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Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells.

Meyers-Needham M, Ponnusamy S, Gencer S, Jiang W, Thomas RJ, Senkal CE, Ogretmen B - EMBO Mol Med (2011)

Bottom Line: Moreover, an alternatively spliced variant CerS1 mRNA (CerS1-2) was detected mainly in cancer cells or primary tumour tissues compared to controls, which was targeted by miR-574-5p for degradation.Interference with HDAC1 and miR-574-5p reconstituted CerS1-2 expression and C(18) -ceramide generation in multiple human cancer cell lines, which subsequently inhibited proliferation and anchorage-independent growth.Accordingly, knockdown of CerS1 partially protected cancer cells from MS-275/miR-574-5p siRNA-mediated growth inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.

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Endogenous Sp1 specifically binds CerS1 promoter at the GC Boxes 2 and 3, whose recruitment to the promoter is modulated by HDAC-dependent histone deacetylationUM-SCC-1 nuclear extracts were isolated as previously described (Ogretmen and Safa, 2000) and pre-incubated with competitor unlabelled oligonucleotides before incubation with P32-Sp1 consensus oligonucleotides.Pre-incubation of nuclear extracts with a-Sp1 antibody, but not non-specific a-IgG antibody, caused a super-shift in the protein/DNA complex, indicating Sp1-specific binding to oligonucleotides creates C1 and C2.Sp1 association with the endogenous CerS1 core promoter region was assessed using Q-ChIP. Relative quantities normalized to 1% input DNA before pull-down with Sp1 or IgG antibody.Cells plated in a 6-well plates (4 × 105 cells/well) were treated with TSA (200 ng/ml) for 24 h, and CerS1 promoter activity was measured by luciferase assay.Sp1 and acetylated histone-H3 (Ac-Histone-H3) association with the core promoter was assed after treatment with 200 ng/ml TSA for 24 h in UM-SCC-1 via Q-ChIP. Sp1 and Ac-Histone-H3 relative quantities normalized to 1% input DNA and IgG controls. These data represent at least two-independent experiments. The error bars represent the standard deviations.
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fig04: Endogenous Sp1 specifically binds CerS1 promoter at the GC Boxes 2 and 3, whose recruitment to the promoter is modulated by HDAC-dependent histone deacetylationUM-SCC-1 nuclear extracts were isolated as previously described (Ogretmen and Safa, 2000) and pre-incubated with competitor unlabelled oligonucleotides before incubation with P32-Sp1 consensus oligonucleotides.Pre-incubation of nuclear extracts with a-Sp1 antibody, but not non-specific a-IgG antibody, caused a super-shift in the protein/DNA complex, indicating Sp1-specific binding to oligonucleotides creates C1 and C2.Sp1 association with the endogenous CerS1 core promoter region was assessed using Q-ChIP. Relative quantities normalized to 1% input DNA before pull-down with Sp1 or IgG antibody.Cells plated in a 6-well plates (4 × 105 cells/well) were treated with TSA (200 ng/ml) for 24 h, and CerS1 promoter activity was measured by luciferase assay.Sp1 and acetylated histone-H3 (Ac-Histone-H3) association with the core promoter was assed after treatment with 200 ng/ml TSA for 24 h in UM-SCC-1 via Q-ChIP. Sp1 and Ac-Histone-H3 relative quantities normalized to 1% input DNA and IgG controls. These data represent at least two-independent experiments. The error bars represent the standard deviations.

Mentions: Then, to assess whether the GC-Boxes 2 and 3 bind Sp1 for the regulation of the CerS1 promoter, we performed EMSA using nuclear extracts isolated from UM-SCC-1 cells, and the [32P]-labelled oligonucleotide (22-mer) containing a conserved Sp1-recognition sequence as a probe with and without unlabelled oligonucleotides containing the Sp1 (cold probe), GC-Boxes 1, 2 or 3 sequences or their mutated variants as competitors. Incubation of the labelled Sp1 probe with the UM-SCC-1 nuclear extracts generated two main shifted bands (complexes 1 and 2), which were competed off when unlabelled wt-probe, but not its mutant, were used as competitors (Fig 4A, lanes 2–4, respectively). As expected, co-incubation of the labelled Sp1 probe with the wt- or mutant GC-Box-1 oligonucleotides had no effect on probe binding with C1 or C2 compared to probe-only control (Fig 4A, lanes 5–6 and 2, respectively). Conversely, while unlabelled oligonucleotides containing wt-GC-Boxes 2 and 3 almost completely prevented the Sp1-C1/C2 binding, their mutated isoforms had no effect compared to probe-only control (Fig 4A, lanes 7–10, compared to lane 2, respectively). We also confirmed that C1/C2 complexes indeed associate with the Sp1 transcription factor using a super-shift assay in the presence of an anti-Sp1 or anti-IgG antibodies in EMSA assays. As seen in Fig 4B, pre-incubation of nuclear extracts with the anti-Sp1 antibody, but not the anti-IgG antibody, caused a super-shift on the mobility of the probe-protein complex (C1), indicating that the C1 contains Sp1-probe complex (Fig 4B). Taken together, both functional (promoter mutations) and biochemical (EMSA) studies suggest that Sp1 induces CerS1 core promoter with the specific involvement of GC-rich sequences within the −177 and −139 regions (GC-Boxes 2 and 3) of the promoter.


Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells.

