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Vol. 19, Issue 9, 3997-4005, September 2008
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*Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Shandong University, Qilu Hospital, Jinan, Shandong 250012, China; and Adult Section of Cardiothoracic Surgery, Texas Heart Institute at St. Luke's Episcopal Hospital, Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030
Submitted November 28, 2007;
Revised May 30, 2008;
Accepted June 26, 2008
Monitoring Editor: William P. Tansey
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Dimmeler and Zeiher, 1999). Adequate NO production is essential for antiatherogenic and antithrombotic processes in the normal arterial wall, and the reduced availability of NO promotes vascular diseases. Decreased basal NO release may predispose to hypertension, thrombosis, vasospasm, and atherosclerosis. In contrast, overproduction of NO can result in excessive oxidative stress and inflammation, both of which promote vascular diseases. Adequate eNOS expression and eNOS enzyme activity are central to maintaining appropriate physiological functions. Tremendous research efforts have recently focused on the regulation of eNOS enzyme activities, either by direct eNOS protein modification (e.g., phosphorylation) or availability of cofactors. However, progress in regulating eNOS expression at the transcriptional, posttranscriptional, and translational levels is relatively slow.
Despite extensive study of the association between eNOS polymorphisms and vascular diseases, the functional roles of variants of eNOS DNA are not clear. A meta-analysis of 12,990 subjects has shown a significant association between vascular diseases and the 27nt repeat polymorphism in eNOS intron 4 (Casas et al., 2004). Studies of ours and others suggest that the 27nt repeat polymorphism of the eNOS gene may regulate eNOS expression. Specifically, we have shown that the 27nt repeats may act as an enhancer/repressor in regulating eNOS expression; the regulatory effect is maintained during in vitro cell replication in ECs of different genotypes (Wang et al., 2002). Second, we have shown that the 27nt repeats in eNOS intron 4 produce 27nt small RNA, which seems to inhibit eNOS expression (Ou et al., 2005; Zhang et al., 2005). In the current study, we investigated the molecular mechanisms responsible for the 27nt small RNA-mediated eNOS suppression. Because 27nt small RNA is found exclusively in the nucleus and has a major effect on transcription, we focused our investigation on changes in histone acetylation and DNA methylation in the eNOS gene. We found that 27nt small RNA differentially altered histone acetylation in eNOS intron 4 and the promoter region. In addition, 27nt induced hypermethylation in the upstream region of the 27nt repeat element in eNOS intron 4.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture, Transfection, and Treatment
Human aortic endothelial cells (HAECs) were cultured in EGM-2 medium (Lonza Walkersville, Walkersville, MD) containing 3% fetal bovine serum (FBS). HeLa cells from American Type Culture Collection (Manassas, VA) were used in transfection assays as a control because HeLa cells do not constitutively express eNOS, but do have the necessary eNOS transcriptional machinery to drive the transfected eNOS constructs. The small RNA duplex or siRNA was transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). 5-Azacytidine (5-aza) (Sigma-Aldrich, St. Louis, MO) was used at a final concentration of 5 nM. Cells were treated every 48 h with 5-aza (a methyltransferase inhibitor) for 7 d. Trichostatin A (TSA) (Sigma-Aldrich), an HDAC inhibitor, was dissolved in 100% ethanol and used at a final concentration of 1 mM. Cells were treated for 24 h with either TSA or an equal amount of ethanol, which was used to dissolve TSA as a vehicle control.
mRNA Stability Assay
To assess the effect of the 27nt small RNA on eNOS mRNA stability, we cultured HAECs in six-well plates up to 90% confluence. We then transfected these cells with 27nt small RNA duplexes by using liposome. The transcription was blocked by adding actinomycin D (2 µg/ml). Treated HAECs were then harvested at 6, 12, 24, 48, and 72 h after the actinomycin D treatment. The relative levels of eNOS mRNA were determined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) adjusted by housekeeping gene β-actin.
