Activation of the growth-differentiation factor 11 gene by the histone deacetylase (HDAC) inhibitor trichostatin A and repression by HDAC3 - PubMed (original) (raw)
Activation of the growth-differentiation factor 11 gene by the histone deacetylase (HDAC) inhibitor trichostatin A and repression by HDAC3
Xiaohong Zhang et al. Mol Cell Biol. 2004 Jun.
Abstract
Histone deacetylase (HDAC) inhibitors inhibit the proliferation of transformed cells in vitro, restrain tumor growth in animals, and are currently being actively exploited as potential anticancer agents. To identify gene targets of the HDAC inhibitor trichostatin A (TSA), we compared the gene expression profiles of BALB/c-3T3 cells treated with or without TSA. Our results show that TSA up-regulates the expression of the gene encoding growth-differentiation factor 11 (Gdf11), a transforming growth factor beta family member that inhibits cell proliferation. Detailed analyses indicated that TSA activates the gdf11 promoter through a conserved CCAAT box element. A comprehensive survey of human HDACs revealed that HDAC3 is necessary and sufficient for the repression of gdf11 promoter activity. Chromatin immunoprecipitation assays showed that treatment of cells with TSA or silencing of HDAC3 expression by small interfering RNA causes the hyperacetylation of Lys-9 in histone H3 on the gdf11 promoter. Together, our results provide a new model in which HDAC inhibitors reverse abnormal cell growth by inactivation of HDAC3, which in turn leads to the derepression of gdf11 expression.
Figures
FIG. 1.
Induction of mouse and human gdf11 by TSA. Northern blot (or Western blot [D]) assays were performed using total RNA prepared from exponentially growing cells. Each blot was stripped and rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA to standardize quality and quantity of RNA. (A) BALB/c-3T3 cells received ethanol or 10 ng of TSA/ml for 12 h. (B) BALB/c-3T3 cells were treated with the indicated concentrations of TSA for 4 h (top panel) or 10 ng of TSA/ml for the indicated times (bottom panel). (C) HeLa and Flow 2000 cells were treated with the indicated concentrations of TSA for 4 h (top panel) or 400 ng of TSA/ml for the indicated times (bottom panel). (D) HeLa cells received 400 ng of TSA/ml for the indicated times. A Western blot assay was performed using an anti-Gdf11 polyclonal antibody. The blot was stripped and reprobed with an anti-β-actin antibody to ensure equal loading and transfer of proteins. (E) HeLa cells were exposed to 400 ng of TSA/ml or 5 mM nicotinamide for 6 h.
FIG. 2.
Activation of the human gdf11 promoter by TSA. (A) Plasmids containing different portions of the human gdf11 promoter were transiently transfected into HeLa cells, and luciferase activity was determined after 12 h of treatment with ethanol (control), 400 ng of TSA/ml, or 5 mM nicotinamide. Arrows represent the putative transcription initiation site on the gdf11 promoter. (B) HeLa cells were stably transfected with either pGL781SX (left panel) or pGL3Basic (right panel). Randomly selected clones were treated with ethanol or 400 ng of TSA/ml for 12 h, and luciferase activities were determined in triplicate. Error bars show standard deviations.
FIG. 3.
Analysis of TSA response elements in the human gdf11 promoter. (A) DNA sequence of a 381-bp human gdf11 promoter. These sequence data have been submitted to the DDBJ, EMBL, and GenBank databases under accession no. BK001652. MatInspector software (
) was used to identify potential TSA response elements (Sp1-binding sites and CCAAT boxes). Potential CCAAT boxes are underlined, and the TATA box is in bold. The arrow indicates the putative transcriptional initiation site. (B) Reporter plasmids containing different deletions and mutations of the gdf11 promoter were transiently transfected into HeLa cells, and luciferase activities were monitored 12 h after TSA treatment. CCAAT boxes on the promoters are illustrated by black ovals. An open oval with an X across it indicates a mutated CCAAT box. Error bars show standard deviations.
FIG. 4.
Repression of human gdf11 promoter by HDAC3. (A and B) pGL191 and plasmids that express Flag-tagged HDACs were cotransfected into HeLa cells. Luciferase assays were performed 72 h after transfection, and HDAC expression levels were monitored by Western blotting with an anti-Flag antibody (representative blots are shown). All transfections were normalized to equal amounts of DNA with parental expression vectors. Results are tabulated from the average of three independent transfections ± the standard deviation. (C) The pBS/U6 vector (control) or plasmids that express siHDAC1, siHDAC2, and siHDAC3 were cotransfected with pGL191 into HeLa cells. Luciferase assays were performed, and the data shown are the averages ± standard deviations from three separate experiments. Western blotting with anti-HDAC1, anti-HDAC2, anti-HDAC3, and anti-β-actin was done to monitor protein expression (representative blots are shown).
FIG. 5.
Enhanced H3K9 acetylation on the gdf11 promoter upon TSA treatment or HDAC3 silencing. (A) Core histones prepared from HeLa cells treated with 400 ng of TSA/ml (left panels) or transfected with plasmids expressing siRNAs (right panels) were Western blotted with antibodies against acetylated H3, H3K9, H3K18, H4, H4K8, and H4K12. A Coomassie-stained gel was prepared in parallel to assess the quality of each histone preparation. (B) Schematic diagram of the human gdf11 promoter (top panel). The arrows facing in opposite directions above the promoter denote the primers used in the ChIP assays. Cross-linked chromatin prepared from HeLa cells treated with (+) or without (−) TSA (middle panels) or transfected with siRNA expression plasmids (bottom panel) was precipitated with the indicated antibodies, and PCR analysis was performed using primers specific for the gdf11 promoter region.
FIG. 6.
Model of gdf11 activation by TSA.
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