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

Qiang Shen, Pomila Singh

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 25bp 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 7bp at -861/-855; binding of the purified Sp3 protein to the 25bp 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.

Original languageEnglish (US)
Pages (from-to)2454-2464
Number of pages11
JournalOncogene
Volume23
Issue number14
DOIs
StatePublished - Apr 1 2004

Fingerprint

Transcription Factor AP-1
Binding Sites
Genes
Mutation
Gene Expression
Insulin-Like Growth Factor Binding Protein 4
Proteins
Colonic Neoplasms
Insects
Up-Regulation
Cell Line
Antibodies

Keywords

  • AP-1 binding site
  • c-Jun
  • Colon cancer cells
  • IGFBP4 promoter
  • JunD
  • Sp3 binding site

ASJC Scopus subject areas

  • Molecular Biology
  • Cancer Research
  • Genetics

Cite this

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. / Shen, Qiang; Singh, Pomila.

In: Oncogene, Vol. 23, No. 14, 01.04.2004, p. 2454-2464.

Research output: Contribution to journalArticle

@article{abd355f55ca6468e95b67048fd0c643a,
title = "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",
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 25bp 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 7bp at -861/-855; binding of the purified Sp3 protein to the 25bp 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.",
keywords = "AP-1 binding site, c-Jun, Colon cancer cells, IGFBP4 promoter, JunD, Sp3 binding site",
author = "Qiang Shen and Pomila Singh",
year = "2004",
month = "4",
day = "1",
doi = "10.1038/sj.onc.1207354",
language = "English (US)",
volume = "23",
pages = "2454--2464",
journal = "Oncogene",
issn = "0950-9232",
publisher = "Nature Publishing Group",
number = "14",

}

TY - JOUR

T1 - Identification of a novel SP3 binding site in the promoter of human IGFBP4 gene

T2 - Role of SP3 and AP-1 in regulating promoter activity in CaCo2 cells

AU - Shen, Qiang

AU - Singh, Pomila

PY - 2004/4/1

Y1 - 2004/4/1

N2 - 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 25bp 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 7bp at -861/-855; binding of the purified Sp3 protein to the 25bp 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.

AB - 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 25bp 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 7bp at -861/-855; binding of the purified Sp3 protein to the 25bp 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.

KW - AP-1 binding site

KW - c-Jun

KW - Colon cancer cells

KW - IGFBP4 promoter

KW - JunD

KW - Sp3 binding site

UR - http://www.scopus.com/inward/record.url?scp=1942468796&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1942468796&partnerID=8YFLogxK

U2 - 10.1038/sj.onc.1207354

DO - 10.1038/sj.onc.1207354

M3 - Article

VL - 23

SP - 2454

EP - 2464

JO - Oncogene

JF - Oncogene

SN - 0950-9232

IS - 14

ER -