Northern Blot Analysis
Total RNAs were extracted with TRIzol (Invitrogen). For eNOS mRNA detection, RNase protection assays (RPA) were performed by using the RPA III kit from Ambion (according to the manufacturer's instruction). Five to 20 µg of total RNA was used per reaction. Cyclophilin antisense control template from Ambion (Austin, TX) was labeled using T7 RNA polymerase as the internal control. 28S rRNA stained with ethidium bromide was used to control the amount of RNA loading.
Antibodies for Western Blot, Coimmunoprecipitation, and ChIP Assays
The ChIP assay kit was purchased from Millipore (Billerica, MA). Anti-acetyl histone H3 antibodies, anti-acetyl histone H4 antibodies, and anti-eNOS antibodies were purchased from Cell Signaling Technology (Danvers, MA). We purchased anti-HDAC3 antibody from Sigma-Aldrich, and the anti-NonO antibody from Abcam (Cambridge, MA).
Cellular Extracts and Western Blot Analysis
For total protein lysate preparation, the cell pellet was homogenized in lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na desoxycholate, 0.1% SDS, 10% glycerol, 0.2 mM EDTA, 200 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaFluorure, and 1 mM Na othovanadate). The cell pellet was suspended in ice-cold lysis buffer before cells were disrupted by sonication. After centrifugation at 12,000 x g for 10 min at 4°C, an aliquot of the supernatant was used for protein determination using the Coomassie Blue Plus protein assay reagent (Pierce Chemical, Rockford, IL), as per manufacturer's instruction. The remaining protein solution was used for Western blot analysis. Electrophoresis was carried out in 10% SDS-polyacrylamide gel, which was transferred to a nitrocellulose membrane (Hybond-C Super; GE Healthcare, Chalfont St. Giles, United Kingdom). The membranes were then blocked in 5% milk and probed with the appropriate specific antibodies. Primary antibodies were incubated at 4°C overnight in 5% milk. Horseradish peroxidase-linked goat anti-rabbit or mouse immunoglobulin G (IgG) (H&L) antibodies (Cell Signaling Technology) were used as secondary antibodies, and detection was performed with the Pierce ECL Western Blotting Substrate solution (Pierce Chemical) according to manufacturer's instruction. Anti-β-actin antibody (Sigma-Aldrich) was used for loading normalization.
Immunofluorescence Confocal Microscope Analysis
HAECs were seeded on plates with coverslips in medium containing 10% FBS for 24 h. Cells were fixed in 4% paraformaldehyde and permeabilized in phosphate-buffered saline (PBS) containing 0.5% Triton X-100. Indirect immunofluorescence was performed using a monoclonal antibody (mAb) against HDAC3 (Sigma-Aldrich), nuclear actin (2G2 antinuclear actin, catalog no. 651132; Progen Biotechnik, Heidelberg, Germany) and NonO (Abcam). Goat anti-mouse cyanine (Cy)2 (green) and goat anti-rabbit Cy5 (red) secondary antibodies (Vector Laboratories, Burlingame, CA) were used to detect the locations of HDAC3, nuclear actin, and NonO in HAECs. Laser-scanning confocal microscopy was performed on an LSM410 (Carl Zeiss, Jena, Germany) with krypton-argon and helium-neon lasers; three-dimensional projections were generated with the accompanying software.
Coimmunoprecipitation
To study the interaction between HDAC3, nuclear actin, and NonO, HAECs were harvested in the lysis buffer for immunoprecipitation with a mouse anti-nuclear actin antibody (anti-actin 2G2) or mouse anti-HDAC3 mAb. The immune complexes were further analyzed by immunoblotting with the HDAC3 or NonO antibody.
Isolation of Nuclear Extracts and Streptavidin-Bead Precipitation Assay
We extracted nuclear protein from 1 x 106 cultured HAECs, according to the instructions of the NE-PER nuclear and cytoplasmic extraction reagents kit (Pierce Chemical). Protease inhibitor cocktail (1:100 dilution) (Sigma-Aldrich) was added to the extracted nuclear proteins, with the protein concentration being determined using a bicinchoninic acid protein assay kit with bovine serum albumin as a standard (Sigma-Aldrich). The 27nt RNA was 5' labeled with biotin was synthesized by Integrated DNA Technologies Inc. (Houston, TX) mixed with 30 µl packed Streptavidin-beads for 3 h at 4°C, and incubated with 500 µg of nuclear protein extracts overnight at 4°C. After washing four times with ice-cold PBS containing 0.5% Triton X-100, 5 mM EDTA, proteins were eluted from the beads by the addition of 2x SDS loading buffer and boiled for 5 min. The eluted nuclear proteins were subjected to Western blot analysis using the anti-HDAC3 mAb.
