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Identification of a novel SP3 binding site in the promoter of human IGFBP4 gene: role of SP3 and AP-1 in regulating promoter activity in CaCo2 cells

Oncogene volume 23, pages 2454–2464 (2004)Cite this article

Abstract

Insulin-like growth factor binding protein 4 (IGFBP4/BP4) gene expression plays an important role in the transition from proliferation to differentiation of a human colon cancer cell line, CaCo2. We recently cloned and identified multiple cis elements (including putative binding sites for activator protein 1 (AP-1) and specificity proteins (Sps)) in the promoter of human BP4 gene, and measured a significant upregulation of the promoter activity in response to c-Jun. We therefore examined the role of the single AP-1 site (−869/−863) and other cis elements, in regulating the expression of hBP4 gene, in the current studies. Deletion of a 25 bp sequence from −872 to −848, which contains the AP-1 site, significantly reduced BP4 promoter activity by approximately 50%. Surprisingly, mutation of the AP-1 site did not produce significant alteration in the activity of the BP4 promoter. However, mutation of 7 bp (5′-TGCTGCA) at the 3′ end of the AP-1 site resulted in significantly decreasing the promoter activity by >50%. Proteins bound to the 25 bp probe (−872/−848) could be supershifted by antibodies specific for JunD and Sp3 in an EMSA. JunD binding was abolished on mutation of the AP-1 site and Sp3 binding was abolished on mutation of the 7 bp at −861/−855; binding of the purified Sp3 protein to the 25 bp probe was similarly abolished on mutation of the newly discovered Sp3 binding site (TGCTGCA). BP4 promoter activity was upregulated in insect cells in response to Sp3 expression, confirming a functional importance of the novel Sp3 binding site. These studies suggest that the Sp3 binding site, rather than the AP-1 site, may be playing a significant role in regulating the expression of IGFBP4 gene in CaCo2 cells.

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Abbreviations

AP-1:

activator protein 1

β-gal:

β-galactosidase

bp:

base pair

BP4:

Insulin-like growth factor binding protein 4

ds:

double stranded

EMSA:

electrophoretic mobility shift assay

IGF:

insulin-like growth factor

IGFBP:

IGF binding protein

luc:

luciferase

mut:

mutant

NE:

nuclear extract

nt:

nucleotide

PCR:

polymerase chain reaction

Sp:

specificity protein

wt:

wild type

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Acknowledgements

These studies were supported by grants from the National Cancer Institute (CA 60087 and CA 72992) to PS. Part of this study was published in its preliminary form as an abstract in Program and Abstracts of the Endocrine Society's 83rd Annual Meeting, June 20–23, 2001, Denver, CO. We thank Drs Powel Brown, Rafeal Herrara and Shiming Jiang from Baylor College of Medicine for their critical comments and valuable suggestions. The technical support of Azar Owlia is gratefully acknowledged and secretarial assistance of Pat Gazzoli is acknowledged.

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  1. Department of Anatomy & Neurosciences, The University of Texas Medical Branch, Galveston, 77555-1043, TX, USA

    Qiang Shen & Pomila Singh

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Correspondence to Pomila Singh.

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Shen, Q., Singh, P. Identification of a novel SP3 binding site in the promoter of human IGFBP4 gene: role of SP3 and AP-1 in regulating promoter activity in CaCo2 cells. Oncogene 23, 2454–2464 (2004). https://doi.org/10.1038/sj.onc.1207354

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