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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 of SATB1 with the chromatin-loop organization and transcription of BCL2 in Jurkat cells. (A) Western blot analysis of SATB1 in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids, respectively indicated that SATB1-shRNA effectively reduced the SATB1 level in the cells. (B) Quantitative ChIP results obtained from Jurkat cells that were transiently transfected with control-shRNA or SATB1-shRNA plasmids showed that knockdown of SATB1 expression significantly reduced the association of SATB1 with SBS1. (C) Quantitative ChIP results obtained from the same cells described above confirmed that knockdown of SATB1 expression also significantly reduced the association of SATB1 with the mbr. (D) Quantitative 3C assay was performed as described in ‘Materials and Methods’ section in parallel with the experiments in B and C. The relative crosslinking frequency between the fragments of SBS1 and mbr indicated that knockdown of SATB1 significantly suppressed the mbr-SBS1 (promter) interaction. (E) RT–PCR analysis of the BCL2 mRNA from Jurkat cells transfected with either control-shRNA or SATB1-shRNA. (F, G and I) The histone acetylation and CREB accessibility were analyzed with quantitative ChIP in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids using antibodies against acetylated histone H3 at K9/14 (F), acetylated histone H4 at K5/8/12/16 (G) and CREB (I). (H) The methylation level of the BCL2 P1 region was evaluated in the Jurkat cells described above using QMSP. (J) Western blot analysis of CREB in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids. The statistical differences 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 3: The Association of SATB1 with the chromatin-loop organization and transcription of BCL2 in Jurkat cells. (A) Western blot analysis of SATB1 in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids, respectively indicated that SATB1-shRNA effectively reduced the SATB1 level in the cells. (B) Quantitative ChIP results obtained from Jurkat cells that were transiently transfected with control-shRNA or SATB1-shRNA plasmids showed that knockdown of SATB1 expression significantly reduced the association of SATB1 with SBS1. (C) Quantitative ChIP results obtained from the same cells described above confirmed that knockdown of SATB1 expression also significantly reduced the association of SATB1 with the mbr. (D) Quantitative 3C assay was performed as described in ‘Materials and Methods’ section in parallel with the experiments in B and C. The relative crosslinking frequency between the fragments of SBS1 and mbr indicated that knockdown of SATB1 significantly suppressed the mbr-SBS1 (promter) interaction. (E) RT–PCR analysis of the BCL2 mRNA from Jurkat cells transfected with either control-shRNA or SATB1-shRNA. (F, G and I) The histone acetylation and CREB accessibility were analyzed with quantitative ChIP in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids using antibodies against acetylated histone H3 at K9/14 (F), acetylated histone H4 at K5/8/12/16 (G) and CREB (I). (H) The methylation level of the BCL2 P1 region was evaluated in the Jurkat cells described above using QMSP. (J) Western blot analysis of CREB in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids. The statistical differences were calculated using t-test. ‘*’ represents P < 0.05 and ‘**’ represents P < 0.01. The error bars represent standard deviation (n = 3).

Mentions: To address the question of whether SATB1 is required for the chromatin loop organization within the BCL2 gene, we determined the correlation between the binding of SATB1 to the two distal elements and the SBS1-mbr interaction. The level of SATB1 in Jurkat cells was knocked down with plasmids expressing short hairpin RNAs (shRNA). Western blot analysis confirmed that shRNA targeted to SATB1 efficiently reduced the expression level of SATB1 (Figure 3A). Quantitative ChIP assays revealed that knockdown of SATB1 significantly reduced the occupation of SATB1 on SBS1 and mbr (Figure 3B and C) and simultaneously decreased the frequency of interaction between SBS1 and mbr as indicated by quantitative 3C assay (Figure 3D). These observations clearly indicate that SATB1 is a critical mediator of long-distance interaction between the mbr and the BCL2 promoter.Figure 3.


