Limits...
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.

Show MeSH
SIRT1 deacetylates SATB1. (A) The 293 cells were transfected with Myc-SATB1 and then treated with TSA and NAM as indicated. Immunoprecipitation was performed using a Myc antibody, and SATB1 acetylation levels were detected by western blotting with an acetylated-lysine antibody. (B) In vitro acetylation assay. Truncated GST-SATB1-1-204 was acetylated in the presence of PCAF and Ac-CoA. The self-acetylated PCAF was used as a positive control, and SATB1 acetylation was detected by western blotting with an acetylated-lysine antibody. The truncated GST-SATB1-1-204 protein was detected by Ponceau S staining. (C) In vitro deacetylation assay. In vitro-acetylated GST-SATB1 1-204 was deacetylated with SIRT1 and NAD+. The self-acetylated PCAF was used as a positive control, and SATB1 acetylation was detected by western blotting with an acetylated-lysine antibody. The truncated GST-SATB1-1-204 protein was detected by Ponceau S staining. (D) The 293 cells were cotransfected with SIRT1 and Myc-SATB1-expressing vectors. Immunoprecipitation was then performed using a Myc antibody, and Myc-SATB1 acetylation levels were detected by western blotting with an acetylated-lysine antibody. The cell lysates were subjected to blotting with the indicated antibodies to show the expression of exogenous SIRT1 and Myc-SATB1. (E) Amino acid sequence of SATB1 containing the acetylated K175. (F) MS/MS spectrum analysis of the SATB1 peptide containing acetylated K136 before and after the in vitro deacetylation assay. (G) MS/MS spectrum analysis of the SATB1 peptide containing acetylated K175 before and after the in vitro deacetylation assay.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3367170&req=5

gks064-F2: SIRT1 deacetylates SATB1. (A) The 293 cells were transfected with Myc-SATB1 and then treated with TSA and NAM as indicated. Immunoprecipitation was performed using a Myc antibody, and SATB1 acetylation levels were detected by western blotting with an acetylated-lysine antibody. (B) In vitro acetylation assay. Truncated GST-SATB1-1-204 was acetylated in the presence of PCAF and Ac-CoA. The self-acetylated PCAF was used as a positive control, and SATB1 acetylation was detected by western blotting with an acetylated-lysine antibody. The truncated GST-SATB1-1-204 protein was detected by Ponceau S staining. (C) In vitro deacetylation assay. In vitro-acetylated GST-SATB1 1-204 was deacetylated with SIRT1 and NAD+. The self-acetylated PCAF was used as a positive control, and SATB1 acetylation was detected by western blotting with an acetylated-lysine antibody. The truncated GST-SATB1-1-204 protein was detected by Ponceau S staining. (D) The 293 cells were cotransfected with SIRT1 and Myc-SATB1-expressing vectors. Immunoprecipitation was then performed using a Myc antibody, and Myc-SATB1 acetylation levels were detected by western blotting with an acetylated-lysine antibody. The cell lysates were subjected to blotting with the indicated antibodies to show the expression of exogenous SIRT1 and Myc-SATB1. (E) Amino acid sequence of SATB1 containing the acetylated K175. (F) MS/MS spectrum analysis of the SATB1 peptide containing acetylated K136 before and after the in vitro deacetylation assay. (G) MS/MS spectrum analysis of the SATB1 peptide containing acetylated K175 before and after the in vitro deacetylation assay.

Mentions: We then tested whether SATB1’s acetylation status changes in response to an inhibitor or activator of SIRT1. 293 cells transfected with Myc-tagged SATB1 were treated with the class I and II histone deacetylase inhibitor TSA (1 μM), SIRT1 inhibitor Nicotinamide (NAM) (5 mM) or both for 10 h. The cell lysates were immunoprecipitated with an anti-Myc antibody, and western blotting was performed with an antibody against acetylated lysine. These analyses indicated that both TSA and NAM increased SATB1 acetylation, and the effect of NAM was greater than that of TSA (Figure 2A). To detect whether SATB1 was directly deacetylated by SIRT1, we performed an in vitro deacetylation assay. It has been reported that SATB1 is acetylated at lysine 136 within its PSD95/Dlg-A/ZO-1 (PDZ)-like domain by PCAF (15); therefore, we purified a truncated SATB1 protein containing amino acids 1–204 that covered the PDZ-like domain and fusion expressed it with a GST tag. GST-SATB1-1-204 was acetylated in vitro by PCAF in the presence of acetyl-coenzyme A (acetyl-CoA). A western blot using an antibody against acetylated lysine confirmed the efficacy of the reaction (Figure 2B). Acetylated GST-SATB-1-204 was used as a substrate for SIRT1 in an in vitro deacetylation assay in the presence of the coenzyme NAD+ (Figure 2C). The auto-acetylated PCAF, which is deacetylated by SIRT1 (23), served as a positive control. We showed that SIRT1 deacetylates GST-SATB1-1-204 in vitro. To determine whether SIRT1 deacetylates SATB1 in vivo, we cotransfected 293 cells with Myc-tagged SATB1, a SIRT1 overexpression construct or an empty vector and then immunoprecipitated the Myc-SATB1. By western blotting with an acetylated-lysine antibody, we found that SIRT1 overexpression decreased the amount of detected acetylated SATB1 (Figure 2D).Figure 2.