ChIP Assay
We used the ChIP assay kit (Millipore) per the manufacturer's protocol. Briefly, 
3 x 106 cells were used per ChIP assay. The cells were fixed in 1% formaldehyde solution for 10 min at 37°C. Sonication was performed on ice using a Sonics and Materials Vibra-Cell sonicator (Misonix, Farmingdale, NY) with a 3-mm tip set at 30% maximum power; chromatin fragments, ranging in size from 200 to 400 bp, were generated by using 5 x 10-s pulses with a 10-s interval between sonications. Samples were diluted 10-fold in ChIP dilution buffer, and a 20-µl aliquot (1% of total) was removed to serve as an input sample. Chromatin was precleared with 80 µl of a mixture of salmon sperm DNA/protein A/protein G at 4°C with rotation for 2 h, followed by the addition of 2 µg of anti-acetyl-histone H3, HDAC3, and NonO antibodies. Immunoprecipitation was performed at 4°C overnight with rotation. To collect immune complexes, 60 µl of a mixture of salmon sperm DNA/protein A/protein G was added and incubated at 4°C with rotation for 2 h. After washing immune complexes, formaldehyde cross-linking was reversed in immunoprecipitated samples and the input chromatin sample by the addition of 20 µl of 5 M or 2 µl of NaCl and incubation at 65°C for 4 h. After proteinase K treatment, phenol/chloroform extraction, and ethanol precipitation, the DNA was resuspended in 25 µl of water. We used yeast tRNA to aid the precipitation process. ChIP analyses were performed at least three times on three independent lots of HAECs. The primers for the ChIP assay are listed in Table 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Previous studies suggested that small RNA can regulate gene transcription by modifying acetylation or methylation of histones. To investigate whether 27nt small RNA affects histone acetylation, we used a quantitative ChIP technique to measure the acetylation status of histone H3 and H4 located in the 27nt repeat element (6340-6516 bp) in eNOS intron 4 and the eNOS promoter region (up to –4917 bp). Chromatin was isolated from HAECs treated with exogenous 27nt microRNA (miRNA) duplex for 24 h. ChIP was performed on soluble chromatin fragments that averaged 200–400 base pairs. We used antibodies against H3 lysine 9 (H3K9), H3 lysine 18 (H3K18), H3 lysine 23 (H3K23), H4 lysine 8 (H4K8), and H4 lysine 12 (H4K12) in the ChIP assay. As shown in Figure 1, levels of acetylated H3K9 and H3K18 in the 27nt repeat element were higher in eNOS-expressing HAECs than in non—eNOS-expressing HeLa cells. When these cells were treated with 27nt RNA, the acetylation status at all five residues increased in the 27nt repeat element. In contrast, H3K9 and H3K23 seemed to be more acetylated in the eNOS core promoter region (–238 to –13 bp; Figure 1) in HAECs than in HeLa cells. On treatment with 27nt RNA, the acetylation status was reduced in all five measured lysine residues in the core promoter. The acetylation changes in other regions of the promoter (–1421/–1304 bp, –891/–79 6bp, –483/–312 bp) and enhancer (–4917/–4626 bp) were not remarkable (data not shown).
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). To study the effect of nuclear actin on the interaction between 27nt RNA and eNOS transcription, we measured histone acetylation status in 27nt RNA-treated HAECs with or without actin overexpression. HAECs were transfected with actin/EGFP plasmids for 24 h and then treated with the 27nt RNA duplex. A systematic ChIP analysis was carried out to examine the intron 4 and promoter region of the eNOS gene. As shown in Figure 2, actin overexpression in ECs did not affect the increased histone acetylation by 27nt RNA at the 27nt repeat element; however, a decreased trend for H4K8 and H4K12 was noted. In contrast, overexpression of β actin increased acetylation status at H3K9, H4K8 and H4K12 in the eNOS core promoter region (–238/–13 bp) in all cells with or without 27nt RNA treatment.
