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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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ChIP analysis of binding of SATB1 at the mbr and the BCL2 promoter region in vivo. The ChIP experiments were performed as described in the ‘Materials and Methods’ section. The results showed that SATB1 bound to SBS1 (A), SBS2 (B) and mbr (D), respectively, but not to SBS3 (C). The input represented 1% of total chromatin used in immunoprecipitation. Non-specific IgG was used as a negative control. The PCR products were visualized by ethidium bromide staining of a 1.5% agarose gel.
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Figure 1: ChIP analysis of binding of SATB1 at the mbr and the BCL2 promoter region in vivo. The ChIP experiments were performed as described in the ‘Materials and Methods’ section. The results showed that SATB1 bound to SBS1 (A), SBS2 (B) and mbr (D), respectively, but not to SBS3 (C). The input represented 1% of total chromatin used in immunoprecipitation. Non-specific IgG was used as a negative control. The PCR products were visualized by ethidium bromide staining of a 1.5% agarose gel.

Mentions: To determine whether SATB1 can bind to these three sequences in vivo, we performed ChIP assays using an anti-SATB1 antibody. SBS1 and SBS2 sequences located −4.1 and −4.7 kb relative to the translational start site (Supplementary Figure S3) were found to be specifically immunoprecipitated with anti-SATB1 (Figure 1A and B), indicating that SATB1 binds to these sequences in vivo. The ChIP assays on the SBS3 sequence did not reveal any binding of SATB1 (Figure 1C). ChIP assays targeting to the mbr were also performed and the binding of SATB1 to the mbr was confirmed in our experimental system (Figure 1D). The binding of SATB1 to both BCL2 promoter and mbr strongly implied that SATB1 might mediate an interaction between the mbr element and the promoter by modification of BCL2 chromatin structure.Figure 1.


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)

ChIP analysis of binding of SATB1 at the mbr and the BCL2 promoter region in vivo. The ChIP experiments were performed as described in the ‘Materials and Methods’ section. The results showed that SATB1 bound to SBS1 (A), SBS2 (B) and mbr (D), respectively, but not to SBS3 (C). The input represented 1% of total chromatin used in immunoprecipitation. Non-specific IgG was used as a negative control. The PCR products were visualized by ethidium bromide staining of a 1.5% agarose gel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113567&req=5

Figure 1: ChIP analysis of binding of SATB1 at the mbr and the BCL2 promoter region in vivo. The ChIP experiments were performed as described in the ‘Materials and Methods’ section. The results showed that SATB1 bound to SBS1 (A), SBS2 (B) and mbr (D), respectively, but not to SBS3 (C). The input represented 1% of total chromatin used in immunoprecipitation. Non-specific IgG was used as a negative control. The PCR products were visualized by ethidium bromide staining of a 1.5% agarose gel.
Mentions: To determine whether SATB1 can bind to these three sequences in vivo, we performed ChIP assays using an anti-SATB1 antibody. SBS1 and SBS2 sequences located −4.1 and −4.7 kb relative to the translational start site (Supplementary Figure S3) were found to be specifically immunoprecipitated with anti-SATB1 (Figure 1A and B), indicating that SATB1 binds to these sequences in vivo. The ChIP assays on the SBS3 sequence did not reveal any binding of SATB1 (Figure 1C). ChIP assays targeting to the mbr were also performed and the binding of SATB1 to the mbr was confirmed in our experimental system (Figure 1D). The binding of SATB1 to both BCL2 promoter and mbr strongly implied that SATB1 might mediate an interaction between the mbr element and the promoter by modification of BCL2 chromatin structure.Figure 1.

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.

Show MeSH