Cloning And Promoter Analysis Of Human Choline Kinase Isoforms
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Date
2012-09
Authors
Yee, Yoke Hiang
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Choline kinase (CK) is the first enzyme in the CDP-choline pathway for the synthesis of phosphatidylcholine in all animal tissues. CK phosphorylates choline into phosphocholine in the presence of ATP and Mg2+. In humans, this enzyme is encoded by two separate genes, ckα and ckβ which produce at least three protein isoforms known as CKα1, CKα2 and CKβ. CK plays an important role in phospholipid synthesis, carcinogenesis and muscular dystrophy as well as bone deformities. The CK promoter region plays a significant role in the regulation of the ck gene expression. This study reports the cloning of ckα and ckβ promoters and the use of a reporter system for evaluating the promoter activity. Promoter sequences of human ckα (2009 bp) and ckβ (2000 bp), located upstream of their respective genes, were cloned into a promoterless luciferase reporter vector, pGL4.10[luc2]. The recombinant plasmid was co-transfected with Renilla luciferase internal control vector, pGL4.73[hRluc/SV40] into the human breast adenocarcinoma, MCF-7, cell line. Its promoter activity was measured using the luciferase assay. Various 5’-terminal deletion mutants were constructed by PCR technique and cloned into pGL4.10[luc2] vector in order to identify the region of the ckα and ckβ promoters that are important for gene transcription. The results showed that the ETS transcription factor is a crucial negative regulator for the ckα promoter while ETS and GATA transcription factors are important negative regulators for the ckβ promoter activity. To confirm the importance of ETS and GATA on the regulation of ckβ gene transcription, several mutations were introduced to the ETS and GATA binding sites in ckβ promoter. The promoter activities of the mutant constructs were dramatically increased. Subsequently, MCF-7 cells transfected with ckβ promoter reporter vector were treated with phorbol 12-myristate 13-acetate (PMA) to explore the role of PMA in ckβ gene regulation via ETS and GATA transcription factors. PMA is the activator of PKC. The activation of PKC increases the binding of negative regulators, ETS and GATA and hence decreases the transcriptional activity of ckβ promoter. Results showed that the activity of the ckβ promoter was significantly reduced after treatment with PMA. Thus, this study has identified ETS and GATA transcription factors as the important repressors in the regulation of ckβ gene transcription.
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Keywords
Cloning And Promoter Analysis