Limits...
High-dose siRNAs upregulate mouse Eri-1 at both transcription and posttranscription levels.

Bian Y, Zhou W, Zhao Y, Li X, Geng W, Hao R, Yang Q, Huang W - PLoS ONE (2011)

Bottom Line: High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency.In addition, hd-siRNAs also abolish the 3' untranslated region (UTR) mediated posttranscriptional repression of meri-1.Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Life Science, Fudan University, Shanghai, China.

ABSTRACT
The eri-1 gene encodes a 3' exonuclease that can negatively regulate RNA interference via siRNase activity. High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency. Here we report that hd-siRNAs induce mouse Eri-1 (meri-1) expression through the recruitment of Sp1, Ets-1, and STAT3 to the meri-1 promoter and the formation of an Sp1-Ets-1-STAT3 complex. In addition, hd-siRNAs also abolish the 3' untranslated region (UTR) mediated posttranscriptional repression of meri-1. Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.

Show MeSH
Necessity of the Sp1 acetylation in the siRNA-stimulated meri-1 Promoter Activity.(A) The histone deacetylase (HDAC) inhibitor TSA enhances the meri-1 promoter activity in a dosage-dependent manner. TSA was added to the culture media of the CHO cells transfected with pPeri-1(−87)-SEAP and Nc siRNA_2. (B) Mutation of the acetylation site interferes with the function of Sp1 as the activator of meri-1 promoter. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA_2 plus pCMV-HA, pCMV-HA-Sp1 (K703A) or pCMV-HA-Sp1 was measured. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples. (C) The acetylation of Sp1 is enhanced by hd-siRNA. Cell extracts were prepared from the CHO cells transfected with pCMV-FLAG-Sp1 or co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2. Immunoprecipitation was performed using M2-anti-FLAG antibody-agarose beads. The immunoprecipitated pellets were analyzed by immunoblotting with anti-acetyl-lysine (upper panel) and anti-FLAG (lower panel) antibodies. The relative intensity of bands was quantified using the ImageGouge software. (D) Acetylation is necessary for the recruitment of Sp1 to the meri-1 promoter. Cell extracts of the CHO cells transfected with pCMV-FLAG-Sp1 or co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2, or with pCMV-FLAG-Sp1 (K703A) and Nc siRNA_2 were prepared. The aliquots were subjected to DAPA with biotinylated Peri-1(−88–−30) oligonucleotides that contain the hd-siRNA elements in the meri-1 promoter (see Table 1). The precipitated complexes (Precipitation) were immunoblotted with anti-FLAG and acetyl-lysine antibodies, and the whole cell lysates (WCL) were monitored for the expression of FLAG-Sp1 or FLAG-Sp1 (K703A). The relative intensity of bands was quantified using the ImageGouge software.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198429&req=5

pone-0026466-g004: Necessity of the Sp1 acetylation in the siRNA-stimulated meri-1 Promoter Activity.(A) The histone deacetylase (HDAC) inhibitor TSA enhances the meri-1 promoter activity in a dosage-dependent manner. TSA was added to the culture media of the CHO cells transfected with pPeri-1(−87)-SEAP and Nc siRNA_2. (B) Mutation of the acetylation site interferes with the function of Sp1 as the activator of meri-1 promoter. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA_2 plus pCMV-HA, pCMV-HA-Sp1 (K703A) or pCMV-HA-Sp1 was measured. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples. (C) The acetylation of Sp1 is enhanced by hd-siRNA. Cell extracts were prepared from the CHO cells transfected with pCMV-FLAG-Sp1 or co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2. Immunoprecipitation was performed using M2-anti-FLAG antibody-agarose beads. The immunoprecipitated pellets were analyzed by immunoblotting with anti-acetyl-lysine (upper panel) and anti-FLAG (lower panel) antibodies. The relative intensity of bands was quantified using the ImageGouge software. (D) Acetylation is necessary for the recruitment of Sp1 to the meri-1 promoter. Cell extracts of the CHO cells transfected with pCMV-FLAG-Sp1 or co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2, or with pCMV-FLAG-Sp1 (K703A) and Nc siRNA_2 were prepared. The aliquots were subjected to DAPA with biotinylated Peri-1(−88–−30) oligonucleotides that contain the hd-siRNA elements in the meri-1 promoter (see Table 1). The precipitated complexes (Precipitation) were immunoblotted with anti-FLAG and acetyl-lysine antibodies, and the whole cell lysates (WCL) were monitored for the expression of FLAG-Sp1 or FLAG-Sp1 (K703A). The relative intensity of bands was quantified using the ImageGouge software.

