatment. Wild type and the Sp1 mutated eNOS promoter/reporter constructs were transfected into HeLa cells. Luciferase activity was measured 24 h after the TSA treatment. WT, wild type eNOS promoter/reporter construct; MT, mutant Sp1 site eNOS promoter/reporter construct; CTR, control.

Fig. 8. Sp1 binding to endogenous eNOS promoter and associating with HDAC1 in HeLa cells

A, occupancy of endogenous eNOS promoter by Sp1 after treatment with TSA and AzadC. ChIP assay was performed with anti-Sp1 antibody for non-treated cells or the cells treated with TSA or AzadC or both. The A pair of primers (as shown in Fig. 3), which covers the proximal Sp1 site, was used to amplify the DNA isolated from the ChIP assay. The data are representative of three separated experiments. B, association of HDAC1 with Sp1 in HeLa cells. Whole cell lysates were immunoprecipitated with anti-Sp1 antibody (left panel) or anti-HDAC1 antibody (right panel) and anti-FLAG antibody as negative control in both panels. The immunocomplexes were analyzed by Western blot with antibodies indicated. Input (40 μg of whole cell lysate) was served for position of HDAC1 or Sp1. The membrane was then stripped for detection with the antibodies used in the immunoprecipitation to show the pull-down proteins (lower panel). C, association of HDAC1 with Sp1 after treatment with TSA or TSA and AzadC. Whole cell lysates from HeLa cells treated with TSA (1 μg/ml, 24 h) or AzadC (7 μm) for 7 days combined with TSA (1 μg/ml) for another 24 h were immunoprecipitated by anti-Sp1 antibody. The immunocomplexes were analyzed by Western blot with anti-HDAC1 antibodies. The membrane was stripped for detection with the Sp1 antibody to show the equal pull-down Sp1 protein in the immunocomplexes (lower panel). IP, immunoprecipitation; WB, Western blot.