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SIRT1 deacetylates SATB1 to facilitate MAR HS2-MAR ε interaction and promote ε-globin expression.

Xue Z, Lv X, Song W, Wang X, Zhao GN, Wang WT, Xiong J, Mao BB, Yu W, Yang B, Wu J, Zhou LQ, Hao DL, Dong WJ, Liu DP, Liang CC - Nucleic Acids Res. (2012)

Bottom Line: SIRT1 expression increased accompanying erythroid differentiation and the strengthening of β-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the β-globin locus, MAR(HS2) and MAR(ε).We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation.Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, PR China.

ABSTRACT
The higher order chromatin structure has recently been revealed as a critical new layer of gene transcriptional control. Changes in higher order chromatin structures were shown to correlate with the availability of transcriptional factors and/or MAR (matrix attachment region) binding proteins, which tether genomic DNA to the nuclear matrix. How posttranslational modification to these protein organizers may affect higher order chromatin structure still pending experimental investigation. The type III histone deacetylase silent mating type information regulator 2, S. cerevisiae, homolog 1 (SIRT1) participates in many physiological processes through targeting both histone and transcriptional factors. We show that MAR binding protein SATB1, which mediates chromatin looping in cytokine, MHC-I and β-globin gene loci, as a new type of SIRT1 substrate. SIRT1 expression increased accompanying erythroid differentiation and the strengthening of β-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the β-globin locus, MAR(HS2) and MAR(ε). We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation. Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.

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SIRT1 promotes ε-globin gene expression in K562 and primary erythroid cells. (A and B) K562 cells were treated with resveratrol or NAM, and ε-globin mRNA levels were detected by real-time PCR. The accompany changes of SATB1 acetylation level were detected by western blotting. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-values are indicated (**P < 0.01, ***P < 0.001). (C) K562 cells were transfected with pcDNA3.1 or pcDNA3.1-SIRT1. ε-globin and SIRT1 mRNA levels were detected by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (**P < 0.01). The SIRT1 protein levels and accompanying SATB1 acetylation levels were detected by western blot. (D) K562 cells were stably infected with a retrovirus carrying control shRNA or SIRT1 shRNA. ε-globin and SIRT1 mRNA levels in the stably infected cells were determined by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (**P < 0.01). The SIRT1 protein levels and accompanying SATB1 acetylation levels were determined by western blotting. (E and F) HSCs were selected from cord blood, cultured in medium for 1 week, and treated with Epo for 3 days to induce erythroid differentiation. The primary erythroid cells were then treated with resveratrol or NAM, and ε-globin mRNA levels were determined by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (*P < 0.05, **P < 0.01). (G) The selected HSCs were infected with a retrovirus carrying control shRNA or SIRT1 shRNA. Polyclones were selected by puromycin before being subjected to Epo induction for 3 days. ε-globin and SIRT1 mRNA levels were detected by real-time PCR. The accompany change of SATB1 acetylation level was detected by western blotting. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (*P < 0.05).
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gks064-F3: SIRT1 promotes ε-globin gene expression in K562 and primary erythroid cells. (A and B) K562 cells were treated with resveratrol or NAM, and ε-globin mRNA levels were detected by real-time PCR. The accompany changes of SATB1 acetylation level were detected by western blotting. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-values are indicated (**P < 0.01, ***P < 0.001). (C) K562 cells were transfected with pcDNA3.1 or pcDNA3.1-SIRT1. ε-globin and SIRT1 mRNA levels were detected by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (**P < 0.01). The SIRT1 protein levels and accompanying SATB1 acetylation levels were detected by western blot. (D) K562 cells were stably infected with a retrovirus carrying control shRNA or SIRT1 shRNA. ε-globin and SIRT1 mRNA levels in the stably infected cells were determined by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (**P < 0.01). The SIRT1 protein levels and accompanying SATB1 acetylation levels were determined by western blotting. (E and F) HSCs were selected from cord blood, cultured in medium for 1 week, and treated with Epo for 3 days to induce erythroid differentiation. The primary erythroid cells were then treated with resveratrol or NAM, and ε-globin mRNA levels were determined by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (*P < 0.05, **P < 0.01). (G) The selected HSCs were infected with a retrovirus carrying control shRNA or SIRT1 shRNA. Polyclones were selected by puromycin before being subjected to Epo induction for 3 days. ε-globin and SIRT1 mRNA levels were detected by real-time PCR. The accompany change of SATB1 acetylation level was detected by western blotting. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (*P < 0.05).