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 of SATB1 with the chromatin-loop organization and transcription of BCL2 in Jurkat cells. (A) Western blot analysis of SATB1 in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids, respectively indicated that SATB1-shRNA effectively reduced the SATB1 level in the cells. (B) Quantitative ChIP results obtained from Jurkat cells that were transiently transfected with control-shRNA or SATB1-shRNA plasmids showed that knockdown of SATB1 expression significantly reduced the association of SATB1 with SBS1. (C) Quantitative ChIP results obtained from the same cells described above confirmed that knockdown of SATB1 expression also significantly reduced the association of SATB1 with the mbr. (D) Quantitative 3C assay was performed as described in ‘Materials and Methods’ section in parallel with the experiments in B and C. The relative crosslinking frequency between the fragments of SBS1 and mbr indicated that knockdown of SATB1 significantly suppressed the mbr-SBS1 (promter) interaction. (E) RT–PCR analysis of the BCL2 mRNA from Jurkat cells transfected with either control-shRNA or SATB1-shRNA. (F, G and I) The histone acetylation and CREB accessibility were analyzed with quantitative ChIP in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids using antibodies against acetylated histone H3 at K9/14 (F), acetylated histone H4 at K5/8/12/16 (G) and CREB (I). (H) The methylation level of the BCL2 P1 region was evaluated in the Jurkat cells described above using QMSP. (J) Western blot analysis of CREB in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids. The statistical differences 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 3: The Association of SATB1 with the chromatin-loop organization and transcription of BCL2 in Jurkat cells. (A) Western blot analysis of SATB1 in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids, respectively indicated that SATB1-shRNA effectively reduced the SATB1 level in the cells. (B) Quantitative ChIP results obtained from Jurkat cells that were transiently transfected with control-shRNA or SATB1-shRNA plasmids showed that knockdown of SATB1 expression significantly reduced the association of SATB1 with SBS1. (C) Quantitative ChIP results obtained from the same cells described above confirmed that knockdown of SATB1 expression also significantly reduced the association of SATB1 with the mbr. (D) Quantitative 3C assay was performed as described in ‘Materials and Methods’ section in parallel with the experiments in B and C. The relative crosslinking frequency between the fragments of SBS1 and mbr indicated that knockdown of SATB1 significantly suppressed the mbr-SBS1 (promter) interaction. (E) RT–PCR analysis of the BCL2 mRNA from Jurkat cells transfected with either control-shRNA or SATB1-shRNA. (F, G and I) The histone acetylation and CREB accessibility were analyzed with quantitative ChIP in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids using antibodies against acetylated histone H3 at K9/14 (F), acetylated histone H4 at K5/8/12/16 (G) and CREB (I). (H) The methylation level of the BCL2 P1 region was evaluated in the Jurkat cells described above using QMSP. (J) Western blot analysis of CREB in Jurkat cells transiently transfected with control-shRNA or SATB1-shRNA plasmids. The statistical differences were calculated using t-test. ‘*’ represents P < 0.05 and ‘**’ represents P < 0.01. The error bars represent standard deviation (n = 3).
Mentions: To address the question of whether SATB1 is required for the chromatin loop organization within the BCL2 gene, we determined the correlation between the binding of SATB1 to the two distal elements and the SBS1-mbr interaction. The level of SATB1 in Jurkat cells was knocked down with plasmids expressing short hairpin RNAs (shRNA). Western blot analysis confirmed that shRNA targeted to SATB1 efficiently reduced the expression level of SATB1 (Figure 3A). Quantitative ChIP assays revealed that knockdown of SATB1 significantly reduced the occupation of SATB1 on SBS1 and mbr (Figure 3B and C) and simultaneously decreased the frequency of interaction between SBS1 and mbr as indicated by quantitative 3C assay (Figure 3D). These observations clearly indicate that SATB1 is a critical mediator of long-distance interaction between the mbr and the BCL2 promoter.Figure 3.

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