Mentions: Several studies have shown that Sp1 is acetylated to regulate its DNA binding affinity or transactivation [12], [51], [52], [53]. In neurons, Sp1 can be acetylated in response to oxidative stress and TSA-induced Sp1 acetylation correlated with an increase in Sp1 DNA binding [12]. To further elucidate the mechanism details of the transcriptional regulation of meri-1, the role of Sp1 acetylation in regulating the meri-1 promoter activity was examined. CHO cells were treated with the prototypic histone deacetylase (HDAC) inhibitor TSA [54], [55], which is an organic hydroxamic acid that can potently inhibit the zinc hydrolase activity of HDACs by chelating zinc [56]. TSA concentration-dependently increased SEAP activity in the CHO cells transfected with hd-siRNA (Figure 4A). Since TSA might also inhibit the deacetylation of proteins other than Sp1, an Sp1 mutant expression vector, pCMV-HA-mSp1K703A, was constructed. It carries a mutation at lysine residue 703 of Sp1 that resides in the DNA binding domain and is the only known acetylated residue of Sp1 [52]. The overexpression of Sp1K703A suppressed the transcriptional activity of meri-1 promoter (Figure 4B). To further determine the role of Sp1 acetylation in the siRNA-stimulated meri-1 promoter activity, the acetylation of Sp1 was assessed in hd-siRNA transfected cells. CHO cells were transfected with hd-siRNA and FLAG-Sp1 expression construct. The cell extracts were immunoprecipitated with M2-anti-FLAG antibody-agarose beads, and the immunoprecipitated pellets were blotted with anti-acetyl-lysine and anti-FLAG antibodies. As shown in Figure 4C, the acetylation of Sp1 in hd-siRNA treated cells was significantly increased compared to the control transfected cells. Next, the influence of Sp1 K703 acetylation on the recruitment of Sp1 to the meri-1 promoter was investigated using cell extracts from the hd-siRNA-treated CHO cells. By DAPAs, as shown in Figure 4D, the mutation of the acetylated residue reduced the binding of Sp1 to the Peri-1(−88–−30) oligonucleotides. This data suggests that the acetylation of Sp1 K703 is critical for the recruitment of Sp1 to the promoter of meri-1 induced by hd-siRNA and that the acetylation of Sp1 is required for its ability to regulate the meri-1 promoter activity.


High-dose siRNAs upregulate mouse Eri-1 at both transcription and posttranscription levels.

Bian Y, Zhou W, Zhao Y, Li X, Geng W, Hao R, Yang Q, Huang W - PLoS ONE (2011)

Necessity of the Sp1 acetylation in the siRNA-stimulated meri-1 Promoter Activity.(A) The histone deacetylase (HDAC) inhibitor TSA enhances the meri-1 promoter activity in a dosage-dependent manner. TSA was added to the culture media of the CHO cells transfected with pPeri-1(−87)-SEAP and Nc siRNA_2. (B) Mutation of the acetylation site interferes with the function of Sp1 as the activator of meri-1 promoter. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA_2 plus pCMV-HA, pCMV-HA-Sp1 (K703A) or pCMV-HA-Sp1 was measured. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples. (C) The acetylation of Sp1 is enhanced by hd-siRNA. Cell extracts were prepared from the CHO cells transfected with pCMV-FLAG-Sp1 or co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2. Immunoprecipitation was performed using M2-anti-FLAG antibody-agarose beads. The immunoprecipitated pellets were analyzed by immunoblotting with anti-acetyl-lysine (upper panel) and anti-FLAG (lower panel) antibodies. The relative intensity of bands was quantified using the ImageGouge software. (D) Acetylation is necessary for the recruitment of Sp1 to the meri-1 promoter. Cell extracts of the CHO cells transfected with pCMV-FLAG-Sp1 or co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2, or with pCMV-FLAG-Sp1 (K703A) and Nc siRNA_2 were prepared. The aliquots were subjected to DAPA with biotinylated Peri-1(−88–−30) oligonucleotides that contain the hd-siRNA elements in the meri-1 promoter (see Table 1). The precipitated complexes (Precipitation) were immunoblotted with anti-FLAG and acetyl-lysine antibodies, and the whole cell lysates (WCL) were monitored for the expression of FLAG-Sp1 or FLAG-Sp1 (K703A). The relative intensity of bands was quantified using the ImageGouge software.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198429&req=5

