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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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The association between SATB1-mediated chromatin looping and response of the BCL2 gene to apoptosis stimulation. For induction of apoptosis, Jurkat cells were treated with 5 μM camptothecin for 2 h and subsequently harvested. The vehicle control for camptothecin is the equal volume of DMSO. For protease inhibition assay, Jurkat cells were preincubated with 10 μM Z-VEID-fmk for 30 min and apoptosis was then induced by addition of camptothecin. (A) RT–PCR analysis of BCL2 mRNA from Jurkat cells described above. (B) Relative crosslinking frequencies between fixed fragments of SBS1 and mbr were determined by 3C assay in cells described above. (C–E) Quantitative ChIP results from Jurkat cells treated with camptothecin and the control, using antibodies against CREB (C), acetylated histone H3 at K9/14 (D) and acetylated histone H4 at K5/8/12/16 (E). (F) The methylation level of BCL2 gene P1 region was evaluated by examining the unmethylation status of this region using QMSP. (G) Western blot analysis of SATB1 from Jurkat cells treated with camptothecin or not. (H and I) Quantitative ChIP results from Jurkat cells with camptothecin treatment or not, using antibodies against SATB1. The statistical differences between the treatment groups were calculated using t-test. ‘*’ represents P < 0.05 and ‘**’ represents P < 0.01.The error bars represent standard deviation (n = 3).
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Figure 6: The association between SATB1-mediated chromatin looping and response of the BCL2 gene to apoptosis stimulation. For induction of apoptosis, Jurkat cells were treated with 5 μM camptothecin for 2 h and subsequently harvested. The vehicle control for camptothecin is the equal volume of DMSO. For protease inhibition assay, Jurkat cells were preincubated with 10 μM Z-VEID-fmk for 30 min and apoptosis was then induced by addition of camptothecin. (A) RT–PCR analysis of BCL2 mRNA from Jurkat cells described above. (B) Relative crosslinking frequencies between fixed fragments of SBS1 and mbr were determined by 3C assay in cells described above. (C–E) Quantitative ChIP results from Jurkat cells treated with camptothecin and the control, using antibodies against CREB (C), acetylated histone H3 at K9/14 (D) and acetylated histone H4 at K5/8/12/16 (E). (F) The methylation level of BCL2 gene P1 region was evaluated by examining the unmethylation status of this region using QMSP. (G) Western blot analysis of SATB1 from Jurkat cells treated with camptothecin or not. (H and I) Quantitative ChIP results from Jurkat cells with camptothecin treatment or not, using antibodies against SATB1. The statistical differences between the treatment groups were calculated using t-test. ‘*’ represents P < 0.05 and ‘**’ represents P < 0.01.The error bars represent standard deviation (n = 3).

Mentions: BCL2 is a key regulator of apoptosis. To address the issue whether SATB1-mediated interaction between mbr and the promoter of the BCL2 is directly involved in response of the gene to apoptosis stimulation, we analyzed the change in chromatin looping, histone acetylation, DNA methylation, CREB recruitment and transcription activity of the gene in parallel in Jurkat cells treated with camptothecin. Cells were incubated with 5 μM camptothecin and harvested after 2 h, when early apoptosis occurred (Supplementary Figure S6). RT–PCR results showed that camptothecin treatment reduced the BCL2 mRNA levels by 50% (Figure 6A). Correspondingly, quantitative 3C assays revealed that the frequency of interaction between SBS1 and the mbr (SATB1-mediated chromatin loop) was reduced (Figure 6B) in camptothecin-treated cells. The diminished BCL2 chromatin looping was accompanied by reductions both in CREB recruitment and acetylation of histone H3 and H4 in the P1 region of BCL2 (Figure 6C–E). Unmethylation of this region slightly decreased in the treated cells (Figure 6F). Corresponding to these changes, the level of SATB1 was clearly decreased in the camptothecin-treated cells as revealed by western blot analysis (Figure 6G). Quantitative ChIP assays further confirmed that less SATB1 occupied the mbr and P1 region (Figure 6H and I), which could explain why SATB1-mediated chromatin looping was diminished upon apoptosis induction. It should be pointed out that genomic integrity was maintained under our experimental conditions, which was confirmed by the slight increase in the frequency of the chromatin loop formed between the mbr and the promoter of the Noxa gene, a pro-apoptotic gene that located 3.4 Mb downstream of the BCL2 gene (Supplementary Figure S7). These data suggested that the SATB1-mediated mbr-promoter interaction was directly involved in regulation of the BCL2 gene in cellular apoptosis response.Figure 6.


