Mapping of Sp1 regulation sites in the promoter of the human alpha1-proteinase inhibitor gene.
Keratoconus is a potentially blinding disease that thins the central cornea. In afflicted corneas, the level of an inhibitor, alpha1-proteinase inhibitor (alpha1-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 alpha1-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 alpha1-PI promoter activity. In this study, 12 DNA segments, a series of 5', 3', and internal deletions of the 1.4-kb alpha1-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 alpha1-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 alpha1-PI. Mapping of the Sp1 sites may help elucidate the molecular pathway leading to the alterations observed in keratoconus.
Maruyama
Yasuhiro
Y
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612, USA.
Li
Yuhong
Y
Zhang
Yan
Y
Wang
Xinping
X
Sugar
Joel
J
Yue
Beatrice Y J T
BY
D000328:Adult; D001665:Binding Sites / Q000502:physiology; D002478:Cells, Cultured; D002648:Child; D003315:Cornea / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D007640:Keratoconus / Q000378:metabolism; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D017154:Stromal Cells / Q000166:cytology; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism
D016329:Sp1 Transcription Factor; D000515:alpha 1-Antitrypsin