pone-0026466-g004: Necessity of the Sp1 acetylation in the siRNA-stimulated meri-1 Promoter Activity.(A) The histone deacetylase (HDAC) inhibitor TSA enhances the meri-1 promoter activity in a dosage-dependent manner. TSA was added to the culture media of the CHO cells transfected with pPeri-1(−87)-SEAP and Nc siRNA_2. (B) Mutation of the acetylation site interferes with the function of Sp1 as the activator of meri-1 promoter. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA_2 plus pCMV-HA, pCMV-HA-Sp1 (K703A) or pCMV-HA-Sp1 was measured. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples. (C) The acetylation of Sp1 is enhanced by hd-siRNA. Cell extracts were prepared from the CHO cells transfected with pCMV-FLAG-Sp1 or co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2. Immunoprecipitation was performed using M2-anti-FLAG antibody-agarose beads. The immunoprecipitated pellets were analyzed by immunoblotting with anti-acetyl-lysine (upper panel) and anti-FLAG (lower panel) antibodies. The relative intensity of bands was quantified using the ImageGouge software. (D) Acetylation is necessary for the recruitment of Sp1 to the meri-1 promoter. Cell extracts of the CHO cells transfected with pCMV-FLAG-Sp1 or co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2, or with pCMV-FLAG-Sp1 (K703A) and Nc siRNA_2 were prepared. The aliquots were subjected to DAPA with biotinylated Peri-1(−88–−30) oligonucleotides that contain the hd-siRNA elements in the meri-1 promoter (see Table 1). The precipitated complexes (Precipitation) were immunoblotted with anti-FLAG and acetyl-lysine antibodies, and the whole cell lysates (WCL) were monitored for the expression of FLAG-Sp1 or FLAG-Sp1 (K703A). The relative intensity of bands was quantified using the ImageGouge software.
Mentions: Several studies have shown that Sp1 is acetylated to regulate its DNA binding affinity or transactivation [12], [51], [52], [53]. In neurons, Sp1 can be acetylated in response to oxidative stress and TSA-induced Sp1 acetylation correlated with an increase in Sp1 DNA binding [12]. To further elucidate the mechanism details of the transcriptional regulation of meri-1, the role of Sp1 acetylation in regulating the meri-1 promoter activity was examined. CHO cells were treated with the prototypic histone deacetylase (HDAC) inhibitor TSA [54], [55], which is an organic hydroxamic acid that can potently inhibit the zinc hydrolase activity of HDACs by chelating zinc [56]. TSA concentration-dependently increased SEAP activity in the CHO cells transfected with hd-siRNA (Figure 4A). Since TSA might also inhibit the deacetylation of proteins other than Sp1, an Sp1 mutant expression vector, pCMV-HA-mSp1K703A, was constructed. It carries a mutation at lysine residue 703 of Sp1 that resides in the DNA binding domain and is the only known acetylated residue of Sp1 [52]. The overexpression of Sp1K703A suppressed the transcriptional activity of meri-1 promoter (Figure 4B). To further determine the role of Sp1 acetylation in the siRNA-stimulated meri-1 promoter activity, the acetylation of Sp1 was assessed in hd-siRNA transfected cells. CHO cells were transfected with hd-siRNA and FLAG-Sp1 expression construct. The cell extracts were immunoprecipitated with M2-anti-FLAG antibody-agarose beads, and the immunoprecipitated pellets were blotted with anti-acetyl-lysine and anti-FLAG antibodies. As shown in Figure 4C, the acetylation of Sp1 in hd-siRNA treated cells was significantly increased compared to the control transfected cells. Next, the influence of Sp1 K703 acetylation on the recruitment of Sp1 to the meri-1 promoter was investigated using cell extracts from the hd-siRNA-treated CHO cells. By DAPAs, as shown in Figure 4D, the mutation of the acetylated residue reduced the binding of Sp1 to the Peri-1(−88–−30) oligonucleotides. This data suggests that the acetylation of Sp1 K703 is critical for the recruitment of Sp1 to the promoter of meri-1 induced by hd-siRNA and that the acetylation of Sp1 is required for its ability to regulate the meri-1 promoter activity.

Bottom Line: High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency.In addition, hd-siRNAs also abolish the 3' untranslated region (UTR) mediated posttranscriptional repression of meri-1.Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Life Science, Fudan University, Shanghai, China.

ABSTRACT
The eri-1 gene encodes a 3' exonuclease that can negatively regulate RNA interference via siRNase activity. High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency. Here we report that hd-siRNAs induce mouse Eri-1 (meri-1) expression through the recruitment of Sp1, Ets-1, and STAT3 to the meri-1 promoter and the formation of an Sp1-Ets-1-STAT3 complex. In addition, hd-siRNAs also abolish the 3' untranslated region (UTR) mediated posttranscriptional repression of meri-1. Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.

Show MeSH