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SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes.

Yang Y, Wang Z, Sun L, Shao L, Yang N, Yu D, Zhang X, Han X, Sun Y - PLoS ONE (2015)

Bottom Line: Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes.The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis.Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing medical University, Nanjing, PR China; Department of Cell Biology, Nanjing Medical University, Nanjing, PR China.

ABSTRACT
Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3'-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways.

No MeSH data available.


Related in: MedlinePlus

The specificity of SATB1 functions in co-regulating the expression of the BCL2 and NOXA genes in the cellular response to apoptosis.Cells were pre-incubated with 10μM Z-VEID-fmk, a specific inhibitor of caspase-6, for 30 min, before camptothecin treatment to inhibit SATB1 degradation. SATB1 levels were determined by Western blot (A). RT–PCR analysis revealed that inhibition of SATB1 degradation reversed the mRNA levels of both BCL2 and NOXA in camptothecin-treated cells (B & C), and significantly suppressed apoptosis (D & E). The specificity of SATB1 function was further confirmed by the expression of mutant SATB1 that was resistant to caspase-6 cleavage. Jurkat cells were transiently transfected with the pEGFP-C1 control vector、wild-type SATB1 expression vector (pEGFP-C1- SATB1), and mutant SATB1 expression vector, respectively, and treated with 5μM camptothecin for 2h to induce apoptosis 24 hours after transfection. SATB1 expression levels were determined by Western blot analysis, which showed that the expression level of mutant SATB1 was consistent in camptothecin-treated cells, and wild-type SATB1 was decreased (F). RT–PCR analysis showed that overexpression of mutant SATB1 completely restored BCL2 expression and inhibited the increase in NOXA expression, and overexpression of the wild-type SATB1 partly reversed mRNA levels of these two genes (G, H). The statistical differences were calculated using t-test. ‘**’ Represents P<0.01. The error bars represent standard deviation (n = 3).
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pone.0139170.g005: The specificity of SATB1 functions in co-regulating the expression of the BCL2 and NOXA genes in the cellular response to apoptosis.Cells were pre-incubated with 10μM Z-VEID-fmk, a specific inhibitor of caspase-6, for 30 min, before camptothecin treatment to inhibit SATB1 degradation. SATB1 levels were determined by Western blot (A). RT–PCR analysis revealed that inhibition of SATB1 degradation reversed the mRNA levels of both BCL2 and NOXA in camptothecin-treated cells (B & C), and significantly suppressed apoptosis (D & E). The specificity of SATB1 function was further confirmed by the expression of mutant SATB1 that was resistant to caspase-6 cleavage. Jurkat cells were transiently transfected with the pEGFP-C1 control vector、wild-type SATB1 expression vector (pEGFP-C1- SATB1), and mutant SATB1 expression vector, respectively, and treated with 5μM camptothecin for 2h to induce apoptosis 24 hours after transfection. SATB1 expression levels were determined by Western blot analysis, which showed that the expression level of mutant SATB1 was consistent in camptothecin-treated cells, and wild-type SATB1 was decreased (F). RT–PCR analysis showed that overexpression of mutant SATB1 completely restored BCL2 expression and inhibited the increase in NOXA expression, and overexpression of the wild-type SATB1 partly reversed mRNA levels of these two genes (G, H). The statistical differences were calculated using t-test. ‘**’ Represents P<0.01. The error bars represent standard deviation (n = 3).

Mentions: SATB1 is the target of caspase-6 and is specifically cleaved by this enzyme during early apoptosis. In order to determine the specificity of the SATB1 function in the cooperative regulation of the BCL2 and NOXA genes during apoptosis, we examined the effects of inhibiting SATB1 degradation on BCL2 and NOXA transcriptional activity. Jurkat cells were pretreated for 30 min with 10 μM Z-VEID-fmk, a caspase-6 inhibitor, before the addition of camptothecin. Western blot analysis showed that pretreatment of cells with 10 μM Z-VEID-fmk completely inhibited camptothecin-induced cleavage of SATB1 (Fig 5). As expected, inhibition of SATB1 degradation reversed the mRNA levels of both BCL2 and NOXA in these cells (Fig 5), significantly suppressing apoptosis (Fig 5). The specificity of SATB1 function was further evaluated by an experiment using a mutant SATB1 expression vector that was resistant to caspase-6 cleavage. Jurkat cells were transfected with a wild-type SATB1 expression vector (EGFP-SATB1), a mutant SATB1 expression vector (EGFP-SATB1-D254A), or a pEGFP-C1 control vector before treatment with camptothecin. The transfection efficiency was examined by Western blot (Fig 5). As expected, overexpression of the mutant undegradable SATB1 almost completely reversed changes in expression levels of the BCL2 and NOXA mRNA in Jurkat cells treated with camptothecin, while overexpression of the wild-type SATB1 had only slight effects on the mRNA levels of these two genes under the same experimental conditions (Fig 5). These data clearly demonstrated that SATB1 plays a specific role in regulating the cooperative response of the BCL2 and NOXA genes to apoptotic stimulation.


SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes.

Yang Y, Wang Z, Sun L, Shao L, Yang N, Yu D, Zhang X, Han X, Sun Y - PLoS ONE (2015)

The specificity of SATB1 functions in co-regulating the expression of the BCL2 and NOXA genes in the cellular response to apoptosis.Cells were pre-incubated with 10μM Z-VEID-fmk, a specific inhibitor of caspase-6, for 30 min, before camptothecin treatment to inhibit SATB1 degradation. SATB1 levels were determined by Western blot (A). RT–PCR analysis revealed that inhibition of SATB1 degradation reversed the mRNA levels of both BCL2 and NOXA in camptothecin-treated cells (B & C), and significantly suppressed apoptosis (D & E). The specificity of SATB1 function was further confirmed by the expression of mutant SATB1 that was resistant to caspase-6 cleavage. Jurkat cells were transiently transfected with the pEGFP-C1 control vector、wild-type SATB1 expression vector (pEGFP-C1- SATB1), and mutant SATB1 expression vector, respectively, and treated with 5μM camptothecin for 2h to induce apoptosis 24 hours after transfection. SATB1 expression levels were determined by Western blot analysis, which showed that the expression level of mutant SATB1 was consistent in camptothecin-treated cells, and wild-type SATB1 was decreased (F). RT–PCR analysis showed that overexpression of mutant SATB1 completely restored BCL2 expression and inhibited the increase in NOXA expression, and overexpression of the wild-type SATB1 partly reversed mRNA levels of these two genes (G, H). The statistical differences were calculated using t-test. ‘**’ Represents P<0.01. The error bars represent standard deviation (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4589335&req=5

pone.0139170.g005: The specificity of SATB1 functions in co-regulating the expression of the BCL2 and NOXA genes in the cellular response to apoptosis.Cells were pre-incubated with 10μM Z-VEID-fmk, a specific inhibitor of caspase-6, for 30 min, before camptothecin treatment to inhibit SATB1 degradation. SATB1 levels were determined by Western blot (A). RT–PCR analysis revealed that inhibition of SATB1 degradation reversed the mRNA levels of both BCL2 and NOXA in camptothecin-treated cells (B & C), and significantly suppressed apoptosis (D & E). The specificity of SATB1 function was further confirmed by the expression of mutant SATB1 that was resistant to caspase-6 cleavage. Jurkat cells were transiently transfected with the pEGFP-C1 control vector、wild-type SATB1 expression vector (pEGFP-C1- SATB1), and mutant SATB1 expression vector, respectively, and treated with 5μM camptothecin for 2h to induce apoptosis 24 hours after transfection. SATB1 expression levels were determined by Western blot analysis, which showed that the expression level of mutant SATB1 was consistent in camptothecin-treated cells, and wild-type SATB1 was decreased (F). RT–PCR analysis showed that overexpression of mutant SATB1 completely restored BCL2 expression and inhibited the increase in NOXA expression, and overexpression of the wild-type SATB1 partly reversed mRNA levels of these two genes (G, H). The statistical differences were calculated using t-test. ‘**’ Represents P<0.01. The error bars represent standard deviation (n = 3).
Mentions: SATB1 is the target of caspase-6 and is specifically cleaved by this enzyme during early apoptosis. In order to determine the specificity of the SATB1 function in the cooperative regulation of the BCL2 and NOXA genes during apoptosis, we examined the effects of inhibiting SATB1 degradation on BCL2 and NOXA transcriptional activity. Jurkat cells were pretreated for 30 min with 10 μM Z-VEID-fmk, a caspase-6 inhibitor, before the addition of camptothecin. Western blot analysis showed that pretreatment of cells with 10 μM Z-VEID-fmk completely inhibited camptothecin-induced cleavage of SATB1 (Fig 5). As expected, inhibition of SATB1 degradation reversed the mRNA levels of both BCL2 and NOXA in these cells (Fig 5), significantly suppressing apoptosis (Fig 5). The specificity of SATB1 function was further evaluated by an experiment using a mutant SATB1 expression vector that was resistant to caspase-6 cleavage. Jurkat cells were transfected with a wild-type SATB1 expression vector (EGFP-SATB1), a mutant SATB1 expression vector (EGFP-SATB1-D254A), or a pEGFP-C1 control vector before treatment with camptothecin. The transfection efficiency was examined by Western blot (Fig 5). As expected, overexpression of the mutant undegradable SATB1 almost completely reversed changes in expression levels of the BCL2 and NOXA mRNA in Jurkat cells treated with camptothecin, while overexpression of the wild-type SATB1 had only slight effects on the mRNA levels of these two genes under the same experimental conditions (Fig 5). These data clearly demonstrated that SATB1 plays a specific role in regulating the cooperative response of the BCL2 and NOXA genes to apoptotic stimulation.

Bottom Line: Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes.The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis.Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing medical University, Nanjing, PR China; Department of Cell Biology, Nanjing Medical University, Nanjing, PR China.

ABSTRACT
Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3'-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways.

No MeSH data available.


Related in: MedlinePlus