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The BCL2 gene is regulated by a special AT-rich sequence binding protein 1-mediated long range chromosomal interaction between the promoter and the distal element located within the 3'-UTR.

Gong F, Sun L, Wang Z, Shi J, Li W, Wang S, Han X, Sun Y - Nucleic Acids Res. (2011)

Bottom Line: During early apoptosis, SATB1 was a key regulator of BCL2 expression.Inhibition of SATB1 cleavage by treatment of cells with a caspase-6 inhibitor or overexpression of mutant SATB1 that was resistant to caspase-6, inhibited disassembly of the SATB1-mediated chromatin loop and restored the BCL2 mRNA level in Jurkat cells.These data revealed a novel mechanism of BCL2 regulation and mechanistically link SATB1-mediated long-range interaction with the regulation of a gene controlling apoptosis pathway for the first time.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Cell Biology and Jiangsu Key Lab of Cancer Biomarkers, Prevention & Treatment, Cancer Center, Nanjing Medical University, Nanjing 210029, PR China.

ABSTRACT
The 279-bp major breakpoint region (mbr) within the 3'-untranslated region (3'-UTR) of the BCL2 gene is a binding site of special AT-rich sequence binding protein 1 (SATB1) that is well known to participate in the long-range regulation of gene transcription. Our previous studies have revealed that the mbr could regulate BCL2 transcription over a 200-kb distance and this regulatory function was closely related to SATB1. This study is to explore the underlying mechanism and its relevance to cellular apoptosis. With chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP) assays we demonstrated that the mbr could physically interact with BCL2 promoter through SATB1-mediated chromatin looping, which was required for epigenetic modifications of the promoter, CREB accessibility and high expression of the BCL2 gene. During early apoptosis, SATB1 was a key regulator of BCL2 expression. Inhibition of SATB1 cleavage by treatment of cells with a caspase-6 inhibitor or overexpression of mutant SATB1 that was resistant to caspase-6, inhibited disassembly of the SATB1-mediated chromatin loop and restored the BCL2 mRNA level in Jurkat cells. These data revealed a novel mechanism of BCL2 regulation and mechanistically link SATB1-mediated long-range interaction with the regulation of a gene controlling apoptosis pathway for the first time.

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Mutation of SATB1 cleavability restored BCL2 transcriptional activity upon apoptosis stimulation. Jurkat cells were transiently transfected with the wild-type SATB1 expression vector (pEGFP-C1-SATB1), mutant SATB1 expression vector (SATB1-D254A, mutated at caspase cleavage site) and pEGFP-C1 control vector, respectively. Twenty-four hours after transfection, cells were treated with 5 μM camptothecin for 2 h to induce cell apoptosis. (A) Western blot analysis of SATB1 from Jurkat cells described above and quantitative analysis. The results confirmed that mutant SATB1 was un-degradable and wild-type SATB1 could be degraded in Jurkat cells treated with camptothecin. (B) RT–PCR analysis showed that camptothecin treatment reduced BCL2 mRNA level. Overexpression of mutant SATB1 completely restored the BCL2 expression, while overexpression of wild-type SATB1 only partially restored BCL2 expression. The statistical differences were calculated using t-test. ‘**’ represents P < 0.01. The error bars represent standard deviation (n = 3).
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Figure 7: Mutation of SATB1 cleavability restored BCL2 transcriptional activity upon apoptosis stimulation. Jurkat cells were transiently transfected with the wild-type SATB1 expression vector (pEGFP-C1-SATB1), mutant SATB1 expression vector (SATB1-D254A, mutated at caspase cleavage site) and pEGFP-C1 control vector, respectively. Twenty-four hours after transfection, cells were treated with 5 μM camptothecin for 2 h to induce cell apoptosis. (A) Western blot analysis of SATB1 from Jurkat cells described above and quantitative analysis. The results confirmed that mutant SATB1 was un-degradable and wild-type SATB1 could be degraded in Jurkat cells treated with camptothecin. (B) RT–PCR analysis showed that camptothecin treatment reduced BCL2 mRNA level. Overexpression of mutant SATB1 completely restored the BCL2 expression, while overexpression of wild-type SATB1 only partially restored BCL2 expression. The statistical differences were calculated using t-test. ‘**’ represents P < 0.01. The error bars represent standard deviation (n = 3).

Mentions: The effects caused by inhibition of SATB1 degradation on the BCL2 activity were further confirmed by the experiment using mutant SATB1 expression vector that was resistant to caspase-6 cleavage. In this experiment, Jurkat cells were transfected with a wild-type SATB1 expression vector (EGFP-SATB1), mutant SATB1 expression vector (EGFP-SATB1-D254A) and pEGFP-C1 control vector, respectively, before treatment with camptothecin. As shown in Figure 7, overexpression of the mutant undegradable SATB1 almost completely restored BCL2 mRNA levels in Jurkat cells treated by camptothecin for two hours, while overexpression of the wild-type SATB1 had only slight effect on BCL2 mRNA level under the same condition (Figure 7B).Figure 7.


