Identification of deoxyribonucleic acid sequences that bind retinoid-X receptor-gamma with high affinity

Dowhan, D. H., Downes, M., Sturm, R. A. and Muscat, G. E. (1994) Identification of deoxyribonucleic acid sequences that bind retinoid-X receptor-gamma with high affinity. Endocrinology, 135 6: 2595-2607. doi:10.1210/en.135.6.2595

Author Dowhan, D. H.
Downes, M.
Sturm, R. A.
Muscat, G. E.
Title Identification of deoxyribonucleic acid sequences that bind retinoid-X receptor-gamma with high affinity
Journal name Endocrinology   Check publisher's open access policy
ISSN 0013-7227
Publication date 1994-12
Sub-type Article (original research)
DOI 10.1210/en.135.6.2595
Volume 135
Issue 6
Start page 2595
End page 2607
Total pages 13
Place of publication Bethesda, MA
Publisher Endocrine Society
Language eng
Subject 060199 Biochemistry and Cell Biology not elsewhere classified
Abstract The retinoid-X receptor (RXR) family (-alpha, -beta and -gamma) forms homodimers that bind to a number of retinoid-X response elements and trans-activate gene expression in a retinoid-dependent manner. Although, the RXRs are known to bind tandem direct repeats (DR) of the hexamer, RGGTCA, separated by 1 nucleotide, it is not known whether these represent the optimal and/or only recognition sequences. We, therefore, used a nonbiased strategy to identify sequences that efficiently bound RXR gamma, an isoform preferentially expressed in cardiac and skeletal muscle tissue. We performed binding site selection with bacterially expressed RXR gamma bound to glutathione-agarose and a pool of random sequences to derive a consensus DNA-binding site for RXR gamma. We analyzed a total of 41 individually selected oligonucleotides and found that RXR gamma bound with high affinity to motifs that were accommodated by the consensus AARGRNCAAAGGTCAA/cR. We observed that the majority of the sequences that formed complexes with RXR gamma in electrophoretic mobility shift analysis were DR-1 motifs; however, DR- motifs separated by 2, 4, and 8 nucleotides and a palindrome-0 motif were also demonstrated to interact with RXR gamma. Mutagenesis of the derived sequences indicated that both RGGTCA motifs were required for high affinity binding to RXR gamma. These derived sequences conferred appropriate 9-cis- and all-trans-retinoic acid (RA) responses to a thymidine kinase promoter. Furthermore, supershift experiments with a RXR antibody verified that these sequences specifically interacted with RXR in nuclear extracts derived from C2C12 muscle cells. In conclusion, this study rigorously defines the range of DR motifs that can recognize RXR and regulate gene expression in a RA-dependent fashion. The derived consensus accommodates retinoid-X response elements that have been identified in a diverse range of genes trans-activated by 9-cis-RA via the RXR family.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: UQ Diamantina Institute Publications
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Created: Tue, 20 Jul 2010, 15:16:28 EST by Laura McTaggart on behalf of UQ Diamantina Institute