Meyers-Needham M, Ponnusamy S, Gencer S, Jiang W, Thomas RJ, Senkal CE, Ogretmen B - EMBO Mol Med (2011)

Endogenous Sp1 specifically binds CerS1 promoter at the GC Boxes 2 and 3, whose recruitment to the promoter is modulated by HDAC-dependent histone deacetylationUM-SCC-1 nuclear extracts were isolated as previously described (Ogretmen and Safa, 2000) and pre-incubated with competitor unlabelled oligonucleotides before incubation with P32-Sp1 consensus oligonucleotides.Pre-incubation of nuclear extracts with a-Sp1 antibody, but not non-specific a-IgG antibody, caused a super-shift in the protein/DNA complex, indicating Sp1-specific binding to oligonucleotides creates C1 and C2.Sp1 association with the endogenous CerS1 core promoter region was assessed using Q-ChIP. Relative quantities normalized to 1% input DNA before pull-down with Sp1 or IgG antibody.Cells plated in a 6-well plates (4 × 105 cells/well) were treated with TSA (200 ng/ml) for 24 h, and CerS1 promoter activity was measured by luciferase assay.Sp1 and acetylated histone-H3 (Ac-Histone-H3) association with the core promoter was assed after treatment with 200 ng/ml TSA for 24 h in UM-SCC-1 via Q-ChIP. Sp1 and Ac-Histone-H3 relative quantities normalized to 1% input DNA and IgG controls. These data represent at least two-independent experiments. The error bars represent the standard deviations.
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Related In: Results  -  Collection

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fig04: Endogenous Sp1 specifically binds CerS1 promoter at the GC Boxes 2 and 3, whose recruitment to the promoter is modulated by HDAC-dependent histone deacetylationUM-SCC-1 nuclear extracts were isolated as previously described (Ogretmen and Safa, 2000) and pre-incubated with competitor unlabelled oligonucleotides before incubation with P32-Sp1 consensus oligonucleotides.Pre-incubation of nuclear extracts with a-Sp1 antibody, but not non-specific a-IgG antibody, caused a super-shift in the protein/DNA complex, indicating Sp1-specific binding to oligonucleotides creates C1 and C2.Sp1 association with the endogenous CerS1 core promoter region was assessed using Q-ChIP. Relative quantities normalized to 1% input DNA before pull-down with Sp1 or IgG antibody.Cells plated in a 6-well plates (4 × 105 cells/well) were treated with TSA (200 ng/ml) for 24 h, and CerS1 promoter activity was measured by luciferase assay.Sp1 and acetylated histone-H3 (Ac-Histone-H3) association with the core promoter was assed after treatment with 200 ng/ml TSA for 24 h in UM-SCC-1 via Q-ChIP. Sp1 and Ac-Histone-H3 relative quantities normalized to 1% input DNA and IgG controls. These data represent at least two-independent experiments. The error bars represent the standard deviations.
Mentions: Then, to assess whether the GC-Boxes 2 and 3 bind Sp1 for the regulation of the CerS1 promoter, we performed EMSA using nuclear extracts isolated from UM-SCC-1 cells, and the [32P]-labelled oligonucleotide (22-mer) containing a conserved Sp1-recognition sequence as a probe with and without unlabelled oligonucleotides containing the Sp1 (cold probe), GC-Boxes 1, 2 or 3 sequences or their mutated variants as competitors. Incubation of the labelled Sp1 probe with the UM-SCC-1 nuclear extracts generated two main shifted bands (complexes 1 and 2), which were competed off when unlabelled wt-probe, but not its mutant, were used as competitors (Fig 4A, lanes 2–4, respectively). As expected, co-incubation of the labelled Sp1 probe with the wt- or mutant GC-Box-1 oligonucleotides had no effect on probe binding with C1 or C2 compared to probe-only control (Fig 4A, lanes 5–6 and 2, respectively). Conversely, while unlabelled oligonucleotides containing wt-GC-Boxes 2 and 3 almost completely prevented the Sp1-C1/C2 binding, their mutated isoforms had no effect compared to probe-only control (Fig 4A, lanes 7–10, compared to lane 2, respectively). We also confirmed that C1/C2 complexes indeed associate with the Sp1 transcription factor using a super-shift assay in the presence of an anti-Sp1 or anti-IgG antibodies in EMSA assays. As seen in Fig 4B, pre-incubation of nuclear extracts with the anti-Sp1 antibody, but not the anti-IgG antibody, caused a super-shift on the mobility of the probe-protein complex (C1), indicating that the C1 contains Sp1-probe complex (Fig 4B). Taken together, both functional (promoter mutations) and biochemical (EMSA) studies suggest that Sp1 induces CerS1 core promoter with the specific involvement of GC-rich sequences within the −177 and −139 regions (GC-Boxes 2 and 3) of the promoter.

Bottom Line: Moreover, an alternatively spliced variant CerS1 mRNA (CerS1-2) was detected mainly in cancer cells or primary tumour tissues compared to controls, which was targeted by miR-574-5p for degradation.Interference with HDAC1 and miR-574-5p reconstituted CerS1-2 expression and C(18) -ceramide generation in multiple human cancer cell lines, which subsequently inhibited proliferation and anchorage-independent growth.Accordingly, knockdown of CerS1 partially protected cancer cells from MS-275/miR-574-5p siRNA-mediated growth inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.

Show MeSH
Related in: MedlinePlus