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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

SATB1 was a critical factor for the chromatin loop conversion and the cooperative expression of BCL2 and NOXA genes in Jurkat cells.Jurkat cells were transiently transfected with control-shRNA or SATB1-shRNA plasmids, and targeting efficiency was confirmed by Western blot analysis (A). SATB1 knockdown significantly reduced the association of SATB1 with NOXA promoter (B), BCL2 promoter (C), and mbr (D), as revealed by q-ChIP. Quantitative 3C assays further showed that reduction of SATB1 binding significantly increased mbr-NOXA interactions and dramatically decreased the mbr-BCL2 interactions (E). RT–PCR analysis confirmed the BCL2 mRNA level was decreased while the NOXA mRNA level was increased, keeping pace with the conversion of the mbr-promoter loops; we normalized all the genes by using actin control for quantity. (F). These results indicated that reduced SATB1 switched the mbr-BCL2 chromatin loop to the mbr-NOXA chromatin loop, which was involved in cooperative regulation of the NOXA and BCL2 genes.
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pone.0139170.g003: SATB1 was a critical factor for the chromatin loop conversion and the cooperative expression of BCL2 and NOXA genes in Jurkat cells.Jurkat cells were transiently transfected with control-shRNA or SATB1-shRNA plasmids, and targeting efficiency was confirmed by Western blot analysis (A). SATB1 knockdown significantly reduced the association of SATB1 with NOXA promoter (B), BCL2 promoter (C), and mbr (D), as revealed by q-ChIP. Quantitative 3C assays further showed that reduction of SATB1 binding significantly increased mbr-NOXA interactions and dramatically decreased the mbr-BCL2 interactions (E). RT–PCR analysis confirmed the BCL2 mRNA level was decreased while the NOXA mRNA level was increased, keeping pace with the conversion of the mbr-promoter loops; we normalized all the genes by using actin control for quantity. (F). These results indicated that reduced SATB1 switched the mbr-BCL2 chromatin loop to the mbr-NOXA chromatin loop, which was involved in cooperative regulation of the NOXA and BCL2 genes.

Mentions: To clarify whether SATB1 was involved in the cooperative regulation of NOXA and BCL2 expression by changing higher-order chromatin structure, we examined the correlation between the SATB1 binding on the gene promoters and mbr enhancer and the frequency of mbr-NOXA and mbr-BCL2 chromatin loops. The expression of SATB1 was knocked down in Jurkat cells with plasmids expressing short hairpin RNAs (shRNA). Targeting efficiency was confirmed by Western blot analysis (Fig 3). The effects of SATB1 knockdown were determined by quantitative ChIP assay (q-ChIP). As indicated in Fig 3, SATB1 knockdown significantly reduced the occupation of SATB1 on NOXA promoter. The knockdown-induced reduction of SATB1 binding on the BCL2 promoter region and the mbr enhancer was confirmed (Fig 3). Notably, the reduced SATB1 binding significantly increased mbr-NOXA interactions, while dramatically decreased the mbr-BCL2 interactions (Fig 3), indicating that SATB1 was involved in the dynamic conversion between the mbr-BCL2 chromatin loop and the mbr-NOXA chromatin loop. Under the same experimental conditions, the transcriptional activities of the BCL2 and NOXA genes were determined by real-time PCR. Our data showed that the change in BCL2 and NOXA mRNA levels kept pace with the conversion of the mbr-promoter loops (Fig 3), indicating that SATB1-mediated mbr-promoter interactions were required for BCL2 and NOXA transcriptional activity. These results suggested that SATB1 was an important determinant for conversion between the mbr-BCL2 chromatin loop and the NOXA chromatin loop, and was involved in cooperative regulation of BCL2 and NOXA gene expression.


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)

SATB1 was a critical factor for the chromatin loop conversion and the cooperative expression of BCL2 and NOXA genes in Jurkat cells.Jurkat cells were transiently transfected with control-shRNA or SATB1-shRNA plasmids, and targeting efficiency was confirmed by Western blot analysis (A). SATB1 knockdown significantly reduced the association of SATB1 with NOXA promoter (B), BCL2 promoter (C), and mbr (D), as revealed by q-ChIP. Quantitative 3C assays further showed that reduction of SATB1 binding significantly increased mbr-NOXA interactions and dramatically decreased the mbr-BCL2 interactions (E). RT–PCR analysis confirmed the BCL2 mRNA level was decreased while the NOXA mRNA level was increased, keeping pace with the conversion of the mbr-promoter loops; we normalized all the genes by using actin control for quantity. (F). These results indicated that reduced SATB1 switched the mbr-BCL2 chromatin loop to the mbr-NOXA chromatin loop, which was involved in cooperative regulation of the NOXA and BCL2 genes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4589335&req=5

pone.0139170.g003: SATB1 was a critical factor for the chromatin loop conversion and the cooperative expression of BCL2 and NOXA genes in Jurkat cells.Jurkat cells were transiently transfected with control-shRNA or SATB1-shRNA plasmids, and targeting efficiency was confirmed by Western blot analysis (A). SATB1 knockdown significantly reduced the association of SATB1 with NOXA promoter (B), BCL2 promoter (C), and mbr (D), as revealed by q-ChIP. Quantitative 3C assays further showed that reduction of SATB1 binding significantly increased mbr-NOXA interactions and dramatically decreased the mbr-BCL2 interactions (E). RT–PCR analysis confirmed the BCL2 mRNA level was decreased while the NOXA mRNA level was increased, keeping pace with the conversion of the mbr-promoter loops; we normalized all the genes by using actin control for quantity. (F). These results indicated that reduced SATB1 switched the mbr-BCL2 chromatin loop to the mbr-NOXA chromatin loop, which was involved in cooperative regulation of the NOXA and BCL2 genes.
Mentions: To clarify whether SATB1 was involved in the cooperative regulation of NOXA and BCL2 expression by changing higher-order chromatin structure, we examined the correlation between the SATB1 binding on the gene promoters and mbr enhancer and the frequency of mbr-NOXA and mbr-BCL2 chromatin loops. The expression of SATB1 was knocked down in Jurkat cells with plasmids expressing short hairpin RNAs (shRNA). Targeting efficiency was confirmed by Western blot analysis (Fig 3). The effects of SATB1 knockdown were determined by quantitative ChIP assay (q-ChIP). As indicated in Fig 3, SATB1 knockdown significantly reduced the occupation of SATB1 on NOXA promoter. The knockdown-induced reduction of SATB1 binding on the BCL2 promoter region and the mbr enhancer was confirmed (Fig 3). Notably, the reduced SATB1 binding significantly increased mbr-NOXA interactions, while dramatically decreased the mbr-BCL2 interactions (Fig 3), indicating that SATB1 was involved in the dynamic conversion between the mbr-BCL2 chromatin loop and the mbr-NOXA chromatin loop. Under the same experimental conditions, the transcriptional activities of the BCL2 and NOXA genes were determined by real-time PCR. Our data showed that the change in BCL2 and NOXA mRNA levels kept pace with the conversion of the mbr-promoter loops (Fig 3), indicating that SATB1-mediated mbr-promoter interactions were required for BCL2 and NOXA transcriptional activity. These results suggested that SATB1 was an important determinant for conversion between the mbr-BCL2 chromatin loop and the NOXA chromatin loop, and was involved in cooperative regulation of BCL2 and NOXA gene expression.

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