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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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Knockdown of SATB1 decreased the occupancy of C/EBPβ and p300 at SBS1 in Jurkat cells. Jurkat cells were transiently transfected with SATB1 RNAi plasmids or control plasmids using an electroporator. Western blot showed that knockdown of SATB1 did not affect expression levels of C/EBPβ and p300 significantly (A). Real-time PCR analysis demonstrated that knockdown of SATB1 significantly reduced the occupancy of C/EBPβ (B) and p300 (C) at SBS1. The statistical differences were calculated using t-test. *P < 0.05 and **P < 0.01. The error bars represent standard deviation (n=3).
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Figure 5: Knockdown of SATB1 decreased the occupancy of C/EBPβ and p300 at SBS1 in Jurkat cells. Jurkat cells were transiently transfected with SATB1 RNAi plasmids or control plasmids using an electroporator. Western blot showed that knockdown of SATB1 did not affect expression levels of C/EBPβ and p300 significantly (A). Real-time PCR analysis demonstrated that knockdown of SATB1 significantly reduced the occupancy of C/EBPβ (B) and p300 (C) at SBS1. The statistical differences were calculated using t-test. *P < 0.05 and **P < 0.01. The error bars represent standard deviation (n=3).

Mentions: To further investigate whether C/EBPβ and p300 could be recruited to SBS1 by SATB1, ChIP experiments were performed using SATB1 knockdown cells to examine if it affected the occupancy of C/EBPβ and p300 at SBS1. Specific SATB1 shRNA plasmids were transiently transfected into Jurkat cells as described above. Western blot analysis showed that knockdown of SATB1 by RNAi did not affect expression of C/EBPβ and p300 significantly (Figure 5A). However, quantitative ChIP assays revealed that the occupancy of C/EBPβ and p300 at SBS1 was significantly decreased when SATB1 was knocked down (Figure 5B and C). These data demonstrated that SATB1 was directly involved in recruitment of C/EBPβ and p300 at SBS1 locus.Figure 5.


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)

Knockdown of SATB1 decreased the occupancy of C/EBPβ and p300 at SBS1 in Jurkat cells. Jurkat cells were transiently transfected with SATB1 RNAi plasmids or control plasmids using an electroporator. Western blot showed that knockdown of SATB1 did not affect expression levels of C/EBPβ and p300 significantly (A). Real-time PCR analysis demonstrated that knockdown of SATB1 significantly reduced the occupancy of C/EBPβ (B) and p300 (C) at SBS1. The statistical differences were calculated using t-test. *P < 0.05 and **P < 0.01. The error bars represent standard deviation (n=3).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Knockdown of SATB1 decreased the occupancy of C/EBPβ and p300 at SBS1 in Jurkat cells. Jurkat cells were transiently transfected with SATB1 RNAi plasmids or control plasmids using an electroporator. Western blot showed that knockdown of SATB1 did not affect expression levels of C/EBPβ and p300 significantly (A). Real-time PCR analysis demonstrated that knockdown of SATB1 significantly reduced the occupancy of C/EBPβ (B) and p300 (C) at SBS1. The statistical differences were calculated using t-test. *P < 0.05 and **P < 0.01. The error bars represent standard deviation (n=3).
Mentions: To further investigate whether C/EBPβ and p300 could be recruited to SBS1 by SATB1, ChIP experiments were performed using SATB1 knockdown cells to examine if it affected the occupancy of C/EBPβ and p300 at SBS1. Specific SATB1 shRNA plasmids were transiently transfected into Jurkat cells as described above. Western blot analysis showed that knockdown of SATB1 by RNAi did not affect expression of C/EBPβ and p300 significantly (Figure 5A). However, quantitative ChIP assays revealed that the occupancy of C/EBPβ and p300 at SBS1 was significantly decreased when SATB1 was knocked down (Figure 5B and C). These data demonstrated that SATB1 was directly involved in recruitment of C/EBPβ and p300 at SBS1 locus.Figure 5.

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