Figure 3
Figure 3. Regulation of HDAC1 promoter by Sp1 transcription factor. (A) Schematic representations of HDAC1, HDAC2, and HDAC3 promoter constructs are shown. Relative locations of the putative binding sites of hematopoietic transcription factors are approximated by the symbols shown in the box. (B) Whole-cell lysates were prepared from MM cell lines and subjected to immunoblotting. HEK293 cells were transduced with expression vectors encoding Sp1, MZF-1, and C/EBPα, and used as positive controls. (C) We transfected 10 μg of pGL4.10 plasmid containing HDAC1 promoter sequences between −1170 and +397 into KMS12-BM cells along with 10 μg of expression vectors encoding Sp1 and MZF-1, and measured luciferase activities after 48 hours. HDAC1 promoter activity was calculated as firefly luciferase activities of cells transfected with an empty expression vector set at 1.0 after normalization of transfection efficiencies using Renilla luciferase activities. Data shown are the means ± SD of 3 independent experiments. P values were calculated by 1-way ANOVA with Student-Newman-Keuls multiple comparisons test. *P < .05. (D) KMS12-BM cells were cultured in the absence or presence of bortezomib for 2 days and subjected to ChIP assays. Chromatin suspensions were immunoprecipitated with the indicated antibodies and corresponding control antibodies. The resulting precipitants were subjected to PCR to amplify the promoter regions of the HDAC genes. The amplified products were visualized by ethidium bromide staining after 2% agarose gel electrophoresis. Representative data of 50 cycles are shown. Input indicates that PCR was performed with genomic DNA. (E) Whole-cell lysates were isolated simultaneously in the experiments described in Figure 2C, and subjected to immunoblotting. Arrows “a” and “b” indicate the intact and cleaved bands of Sp1, respectively. The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values setting day 0 controls to 1.0. (F) KMS12-BM cells were cultured with the indicated combinations of 8nM bortezomib (Bort), 100μM z-IETD-FMK (caspase-8 inhibitor), 100μM z-LEHD-FMK (caspase-9 inhibitor), 50μM z-DEVD-FMK (caspase-3 inhibitor), and 20μM z-ATAD-FMK (caspase-12 inhibitor) for 48 hours. Whole-cell lysates were subjected to immunoblotting. (G) Cell viability was determined with a Cell Counting Kit (WAKO) after culturing MM cells in the absence or presence of 8nM bortezomib (Bort) with or without either 100μM z-IETD-FMK (IETD) or 100μM z-LEHD-FMK (LEHD) for 48 hours. Absorbance at 450 nm was measured with a microplate reader, and expressed as a percentage of the value of the corresponding untreated cells. The means ± SD (bars) of 3 independent experiments are shown. P values were calculated by 1-way ANOVA with the Student-Newman-Keuls multiple comparisons test. *P < .05.

Regulation of HDAC1 promoter by Sp1 transcription factor. (A) Schematic representations of HDAC1, HDAC2, and HDAC3 promoter constructs are shown. Relative locations of the putative binding sites of hematopoietic transcription factors are approximated by the symbols shown in the box. (B) Whole-cell lysates were prepared from MM cell lines and subjected to immunoblotting. HEK293 cells were transduced with expression vectors encoding Sp1, MZF-1, and C/EBPα, and used as positive controls. (C) We transfected 10 μg of pGL4.10 plasmid containing HDAC1 promoter sequences between −1170 and +397 into KMS12-BM cells along with 10 μg of expression vectors encoding Sp1 and MZF-1, and measured luciferase activities after 48 hours. HDAC1 promoter activity was calculated as firefly luciferase activities of cells transfected with an empty expression vector set at 1.0 after normalization of transfection efficiencies using Renilla luciferase activities. Data shown are the means ± SD of 3 independent experiments. P values were calculated by 1-way ANOVA with Student-Newman-Keuls multiple comparisons test. *P < .05. (D) KMS12-BM cells were cultured in the absence or presence of bortezomib for 2 days and subjected to ChIP assays. Chromatin suspensions were immunoprecipitated with the indicated antibodies and corresponding control antibodies. The resulting precipitants were subjected to PCR to amplify the promoter regions of the HDAC genes. The amplified products were visualized by ethidium bromide staining after 2% agarose gel electrophoresis. Representative data of 50 cycles are shown. Input indicates that PCR was performed with genomic DNA. (E) Whole-cell lysates were isolated simultaneously in the experiments described in Figure 2C, and subjected to immunoblotting. Arrows “a” and “b” indicate the intact and cleaved bands of Sp1, respectively. The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values setting day 0 controls to 1.0. (F) KMS12-BM cells were cultured with the indicated combinations of 8nM bortezomib (Bort), 100μM z-IETD-FMK (caspase-8 inhibitor), 100μM z-LEHD-FMK (caspase-9 inhibitor), 50μM z-DEVD-FMK (caspase-3 inhibitor), and 20μM z-ATAD-FMK (caspase-12 inhibitor) for 48 hours. Whole-cell lysates were subjected to immunoblotting. (G) Cell viability was determined with a Cell Counting Kit (WAKO) after culturing MM cells in the absence or presence of 8nM bortezomib (Bort) with or without either 100μM z-IETD-FMK (IETD) or 100μM z-LEHD-FMK (LEHD) for 48 hours. Absorbance at 450 nm was measured with a microplate reader, and expressed as a percentage of the value of the corresponding untreated cells. The means ± SD (bars) of 3 independent experiments are shown. P values were calculated by 1-way ANOVA with the Student-Newman-Keuls multiple comparisons test. *P < .05.

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