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). The recruitment of the NonO protein to the HDAC3 and nuclear actin complex in the 27nt repeat region could be responsible for the suppressive effects of the 27nt small RNA on eNOS transcription. Using confocal microscopy, we further illustrated the colocalization of HDAC3, actin, and NonO within the nucleus (Figure 3B, right). Most of the actin was present in the cytoplasm, but a small amount was visible within the nucleus (Figure 3B, top and bottom right). Most of the HDAC3 was found within the nucleus, but a trace amount was seen in the cytoplasm. NonO was found predominantly within the nucleus. To determine whether the HDAC3 and NonO interaction occurred in the 27nt repeat element in eNOS intron 4, we performed a ChIP assay by using anti-HDAC3 or anti-NonO antibodies to pull down the nucleosomes extracted from HAECs treated with either 27nt RNA duplex or luciferase gene-specific siRNA as a control. We amplified the genomic DNA using the primers covering the 27nt repeat element. As shown in Figure 4A, HDAC3 and NonO did bind on the 27nt repeat element in eNOS intron 4. Treatment of HAECs with 27nt RNA significantly increased the binding between the HDAC3 or NonO and the 27nt repeat element (Figure 4A, p < 0.01). The role of HDAC in 27nt RNA induced eNOS suppression was further supported in cells treated with 1 mM TSA (Figure 4B).
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Effects of 27nt RNA on eNOS DNA Methylation
Because DNA methylation may be a mechanism for small RNA-induced transcription suppression (Klenov and Gvozdev, 2005; Klenov et al., 2007), we looked for changes in methylation status of the CpG islands located in the eNOS DNA sequence upstream from the 27nt repeat element. 27nt RNA treatment did not affect CpG methylation status in the eNOS promoter region (data not shown); however, HAECs treated with the 27nt RNA had significantly increased methylation of CpG immediately upstream from the 27nt repeat element in intron 4. Using sodium bisulfite genomic sequencing, we found that nine CpG dinucleotides located within the +4567/+4940 region were highly methylated by treatment with 27nt RNA (Figure 6A). The methylation rate at site 4567 increased from 10 to 80%, at site 4594 from 12 to 89%, at site 4608 from 12 to 92%, at site 4708 from 13 to 76%, at site 4801 from 8 to 83%, at site 4810 from 15 to 90%, at site 4922 from 7 to 81%, at site 4929 from 10 to 87%, and at site 4940 from 12 to 82%.
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Effects of 27nt RNA on eNOS mRNA Stability
Our previous study has shown that more 27nt repeats the eNOS gene have the more 27nt small RNA is produced and the lower the eNOS expression levels (Zhang et al., 2008). Our current study further demonstrated that human endothelial cells with 5x27nt repeats produced less eNOS mRNA levels (Figure 7A). Although our experiments showed that the 27nt RNA could affect eNOS transcript at DNA level, we further examined whether 27nt RNA could also affect the eNOS mRNA stability. Using actinomycin D to block the eNOS transcription, our experiment showed that eNOS mRNA in endothelial cells treated with 27nt RNA degraded significantly faster than the endothelial cells treated with the nonspecific small RNA (Figure 7B). These findings suggest that the 27nt small RNA may also reduce the eNOS mRNA stability.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Through protein–protein interaction, nuclear actin, which binds on the 27nt repeat element, recruits HDAC3 to the site. Then, HDAC3 induces histone deacetylation at the core promoter region of the eNOS, which represses eNOS transcriptional activity.
