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Mapping of Sp1 regulation sites in the promoter of the human α1‐proteinase inhibitor gene
Author(s) -
Maruyama Yasuhiro,
Li Yuhong,
Zhang Yan,
Wang Xinping,
Sugar Joel,
Y.J.T. Yue Beatrice
Publication year - 2002
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.10149
Subject(s) - microbiology and biotechnology , biology , transfection , sp1 transcription factor , gene , reporter gene , promoter , transcription (linguistics) , expression vector , transcriptional regulation , gene expression , genetics , recombinant dna , linguistics , philosophy
Keratoconus is a potentially blinding disease that thins the central cornea. In afflicted corneas, the level of an inhibitor, α1‐proteinase inhibitor (α1‐PI), is found reduced. An increased expression of transcription factor Sp1 is also demonstrated. To examine the role of Sp1 in regulation of the human α1‐PI gene, a 1.4‐kb (−1397/+9) 5′‐flanking promoter sequence that contains 10 Sp1 sites was cloned. Previous transient transfection experiments showed that Sp1 expression indeed repressed the α1‐PI promoter activity. In this study, 12 DNA segments, a series of 5′, 3′, and internal deletions of the 1.4‐kb α1‐PI promoter sequence, were ligated into the SEAP (secreted alkaline phosphatase) reporter gene vector and transfected into human corneal stromal cells. Co‐transfection with a Sp1 expression vector pPacSp1 was also performed in parallel. The SEAP enzyme activity was assayed. A fragment with 489 bp (−480/+9) of the 3′ sequence, and three fragments with internal deletions, were found to confer a majority of the full promoter activity. Other deletions significantly abolished the promoter activity. Site‐directed mutagenesis experiments further revealed that the most proximal Sp1 site (−100/−87) may be an essential element involved in the negative regulation of α1‐PI promoter activity by Sp1. Interaction between the proximal and distal Sp1 sites also seemed to be important. These results provide the first in‐depth characterization of the transcription mechanisms regulating the expression of α1‐PI. Mapping of the Sp1 sites may help elucidate the molecular pathway leading to the alterations observed in keratoconus. J. Cell. Biochem. 85: 482–489, 2002. © 2002 Wiley‐Liss, Inc.