Mentions: Given that SATB1 can activate the expression of the ε-globin gene in K562 cells (12–14), we hypothesized that SIRT1, which interacts with and deacetylases SATB1, could also influence ε-globin gene expression. K562 cells were treated with the SIRT1 activator resveratrol for 24 h or with the SIRT1 inhibitor NAM for 10 h. The expression of the ε-globin gene was monitored using real-time PCR. We found that resveratrol activated ε-globin gene expression in a concentration-dependent manner (Figure 3A), whereas NAM decreased the ε-globin gene expression (Figure 3B), accompanied with decreased SATB1 acetylation level upon resveratrol challenge and increased SATB1 acetylation level after NAM treatment (Figure 3A and B). We then determined whether overexpression or knockdown of SIRT1 could affect the mRNA abundance of the ε-globin gene. In agreement with the results obtained with resveratrol treatment, SIRT1 overexpression in K562 cells promoted ε-globin gene expression dramatically (Figure 3C). In contrast, the mRNA level of the ε-globin gene decreased significantly in K562 cells with stable knockdown of SIRT1 (Figure 3D), concomitant SATB1 acetylation level was shown decreased upon SIRT1 overexpression and increased in SIRT1-knockdown K562 cells (Figure 3C and D).Figure 3.


SIRT1 deacetylates SATB1 to facilitate MAR HS2-MAR ε interaction and promote ε-globin expression.

Xue Z, Lv X, Song W, Wang X, Zhao GN, Wang WT, Xiong J, Mao BB, Yu W, Yang B, Wu J, Zhou LQ, Hao DL, Dong WJ, Liu DP, Liang CC - Nucleic Acids Res. (2012)