Currently, several types of small RNAs have been described in plants and animals: miRNA, piwiRNA (Aravin et al., 2006), siRNA, tiny noncoding RNA, heterochromatic siRNAs, and repeat associated small interfering RNAs (rasiRNAs) (Reinhart and Bartel, 2002; Ambros et al., 2003; Aravin et al., 2003). These small RNAs are usually 21–25 nt; rasiRNAs may be longer (Aravin et al., 2003). Although siRNAs usually arise from an exogenous source or during virus infection, miRNAs are mainly transcribed through designated genomic sequences (Cullen, 2004). Small RNAs usually inhibit gene expression by promoting the degradation of sequence-matched target mRNA in the cytoplasm via the RNA-induced silencing complex. The finding of endogenous miRNAs further indicates that miRNA can induce translational repression through imperfect match-binding at 3' untranslated region (Cullen, 2004). In contrast, rasiRNAs have been reported to be mostly derived from centromere heterochromatic repeats and tend to be a few nucleotides longer than a typical mature miRNA or siRNA (Pelissier and Wassenegger, 2000; Cao et al., 2003; Matzke et al., 2004; Rana, 2007). One of the critical differences between rasiRNAs and miRNAs is that rasiRNAs regulate gene expression by modifying histones and DNA of the target regions (Aravin et al., 2001; Gvozdev et al., 2003; Lippman et al., 2004) (e.g., histone methylation and acetylation) (Volpe et al., 2002; Hall et al., 2003); this effect seems to be reversible with the deacetylase inhibitor TSA (Hall et al., 2002). The rasiRNAs in conjunction with one or more DNA methyltransferases and possibly chromatin-modifying factors are thought to trigger the de novo methylation and transcriptional silencing of the homologous promoter at the target locus (Aufsatz et al., 2002).
In comparison with the known classes of small RNAs, the 27nt small RNA could be either a new class of small RNA or an atypical form of the miRNA. Because the 27nt small RNA is transcribed from the host gene eNOS, we hypothesize that the intron 4-derived 27nt small RNA may function as a negative feedback regulator and maintain the stable transcription of eNOS. The more the host gene is transcribed, the more gene-specific repeat element derived-RNA will be produced, which forms a negative feedback regulatory loop to fine-tune gene expression in the host. Variable numbers of the repeats in the population may be responsible for quantitative interindividual differences in the expressed genes and disease susceptibility. More specifically, the 27nt repeats in the eNOS intron 4 can be converted to short intronic repeat RNA (sir-RNA) and can function as a negative feedback regulator of eNOS expression. Individuals with different numbers of 27nt repeats will have different levels of 27nt sir-RNA, which is responsible for between-individual differences in eNOS expression, and hence susceptibility to vascular diseases.
The current study is limited by the relatively preliminary nature of the evidence. Although our novel findings suggest that intron-derived 27nt small RNA suppresses the host gene via alteration of histone acetylation and DNA methylation at the transcriptional level, we do not have mechanistic data regarding the nuclear proteins involved in this process. Further experiments are necessary to completely understand how 27nt RNA induces hyperacetylation in histones located at the site of the 27nt repeat element and how the subsequent recruitment of HDAC3 causes histone deacetylation in the upstream promoter region rather than deacetylation of the histones in the 27nt repeat region. Furthermore, whether NonO directly inhibits eNOS transcription or merely facilitates changes in histone acetylation is worth investigating. The findings that 27nt RNA also decreases the eNOS mRNA stability suggest that the intron derived small RNA could also regulate the host gene posttranscriptionally. However, it is not clear whether this posttranscriptional regulation targets at the eNOS pre-mRNA or mature mRNA. Despite these unknowns, the finding that 27nt RNA site-specifically alters the eNOS histone acetylation and methylation status and eNOS mRNA stability is novel. Our findings may lead to the establishment of a new negative feedback regulatory mechanism at the transcriptional level—one that can more efficiently and quickly maintain the host gene within physiological range. This finding may have genome-wide implications for genes sharing the similar repeat structure.
In summary, we have established that the 27nt repeat element-derived small RNA can suppress transcription of the host gene by modifying histone acetylation and DNA methylation of the eNOS gene. Hyperacetylation at the repressor region (i.e., the 27nt repeat element) results in recruitment of a negative transcription factor, such as NonO. Hypoacetylation at the eNOS core promoter induced by the 27nt small RNA may inhibit eNOS suppression.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Xing Li Wang (xlwang{at}bcm.edu) or Ming-Xiang Zhang (zhangmingxiang123{at}hotmail.com)
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