Ligand-dependent occupancy of the retinoic acid receptor β2 promoter in vivo

Anup Dey, Saverio Minucci, Keiko Ozato

Research output: Contribution to journalArticlepeer-review

Abstract

Retinoic acid (RA) activates transcription of the RA receptor β2 (RARβ2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (βRARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the βRARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXRβ, in which RXR heterodimers are unable to bind to the βRARE. Results indicate that binding of a liganded heterodimer receptor to the βRARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the βRARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RARβ2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.

Original languageEnglish
Pages (from-to)8191-8201
Number of pages11
JournalMolecular and Cellular Biology
Volume14
Issue number12
Publication statusPublished - Dec 1994

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

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