Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells

Abstract

Background: Ethanolamine kinase (EK) catalyzes the phosphorylation of ethanolamine, the first step in the CDP-ethanolamine pathway for the biosynthesis of phosphatidylethanolamine (PE). Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied.

Methodology/principal findings: In this report, we mapped the important regulatory regions in the human ek1 promoter. 5' deletion analysis and site-directed mutagenesis identified a Sp site at position (-40/-31) that was essential for the basal transcription of this gene. Treatment of HCT116 cells with trichostatin A (TSA), a histone deacetylase inhibitor, significantly upregulated the ek1 promoter activity through the Sp(-40/-31) site and increased the endogenous expression of ek1. Chromatin immunoprecipitation assay revealed that TSA increased the binding of Sp1, Sp3 and RNA polymerase II to the ek1 promoter in HCT116 cells. The effect of TSA on ek1 promoter activity was cell-line specific as TSA treatment did not affect ek1 promoter activity in HepG2 cells.

Conclusion/significance: In conclusion, we showed that Sp1 and Sp3 are not only essential for the basal transcription of the ek1 gene, their accessibility to the target site on the ek1 promoter is regulated by histone protein modification in a cell line dependent manner.

Fig 1. Sequence analysis of ek1 5’-flanking region.

Underlined sequences are the transcription factor binding sites predicted by TFSEARCH and MatInspector 8.0. Translation start codon ATG is indicated with boldface and boxed.

Fig 2. Promoter activities of pGL4.10-ek1(-1966/+1) construct in HepG2, HCT116 and MCF-7 cells.

Each bar represents the mean ± SEM of triplicate samples from three independent experiments. (** p < 0.01; ***p < 0.001 vs. promoterless pGL4.10[luc2]).

Fig 3. Identification of important region for the ek1 promoter activity.

Promoter activities of luciferase reporter vector containing the ek1 promoter ranged from (A) -1966 to -229 and (B) -229 to -69. (C) The effect of Sp binding sites mutations in the pGL4.10-ek1(-69/+1) reporter construct. Schematic structures of the reporter construct are shown on the left. Binding sites for Sp transcription factors are indicated with open circles. The mutations introduced into the Sp binding sites are underlined and represented as closed circle. Each bar represents the mean ± SEM of triplicate samples from three independent experiments. (**p < 0.01; ***p < 0.001).

Fig 4. Effect of TSA on the activities of wild type and Sp(-40/-31) mutated ek1 minimal promoters.

A. Promoter activity of pGL4.10-ek1(-69/+1) reporter construct in HCT116 cells treated with the indicated concentrations of TSA for 24 hours (*p < 0.001; **p < 0.01; ***p < 0.05 vs. DMSO control; #p < 0.05, significant within TSA treatment group). B. Promoter activity of pGL4.10-ek1(-69/+1) reporter construct in HCT116 cells treated with 1 μM of TSA for the indicated time points (*p < 0.05 vs. DMSO control). C. Activities of wild type ek1 minimal promoter and Sp(-40/-31)-mutated ek1 minimal promoter after treatment with 1 μM of TSA for 24 hours. Each bar represents the mean ± SEM of triplicate samples from three independent experiments. D. Effect of TSA on ek1 gene expression in HCT116 cells. HCT116 cells were treated with 1 μM of TSA for 24 hours (**p < 0.01 vs. DMSO control). Each bar represents the mean ± SEM of triplicate samples from three independent experiments.

Fig 5. Effects TSA treatment on the binding of Sp1, Sp3 and RNA polymerase II to the ek1 minimal promoter region.

ChIP analysis was performed to confirm the interaction of (A) Sp proteins and (B) RNA polymerase II with the promoter under 1 μM TSA treatment for 24 hours. PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler^™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH primers) and N: pre-immune normal rabbit IgG (negative control).

Fig 6. Effect of TSA on the activity of wild type ek1 minimal promoter in HepG2 and HCT116 cells.

Cells were treated with 1 μM of TSA for 24 hours (**p < 0.01 vs. DMSO control). Each bar represents the mean ± SEM of triplicate samples from three independent experiments.

Fig 7. ChIP analysis of ek1 minimal promoter region for the binding of Sp1 and Sp3 in HCT116 and HepG2 cells.

PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler^™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH Primers) and N: pre-immune normal rabbit IgG (negative control).