Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo.
Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE 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 RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
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Dey
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Minucci
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Ozato
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D005786:Gene Expression Regulation / Q000187:drug effects; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014212:Tretinoin / Q000494:pharmacology*
D017931:DNA Primers; D008024:Ligands; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014212:Tretinoin