The BCL2 gene is regulated by a special AT-rich sequence binding protein 1-mediated long range chromosomal interaction between the promoter and the distal element located within the 3'-UTR.

Gong F, Sun L, Wang Z, Shi J, Li W, Wang S, Han X, Sun Y - Nucleic Acids Res. (2011)

Mutation of SATB1 cleavability restored BCL2 transcriptional activity upon apoptosis stimulation. Jurkat cells were transiently transfected with the wild-type SATB1 expression vector (pEGFP-C1-SATB1), mutant SATB1 expression vector (SATB1-D254A, mutated at caspase cleavage site) and pEGFP-C1 control vector, respectively. Twenty-four hours after transfection, cells were treated with 5 μM camptothecin for 2 h to induce cell apoptosis. (A) Western blot analysis of SATB1 from Jurkat cells described above and quantitative analysis. The results confirmed that mutant SATB1 was un-degradable and wild-type SATB1 could be degraded in Jurkat cells treated with camptothecin. (B) RT–PCR analysis showed that camptothecin treatment reduced BCL2 mRNA level. Overexpression of mutant SATB1 completely restored the BCL2 expression, while overexpression of wild-type SATB1 only partially restored BCL2 expression. The statistical differences were calculated using t-test. ‘**’ represents P < 0.01. The error bars represent standard deviation (n = 3).
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Related In: Results  -  Collection

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Figure 7: Mutation of SATB1 cleavability restored BCL2 transcriptional activity upon apoptosis stimulation. Jurkat cells were transiently transfected with the wild-type SATB1 expression vector (pEGFP-C1-SATB1), mutant SATB1 expression vector (SATB1-D254A, mutated at caspase cleavage site) and pEGFP-C1 control vector, respectively. Twenty-four hours after transfection, cells were treated with 5 μM camptothecin for 2 h to induce cell apoptosis. (A) Western blot analysis of SATB1 from Jurkat cells described above and quantitative analysis. The results confirmed that mutant SATB1 was un-degradable and wild-type SATB1 could be degraded in Jurkat cells treated with camptothecin. (B) RT–PCR analysis showed that camptothecin treatment reduced BCL2 mRNA level. Overexpression of mutant SATB1 completely restored the BCL2 expression, while overexpression of wild-type SATB1 only partially restored BCL2 expression. The statistical differences were calculated using t-test. ‘**’ represents P < 0.01. The error bars represent standard deviation (n = 3).
Mentions: The effects caused by inhibition of SATB1 degradation on the BCL2 activity were further confirmed by the experiment using mutant SATB1 expression vector that was resistant to caspase-6 cleavage. In this experiment, Jurkat cells were transfected with a wild-type SATB1 expression vector (EGFP-SATB1), mutant SATB1 expression vector (EGFP-SATB1-D254A) and pEGFP-C1 control vector, respectively, before treatment with camptothecin. As shown in Figure 7, overexpression of the mutant undegradable SATB1 almost completely restored BCL2 mRNA levels in Jurkat cells treated by camptothecin for two hours, while overexpression of the wild-type SATB1 had only slight effect on BCL2 mRNA level under the same condition (Figure 7B).Figure 7.

Bottom Line: During early apoptosis, SATB1 was a key regulator of BCL2 expression.Inhibition of SATB1 cleavage by treatment of cells with a caspase-6 inhibitor or overexpression of mutant SATB1 that was resistant to caspase-6, inhibited disassembly of the SATB1-mediated chromatin loop and restored the BCL2 mRNA level in Jurkat cells.These data revealed a novel mechanism of BCL2 regulation and mechanistically link SATB1-mediated long-range interaction with the regulation of a gene controlling apoptosis pathway for the first time.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Cell Biology and Jiangsu Key Lab of Cancer Biomarkers, Prevention & Treatment, Cancer Center, Nanjing Medical University, Nanjing 210029, PR China.

ABSTRACT
The 279-bp major breakpoint region (mbr) within the 3'-untranslated region (3'-UTR) of the BCL2 gene is a binding site of special AT-rich sequence binding protein 1 (SATB1) that is well known to participate in the long-range regulation of gene transcription. Our previous studies have revealed that the mbr could regulate BCL2 transcription over a 200-kb distance and this regulatory function was closely related to SATB1. This study is to explore the underlying mechanism and its relevance to cellular apoptosis. With chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP) assays we demonstrated that the mbr could physically interact with BCL2 promoter through SATB1-mediated chromatin looping, which was required for epigenetic modifications of the promoter, CREB accessibility and high expression of the BCL2 gene. During early apoptosis, SATB1 was a key regulator of BCL2 expression. Inhibition of SATB1 cleavage by treatment of cells with a caspase-6 inhibitor or overexpression of mutant SATB1 that was resistant to caspase-6, inhibited disassembly of the SATB1-mediated chromatin loop and restored the BCL2 mRNA level in Jurkat cells. These data revealed a novel mechanism of BCL2 regulation and mechanistically link SATB1-mediated long-range interaction with the regulation of a gene controlling apoptosis pathway for the first time.

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