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

Chee Sian Kuan,Wei Cun See Too,Ling Ling Few,Klaus Roemer

doi:10.1371/journal.pone.0147886

Abstract

BackgroundEthanolamine 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 FindingsIn 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/SignificanceIn 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.

Highlights

Phosphatidylethanolamine (PE) is the second most abundant phospholipid in eukaryotic cells [1]
The most striking feature of the ek1 promoter is the lack of a typical CAAT or TATA box, which is a common characteristic of GC-rich promoters
The results showed that in HepG2 cells, Sp1 and Sp3 transcription factors were already bound to the basal ek1 promoter without trichostatin A (TSA) treatment and this explained why the promoter activity was not affected by TSA as in the case of HCT116 cells

Summary

Introduction

Phosphatidylethanolamine (PE) is the second most abundant phospholipid in eukaryotic cells [1]. EK exists as three isoforms, encoded by two separate genes, named ek1 [NCBI Gene ID: 55500] and ek2 [NCBI Gene ID: 55224]. Induction of EK activity by oncogenes and carcinogens suggested the involvement of EK in carcinogenesis by promoting cell growth and/or survival [4,5,6]. Increased phosphorylation of ethanolamine and its analogs (methylethanolamine and dimethylethanolamine) in EK overexpressor cells increases cell survival in serum-free medium but decreases the insulin-dependent DNA synthesis [8]. 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 ek and ek. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied

Objectives

Methods

Results

Conclusion