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J. Biol. Chem., Vol. 282, Issue 35, 25322-25337, August 31, 2007

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Cloning and Characterization of the Rat Lysyl Oxidase Gene Promoter

IDENTIFICATION OF CORE PROMOTER ELEMENTS AND FUNCTIONAL NUCLEAR FACTOR I-BINDING SITES^*

Song Gao, Yinzhi Zhao, Lingfa Kong, Paul Toselli, Iih-Nan Chou, Phillip Stone, and Wande Li¹

From the Departments of Biochemistry and Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118

Lysyl oxidase (LO) stabilizes the extracellular matrix by cross-linking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5'-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from –78 to –51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5' Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region –709/–598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment –612/–580, which contains a nuclear factor I (NFI)-binding site at the region –594/–580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at –147/–133, were identified in the LO promoter region –804/–1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation.

Received for publication, October 30, 2006 , and in revised form, April 30, 2007.

^* This work was supported by National Institutes of Health Research Grant R01-ES 11340 and the External Research Program of Philip Morris USA and Philip Morris International. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, 715 Albany St., K121, Boston, MA 02118. Tel.: 617-638-5485; Fax: 617-6385339; E-mail: wandeli{at}bu.edu.

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