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 deacetylates SATB1. (A) The 293 cells were transfected with Myc-SATB1 and then treated with TSA and NAM as indicated. Immunoprecipitation was performed using a Myc antibody, and SATB1 acetylation levels were detected by western blotting with an acetylated-lysine antibody. (B) In vitro acetylation assay. Truncated GST-SATB1-1-204 was acetylated in the presence of PCAF and Ac-CoA. The self-acetylated PCAF was used as a positive control, and SATB1 acetylation was detected by western blotting with an acetylated-lysine antibody. The truncated GST-SATB1-1-204 protein was detected by Ponceau S staining. (C) In vitro deacetylation assay. In vitro-acetylated GST-SATB1 1-204 was deacetylated with SIRT1 and NAD+. The self-acetylated PCAF was used as a positive control, and SATB1 acetylation was detected by western blotting with an acetylated-lysine antibody. The truncated GST-SATB1-1-204 protein was detected by Ponceau S staining. (D) The 293 cells were cotransfected with SIRT1 and Myc-SATB1-expressing vectors. Immunoprecipitation was then performed using a Myc antibody, and Myc-SATB1 acetylation levels were detected by western blotting with an acetylated-lysine antibody. The cell lysates were subjected to blotting with the indicated antibodies to show the expression of exogenous SIRT1 and Myc-SATB1. (E) Amino acid sequence of SATB1 containing the acetylated K175. (F) MS/MS spectrum analysis of the SATB1 peptide containing acetylated K136 before and after the in vitro deacetylation assay. (G) MS/MS spectrum analysis of the SATB1 peptide containing acetylated K175 before and after the in vitro deacetylation assay.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367170&req=5

gks064-F2: SIRT1 deacetylates SATB1. (A) The 293 cells were transfected with Myc-SATB1 and then treated with TSA and NAM as indicated. Immunoprecipitation was performed using a Myc antibody, and SATB1 acetylation levels were detected by western blotting with an acetylated-lysine antibody. (B) In vitro acetylation assay. Truncated GST-SATB1-1-204 was acetylated in the presence of PCAF and Ac-CoA. The self-acetylated PCAF was used as a positive control, and SATB1 acetylation was detected by western blotting with an acetylated-lysine antibody. The truncated GST-SATB1-1-204 protein was detected by Ponceau S staining. (C) In vitro deacetylation assay. In vitro-acetylated GST-SATB1 1-204 was deacetylated with SIRT1 and NAD+. The self-acetylated PCAF was used as a positive control, and SATB1 acetylation was detected by western blotting with an acetylated-lysine antibody. The truncated GST-SATB1-1-204 protein was detected by Ponceau S staining. (D) The 293 cells were cotransfected with SIRT1 and Myc-SATB1-expressing vectors. Immunoprecipitation was then performed using a Myc antibody, and Myc-SATB1 acetylation levels were detected by western blotting with an acetylated-lysine antibody. The cell lysates were subjected to blotting with the indicated antibodies to show the expression of exogenous SIRT1 and Myc-SATB1. (E) Amino acid sequence of SATB1 containing the acetylated K175. (F) MS/MS spectrum analysis of the SATB1 peptide containing acetylated K136 before and after the in vitro deacetylation assay. (G) MS/MS spectrum analysis of the SATB1 peptide containing acetylated K175 before and after the in vitro deacetylation assay.
Mentions: We then tested whether SATB1’s acetylation status changes in response to an inhibitor or activator of SIRT1. 293 cells transfected with Myc-tagged SATB1 were treated with the class I and II histone deacetylase inhibitor TSA (1 μM), SIRT1 inhibitor Nicotinamide (NAM) (5 mM) or both for 10 h. The cell lysates were immunoprecipitated with an anti-Myc antibody, and western blotting was performed with an antibody against acetylated lysine. These analyses indicated that both TSA and NAM increased SATB1 acetylation, and the effect of NAM was greater than that of TSA (Figure 2A). To detect whether SATB1 was directly deacetylated by SIRT1, we performed an in vitro deacetylation assay. It has been reported that SATB1 is acetylated at lysine 136 within its PSD95/Dlg-A/ZO-1 (PDZ)-like domain by PCAF (15); therefore, we purified a truncated SATB1 protein containing amino acids 1–204 that covered the PDZ-like domain and fusion expressed it with a GST tag. GST-SATB1-1-204 was acetylated in vitro by PCAF in the presence of acetyl-coenzyme A (acetyl-CoA). A western blot using an antibody against acetylated lysine confirmed the efficacy of the reaction (Figure 2B). Acetylated GST-SATB-1-204 was used as a substrate for SIRT1 in an in vitro deacetylation assay in the presence of the coenzyme NAD+ (Figure 2C). The auto-acetylated PCAF, which is deacetylated by SIRT1 (23), served as a positive control. We showed that SIRT1 deacetylates GST-SATB1-1-204 in vitro. To determine whether SIRT1 deacetylates SATB1 in vivo, we cotransfected 293 cells with Myc-tagged SATB1, a SIRT1 overexpression construct or an empty vector and then immunoprecipitated the Myc-SATB1. By western blotting with an acetylated-lysine antibody, we found that SIRT1 overexpression decreased the amount of detected acetylated SATB1 (Figure 2D).Figure 2.

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