DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells.

Siti Aisyah Faten Mohamed Sa’Dom,Shaharum Shamsuddin,Ling Ling Few,Wei Cun See Too,Sweta Raikundalia

doi:10.3390/genes12060853

Siti Aisyah Faten Mohamed Sa’Dom, Shaharum Shamsuddin + Show 3 more

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https://doi.org/10.3390/genes12060853

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Journal: Genes	Publication Date: Jun 1, 2021
Citations: 1	License type: CC BY 4.0

Affiliation: Universiti Sains Malaysia

Abstract

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

Highlights

Choline kinase (CK) is a cytosolic enzyme in the CDP-choline pathway that catalyzes the phosphorylation of choline to phosphocholine for the biosynthesis of phosphatidylcholine, the major phospholipid of eukaryotic cell membranes [1,2]
The results suggest that the transcriptional control of cka gene involves DNA methylation of CpG islands located in the promoter region
Our analysis showed that cka promoter lacks CAAT box and TATA box and contains two core promoter elements, namely the downstream promoter element (DPE) and Bridge element, which are a typical characteristic of GC-rich promoters (Figure 1d)

Summary

Introduction

Choline kinase (CK) is a cytosolic enzyme in the CDP-choline pathway that catalyzes the phosphorylation of choline to phosphocholine for the biosynthesis of phosphatidylcholine, the major phospholipid of eukaryotic cell membranes [1,2]. Increased activity of CKα isoform and higher levels of PCho have been implicated in human carcinogenesis. Ckα gene transcription was first reported to be regulated by hypoxia via the binding of HIF-1α transcription factor to the hypoxia response element-7 (HRE7) in the promoter [11]. It was demonstrated that KDM2B binding to ckα promoter represses the expression of this gene [13]. Post-transcriptional regulation of ckα gene expression involving miRNAs has been reported. DNA methylation is the most well-studied epigenetic mechanism that is involved in diverse cellular functions, including the silencing of transposable elements, inactivation of viral sequences, maintenance of chromosomal integrity, X-chromosome inactivation, and transcriptional suppression of a large number of genes [17,18]. Aberrant methylation levels have been postulated to inactivate tumor suppressors and activate oncogenes, which lead to carcinogenesis [21]

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