SIRT1 promotes ε-globin gene expression in K562 and primary erythroid cells. (A and B) K562 cells were treated with resveratrol or NAM, and ε-globin mRNA levels were detected by real-time PCR. The accompany changes of SATB1 acetylation level were detected by western blotting. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-values are indicated (**P < 0.01, ***P < 0.001). (C) K562 cells were transfected with pcDNA3.1 or pcDNA3.1-SIRT1. ε-globin and SIRT1 mRNA levels were detected by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (**P < 0.01). The SIRT1 protein levels and accompanying SATB1 acetylation levels were detected by western blot. (D) K562 cells were stably infected with a retrovirus carrying control shRNA or SIRT1 shRNA. ε-globin and SIRT1 mRNA levels in the stably infected cells were determined by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (**P < 0.01). The SIRT1 protein levels and accompanying SATB1 acetylation levels were determined by western blotting. (E and F) HSCs were selected from cord blood, cultured in medium for 1 week, and treated with Epo for 3 days to induce erythroid differentiation. The primary erythroid cells were then treated with resveratrol or NAM, and ε-globin mRNA levels were determined by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (*P < 0.05, **P < 0.01). (G) The selected HSCs were infected with a retrovirus carrying control shRNA or SIRT1 shRNA. Polyclones were selected by puromycin before being subjected to Epo induction for 3 days. ε-globin and SIRT1 mRNA levels were detected by real-time PCR. The accompany change of SATB1 acetylation level was detected by western blotting. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (*P < 0.05).
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gks064-F3: SIRT1 promotes ε-globin gene expression in K562 and primary erythroid cells. (A and B) K562 cells were treated with resveratrol or NAM, and ε-globin mRNA levels were detected by real-time PCR. The accompany changes of SATB1 acetylation level were detected by western blotting. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-values are indicated (**P < 0.01, ***P < 0.001). (C) K562 cells were transfected with pcDNA3.1 or pcDNA3.1-SIRT1. ε-globin and SIRT1 mRNA levels were detected by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (**P < 0.01). The SIRT1 protein levels and accompanying SATB1 acetylation levels were detected by western blot. (D) K562 cells were stably infected with a retrovirus carrying control shRNA or SIRT1 shRNA. ε-globin and SIRT1 mRNA levels in the stably infected cells were determined by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (**P < 0.01). The SIRT1 protein levels and accompanying SATB1 acetylation levels were determined by western blotting. (E and F) HSCs were selected from cord blood, cultured in medium for 1 week, and treated with Epo for 3 days to induce erythroid differentiation. The primary erythroid cells were then treated with resveratrol or NAM, and ε-globin mRNA levels were determined by real-time PCR. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (*P < 0.05, **P < 0.01). (G) The selected HSCs were infected with a retrovirus carrying control shRNA or SIRT1 shRNA. Polyclones were selected by puromycin before being subjected to Epo induction for 3 days. ε-globin and SIRT1 mRNA levels were detected by real-time PCR. The accompany change of SATB1 acetylation level was detected by western blotting. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student’s t-test was used for statistical analysis and P-value is indicated (*P < 0.05).
Mentions: Given that SATB1 can activate the expression of the ε-globin gene in K562 cells (12–14), we hypothesized that SIRT1, which interacts with and deacetylases SATB1, could also influence ε-globin gene expression. K562 cells were treated with the SIRT1 activator resveratrol for 24 h or with the SIRT1 inhibitor NAM for 10 h. The expression of the ε-globin gene was monitored using real-time PCR. We found that resveratrol activated ε-globin gene expression in a concentration-dependent manner (Figure 3A), whereas NAM decreased the ε-globin gene expression (Figure 3B), accompanied with decreased SATB1 acetylation level upon resveratrol challenge and increased SATB1 acetylation level after NAM treatment (Figure 3A and B). We then determined whether overexpression or knockdown of SIRT1 could affect the mRNA abundance of the ε-globin gene. In agreement with the results obtained with resveratrol treatment, SIRT1 overexpression in K562 cells promoted ε-globin gene expression dramatically (Figure 3C). In contrast, the mRNA level of the ε-globin gene decreased significantly in K562 cells with stable knockdown of SIRT1 (Figure 3D), concomitant SATB1 acetylation level was shown decreased upon SIRT1 overexpression and increased in SIRT1-knockdown K562 cells (Figure 3C and D).Figure 3.

Bottom Line: SIRT1 expression increased accompanying erythroid differentiation and the strengthening of β-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the β-globin locus, MAR(HS2) and MAR(ε).We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation.Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, PR China.

ABSTRACT
The higher order chromatin structure has recently been revealed as a critical new layer of gene transcriptional control. Changes in higher order chromatin structures were shown to correlate with the availability of transcriptional factors and/or MAR (matrix attachment region) binding proteins, which tether genomic DNA to the nuclear matrix. How posttranslational modification to these protein organizers may affect higher order chromatin structure still pending experimental investigation. The type III histone deacetylase silent mating type information regulator 2, S. cerevisiae, homolog 1 (SIRT1) participates in many physiological processes through targeting both histone and transcriptional factors. We show that MAR binding protein SATB1, which mediates chromatin looping in cytokine, MHC-I and β-globin gene loci, as a new type of SIRT1 substrate. SIRT1 expression increased accompanying erythroid differentiation and the strengthening of β-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the β-globin locus, MAR(HS2) and MAR(ε). We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation. Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.

Show MeSH
Related in: MedlinePlus