Cytochrome P450RAI(CYP26) promoter: A distinct composite retinoic acid response element underlies the complex regulation of retinoic acid metabolism

Olivier Loudig, Charolyn Babichuk, Jay White, Suzan Abu-Abed, Chris Mueller, Martin Petkovich

Research output: Contribution to journalArticle

149 Citations (Scopus)

Abstract

The catabolism of retinoic acid (RA) is an essential mechanism for restricting the exposure of specific tissues and cells to RA. We recently reported the identification of a RA-inducible cytochrome P450 [P450RAI(CYP26)], in zebrafish, mouse, and human, which was shown to be responsible for RA catabolism. P450RAI exhibits a complex spatiotemporal pattern of expression during development and is highly inducible by exogenous RA treatment in certain tissues and cell lines. Sequence analysis of the proximal upstream region of the P450RAI promoter revealed a high degree of conservation between zebrafish, mouse, and human. This region of the promoter contains a canonical retinoic acid response element (5'-AGT-TCA-(n)5-AGTTCA-3'), embedded within a 32-bp region (designated R1), which is conserved among all three species. Electrophoretic mobility shift assays using this element demonstrated the specific binding of routine retinoic acid receptor-γ (RARγ) and retinoid X receptor-α (RXRα) proteins. Transient transfection experiments with the mouse P450RAI promoter fused to a luciferase reporter gene showed transcriptional activation in the presence of RA in HeLa, Cos-1, and F9 wild-type cells. This activation, as well as basal promoter activity, was abolished upon mutation of the RARE. Deletion and mutational analyses of the P450RAI promoter, as well as DNase I footprinting studies, revealed potential binding sites for several other proteins in conserved regions of the promoter. Also, two conserved 5'-TAAT-3' sequences flanking the RARE were investigated for their potential importance in P450RAI promoter activity. Moreover, these studies revealed an essential requirement for a G-rich element (designated GGRE), located just upstream of the RARE, for RA inducibility. This element was demonstrated to form complexes with Sp1 and Sp3 using nuclear extracts from either murine F9 or P19 cells. Together, these results indicate that the P450RAI-RARE is atypical in that conserved flanking sequences may play a very important role in regulating RA inducibility and expression of P450RAI(CYP26).

Original languageEnglish (US)
Pages (from-to)1483-1497
Number of pages15
JournalMolecular Endocrinology
Volume14
Issue number9
StatePublished - 2000
Externally publishedYes

Fingerprint

Response Elements
Cytochromes
Tretinoin
Genetic Promoter Regions
Zebrafish
3' Flanking Region
Retinoid X Receptors
Retinoic Acid 4-Hydroxylase
Retinoic Acid Receptors
Conserved Sequence
Deoxyribonuclease I
Electrophoretic Mobility Shift Assay
Luciferases
Reporter Genes
Transcriptional Activation
Transfection
Sequence Analysis
Proteins
Binding Sites
Cell Line

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Cytochrome P450RAI(CYP26) promoter : A distinct composite retinoic acid response element underlies the complex regulation of retinoic acid metabolism. / Loudig, Olivier; Babichuk, Charolyn; White, Jay; Abu-Abed, Suzan; Mueller, Chris; Petkovich, Martin.

In: Molecular Endocrinology, Vol. 14, No. 9, 2000, p. 1483-1497.

Research output: Contribution to journalArticle

Loudig, O, Babichuk, C, White, J, Abu-Abed, S, Mueller, C & Petkovich, M 2000, 'Cytochrome P450RAI(CYP26) promoter: A distinct composite retinoic acid response element underlies the complex regulation of retinoic acid metabolism', Molecular Endocrinology, vol. 14, no. 9, pp. 1483-1497.
Loudig, Olivier ; Babichuk, Charolyn ; White, Jay ; Abu-Abed, Suzan ; Mueller, Chris ; Petkovich, Martin. / Cytochrome P450RAI(CYP26) promoter : A distinct composite retinoic acid response element underlies the complex regulation of retinoic acid metabolism. In: Molecular Endocrinology. 2000 ; Vol. 14, No. 9. pp. 1483-1497.
@article{ef9d661437394b7aadf0836107115868,
title = "Cytochrome P450RAI(CYP26) promoter: A distinct composite retinoic acid response element underlies the complex regulation of retinoic acid metabolism",
abstract = "The catabolism of retinoic acid (RA) is an essential mechanism for restricting the exposure of specific tissues and cells to RA. We recently reported the identification of a RA-inducible cytochrome P450 [P450RAI(CYP26)], in zebrafish, mouse, and human, which was shown to be responsible for RA catabolism. P450RAI exhibits a complex spatiotemporal pattern of expression during development and is highly inducible by exogenous RA treatment in certain tissues and cell lines. Sequence analysis of the proximal upstream region of the P450RAI promoter revealed a high degree of conservation between zebrafish, mouse, and human. This region of the promoter contains a canonical retinoic acid response element (5'-AGT-TCA-(n)5-AGTTCA-3'), embedded within a 32-bp region (designated R1), which is conserved among all three species. Electrophoretic mobility shift assays using this element demonstrated the specific binding of routine retinoic acid receptor-γ (RARγ) and retinoid X receptor-α (RXRα) proteins. Transient transfection experiments with the mouse P450RAI promoter fused to a luciferase reporter gene showed transcriptional activation in the presence of RA in HeLa, Cos-1, and F9 wild-type cells. This activation, as well as basal promoter activity, was abolished upon mutation of the RARE. Deletion and mutational analyses of the P450RAI promoter, as well as DNase I footprinting studies, revealed potential binding sites for several other proteins in conserved regions of the promoter. Also, two conserved 5'-TAAT-3' sequences flanking the RARE were investigated for their potential importance in P450RAI promoter activity. Moreover, these studies revealed an essential requirement for a G-rich element (designated GGRE), located just upstream of the RARE, for RA inducibility. This element was demonstrated to form complexes with Sp1 and Sp3 using nuclear extracts from either murine F9 or P19 cells. Together, these results indicate that the P450RAI-RARE is atypical in that conserved flanking sequences may play a very important role in regulating RA inducibility and expression of P450RAI(CYP26).",
author = "Olivier Loudig and Charolyn Babichuk and Jay White and Suzan Abu-Abed and Chris Mueller and Martin Petkovich",
year = "2000",
language = "English (US)",
volume = "14",
pages = "1483--1497",
journal = "Molecular Endocrinology",
issn = "0888-8809",
publisher = "The Endocrine Society",
number = "9",