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)

The association between SATB1-mediated chromatin looping and response of the BCL2 gene to apoptosis stimulation. For induction of apoptosis, Jurkat cells were treated with 5 μM camptothecin for 2 h and subsequently harvested. The vehicle control for camptothecin is the equal volume of DMSO. For protease inhibition assay, Jurkat cells were preincubated with 10 μM Z-VEID-fmk for 30 min and apoptosis was then induced by addition of camptothecin. (A) RT–PCR analysis of BCL2 mRNA from Jurkat cells described above. (B) Relative crosslinking frequencies between fixed fragments of SBS1 and mbr were determined by 3C assay in cells described above. (C–E) Quantitative ChIP results from Jurkat cells treated with camptothecin and the control, using antibodies against CREB (C), acetylated histone H3 at K9/14 (D) and acetylated histone H4 at K5/8/12/16 (E). (F) The methylation level of BCL2 gene P1 region was evaluated by examining the unmethylation status of this region using QMSP. (G) Western blot analysis of SATB1 from Jurkat cells treated with camptothecin or not. (H and I) Quantitative ChIP results from Jurkat cells with camptothecin treatment or not, using antibodies against SATB1. The statistical differences between the treatment groups were calculated using t-test. ‘*’ represents P < 0.05 and ‘**’ represents P < 0.01.The error bars represent standard deviation (n = 3).
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Figure 6: The association between SATB1-mediated chromatin looping and response of the BCL2 gene to apoptosis stimulation. For induction of apoptosis, Jurkat cells were treated with 5 μM camptothecin for 2 h and subsequently harvested. The vehicle control for camptothecin is the equal volume of DMSO. For protease inhibition assay, Jurkat cells were preincubated with 10 μM Z-VEID-fmk for 30 min and apoptosis was then induced by addition of camptothecin. (A) RT–PCR analysis of BCL2 mRNA from Jurkat cells described above. (B) Relative crosslinking frequencies between fixed fragments of SBS1 and mbr were determined by 3C assay in cells described above. (C–E) Quantitative ChIP results from Jurkat cells treated with camptothecin and the control, using antibodies against CREB (C), acetylated histone H3 at K9/14 (D) and acetylated histone H4 at K5/8/12/16 (E). (F) The methylation level of BCL2 gene P1 region was evaluated by examining the unmethylation status of this region using QMSP. (G) Western blot analysis of SATB1 from Jurkat cells treated with camptothecin or not. (H and I) Quantitative ChIP results from Jurkat cells with camptothecin treatment or not, using antibodies against SATB1. The statistical differences between the treatment groups were calculated using t-test. ‘*’ represents P < 0.05 and ‘**’ represents P < 0.01.The error bars represent standard deviation (n = 3).
Mentions: BCL2 is a key regulator of apoptosis. To address the issue whether SATB1-mediated interaction between mbr and the promoter of the BCL2 is directly involved in response of the gene to apoptosis stimulation, we analyzed the change in chromatin looping, histone acetylation, DNA methylation, CREB recruitment and transcription activity of the gene in parallel in Jurkat cells treated with camptothecin. Cells were incubated with 5 μM camptothecin and harvested after 2 h, when early apoptosis occurred (Supplementary Figure S6). RT–PCR results showed that camptothecin treatment reduced the BCL2 mRNA levels by 50% (Figure 6A). Correspondingly, quantitative 3C assays revealed that the frequency of interaction between SBS1 and the mbr (SATB1-mediated chromatin loop) was reduced (Figure 6B) in camptothecin-treated cells. The diminished BCL2 chromatin looping was accompanied by reductions both in CREB recruitment and acetylation of histone H3 and H4 in the P1 region of BCL2 (Figure 6C–E). Unmethylation of this region slightly decreased in the treated cells (Figure 6F). Corresponding to these changes, the level of SATB1 was clearly decreased in the camptothecin-treated cells as revealed by western blot analysis (Figure 6G). Quantitative ChIP assays further confirmed that less SATB1 occupied the mbr and P1 region (Figure 6H and I), which could explain why SATB1-mediated chromatin looping was diminished upon apoptosis induction. It should be pointed out that genomic integrity was maintained under our experimental conditions, which was confirmed by the slight increase in the frequency of the chromatin loop formed between the mbr and the promoter of the Noxa gene, a pro-apoptotic gene that located 3.4 Mb downstream of the BCL2 gene (Supplementary Figure S7). These data suggested that the SATB1-mediated mbr-promoter interaction was directly involved in regulation of the BCL2 gene in cellular apoptosis response.Figure 6.

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