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Journal of Biochemistry Advance Access originally published online on October 3, 2008
Journal of Biochemistry 2008 144(6):741-752; doi:10.1093/jb/mvn129
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Identification and Characterization of a Positive Regulatory cis-element Within the Upstream Region of c-jun

Shivani Agarwal, Raj Kumar*, Preeti Gupta and Aparna Dixit{dagger}

Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

{dagger}To whom correspondence should be addressed. Tel: +91-11-26742164/26704085, Fax: +91-11-26717561, E-mail: adix2100{at}mail.jnu.ac.in; adixit7{at}yahoo.com; adixit7{at}hotmail.com

Received August 31, 2008; Accepted September 15, 2008


  Abstract

Jun oncoprotein, a component of transcription factor AP-1, is responsible for expression of multiple genes which trigger cell proliferation, differentiation and apoptosis. The diverse regulatory roles that c-Jun plays point towards its varied and complex transcriptional regulation under different physiological conditions. This could possibly involve an interplay of different trans-acting factors and multiple cis-acting elements present in the c-jun. It is, therefore, important to identify such regulatory elements that could be involved in its stringent up- or down-regulation. In the present study, transient transfection analysis with stepwise deletion variants of the upstream region (–563 to –273), linked to basal c-jun promoter in a reporter plasmid revealed the existence of multiple regulatory regions that modulated gene expression. Further delineation of the region, conferring positive regulation of transcription from the c-jun promoter, led to the identification of a cis-acting element spanning –538 to –514 region. The trans-acting factor(s) present in rat liver nuclear extract interacted specifically only in the phosphorylated form, and with a high affinity to the recognition sequence. UV cross-linking and South-western blotting, in conjunction with the analysis of affinity purified proteins interacting with this region, revealed that at least two protein factors of ~45 kDa and ~34 kDa bind specifically to this region.

Key Words: c-jun, cis-acting element, EMSA, positive regulatory factor, transcriptional regulation

Abbreviations: AP-1, activator protein-1; RNE-d, rat liver nuclear extract fraction-d; EMSA, electrophoretic mobility shift assay; SS-DNA, salmon sperm DNA; CIAP, calf intestinal alkaline phosphatase; RLjunRP, rat liver jun regulatory protein

*Present address: Raj Kumar, Division of Health and Reproductive Sciences, Lexington, KY 40202, USA


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