}

TY - JOUR

T1 - Cytochrome P450RAI(CYP26) promoter

T2 - A distinct composite retinoic acid response element underlies the complex regulation of retinoic acid metabolism

AU - Loudig, Olivier

AU - Babichuk, Charolyn

AU - White, Jay

AU - Abu-Abed, Suzan

AU - Mueller, Chris

AU - Petkovich, Martin

PY - 2000

Y1 - 2000

N2 - The catabolism of retinoic acid (RA) is an essential mechanism for restricting the exposure of specific tissues and cells to RA. We recently reported the identification of a RA-inducible cytochrome P450 [P450RAI(CYP26)], in zebrafish, mouse, and human, which was shown to be responsible for RA catabolism. P450RAI exhibits a complex spatiotemporal pattern of expression during development and is highly inducible by exogenous RA treatment in certain tissues and cell lines. Sequence analysis of the proximal upstream region of the P450RAI promoter revealed a high degree of conservation between zebrafish, mouse, and human. This region of the promoter contains a canonical retinoic acid response element (5'-AGT-TCA-(n)5-AGTTCA-3'), embedded within a 32-bp region (designated R1), which is conserved among all three species. Electrophoretic mobility shift assays using this element demonstrated the specific binding of routine retinoic acid receptor-γ (RARγ) and retinoid X receptor-α (RXRα) proteins. Transient transfection experiments with the mouse P450RAI promoter fused to a luciferase reporter gene showed transcriptional activation in the presence of RA in HeLa, Cos-1, and F9 wild-type cells. This activation, as well as basal promoter activity, was abolished upon mutation of the RARE. Deletion and mutational analyses of the P450RAI promoter, as well as DNase I footprinting studies, revealed potential binding sites for several other proteins in conserved regions of the promoter. Also, two conserved 5'-TAAT-3' sequences flanking the RARE were investigated for their potential importance in P450RAI promoter activity. Moreover, these studies revealed an essential requirement for a G-rich element (designated GGRE), located just upstream of the RARE, for RA inducibility. This element was demonstrated to form complexes with Sp1 and Sp3 using nuclear extracts from either murine F9 or P19 cells. Together, these results indicate that the P450RAI-RARE is atypical in that conserved flanking sequences may play a very important role in regulating RA inducibility and expression of P450RAI(CYP26).

AB - The catabolism of retinoic acid (RA) is an essential mechanism for restricting the exposure of specific tissues and cells to RA. We recently reported the identification of a RA-inducible cytochrome P450 [P450RAI(CYP26)], in zebrafish, mouse, and human, which was shown to be responsible for RA catabolism. P450RAI exhibits a complex spatiotemporal pattern of expression during development and is highly inducible by exogenous RA treatment in certain tissues and cell lines. Sequence analysis of the proximal upstream region of the P450RAI promoter revealed a high degree of conservation between zebrafish, mouse, and human. This region of the promoter contains a canonical retinoic acid response element (5'-AGT-TCA-(n)5-AGTTCA-3'), embedded within a 32-bp region (designated R1), which is conserved among all three species. Electrophoretic mobility shift assays using this element demonstrated the specific binding of routine retinoic acid receptor-γ (RARγ) and retinoid X receptor-α (RXRα) proteins. Transient transfection experiments with the mouse P450RAI promoter fused to a luciferase reporter gene showed transcriptional activation in the presence of RA in HeLa, Cos-1, and F9 wild-type cells. This activation, as well as basal promoter activity, was abolished upon mutation of the RARE. Deletion and mutational analyses of the P450RAI promoter, as well as DNase I footprinting studies, revealed potential binding sites for several other proteins in conserved regions of the promoter. Also, two conserved 5'-TAAT-3' sequences flanking the RARE were investigated for their potential importance in P450RAI promoter activity. Moreover, these studies revealed an essential requirement for a G-rich element (designated GGRE), located just upstream of the RARE, for RA inducibility. This element was demonstrated to form complexes with Sp1 and Sp3 using nuclear extracts from either murine F9 or P19 cells. Together, these results indicate that the P450RAI-RARE is atypical in that conserved flanking sequences may play a very important role in regulating RA inducibility and expression of P450RAI(CYP26).

UR - http://www.scopus.com/inward/record.url?scp=0033743405&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033743405&partnerID=8YFLogxK

M3 - Article

C2 - 10976925

AN - SCOPUS:0033743405

VL - 14

SP - 1483

EP - 1497

JO - Molecular Endocrinology

JF - Molecular Endocrinology

SN - 0888-8809

IS - 9

ER -