Identification of a retinoic acid responsive element in the retinoic acid receptor & beta;gene

Abstract

Retinoic acid, the first morphogen described so far in vertebrates, is a vitamin A derivative which exerts striking effects on development and differentiation. The identification of three retinoic acid receptors as members of the nuclear receptor super-family provides an explantation for the molecular action of morphogens on gene expression. Functional analysis of the receptors requires the identification of target genes and of their cis-acting retinoic acid-responsive elements. We have previously shown that the retinoic acid receptor beta gene is transcriptionally up-regulated by retinoic acid and now report the characterization of a functional retinoic acid responsive element in the beta gene that mediates trans-activation by retinoic acid. Using deletion mapping, we have identified a 27-base pair fragment, located 59 base pairs upstream of the transcriptional start, which confers retinoic acid responsiveness on the herpes virus thymidine kinase promoter. This sequence contains a perfect direct repeat of the motif GTTCAC, which is reminiscent of the 5' half-palindrome of the thyroid and oestrogen hormone responsive elements. Specific binding of the beta protein to the retinoic acid responsive element is demonstrated and is independent of the presence of retinoic acid. Both alpha and beta receptors enhance retinoic acid response in CV1 cells, indicating that they can both act through the same DNA sequence.

Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor β2 promoter in breast cancer cells

“…The RARb2 promoter is methylated in breast cancer cell lines independently of their ER status and RA-inducibility RARb transcription was ®rst tested in a panel of breast cancer cell lines grown in the absence of exogenous RA, by reverse transcriptase-PCR (RT ± PCR), using primers encompassing exons 5 and 6 (de The ' et al, 1990;van der Leede et al, 1992;Toulouse et al, 1997). Under these conditions, only one cell line, Hs578t, produced a detectable 256 bp RT ± PCR product ( Figure 4a).…”

“…The exon 5 (sense primer 5'-GAC TGT ATG GAT GTT CTG TCA G-3') and exon 6 (antisense primer 5'-ATT TGT CCT GGC AGA CGA AGC A-3') were designed on the basis of published RARb2 transcript (de The' et al, 1990;van der Leede et al, 1992) and used to amplify 50 ng of DNase treated total RNA using the Superscript One-Step RT ± PCR System (Life Technologies). RT ± PCR with actin primers (sense primer 5'-ACC ATG GAT GAT GAT ATC G-3' and antisense primer 5'-ACA TGG CTG GGG TGT TGA AG-3' was used as an internal RNA control.…”

“…A complex regulatory region, with two promoters regulates RARb gene expression. Only one promoter, RARb2, containing several RA-response elements, including a canonical and an auxiliary RA response element, bRARE (de The ' et al, 1990;Valcarel et al, 1994) is active in human mammary epithelial cells (HMEC). The transcription of the RARb2 promoter is mediated by multiple RARs including, RARa and RARb itself (Chiba et al, 1997) able to recruit coactivator and corepressor protein complexes with HAT/HDAC activities, respectively (Chambon, 1996).…”

Functional specificity of the two retinoic acid receptor RAR and RXR families in myogenesis

“…Before using these mutant receptors (designated in this work as dnRAR and dnRXR) to block the action of endogenous retinoic acid receptors in myogenesis, we ®rst tested their dominant negative activity following transient transfection into C2 myogenic cells. For this purpose, C2 cells were cotransfected with the expression vectors encoding dnRAR or dnRXR receptors (pSG5-dnRAR or pSG5-dnRXR) and a reporter construct containing the classical retinoic-acid response element (RAREb) (de The et al, 1990;Ho man et al, 1990) in front of the thymidine kinase promoter linked to the chloramphenicol-acetyl transferase gene (tk-CAT) (see Materials and methods) ( Figure 1). As shown in Figure 1, treatment of pSG5-transfected C2 cells with RA increased RAREb-tk-CAT reporter activity by 2 ± 3-fold.…”

“…Furthermore, the RAREb response element has been described and characterized in the regulatory region of RARb gene (de The et al, 1990;Ho man et al, 1990). In C2-neo control cells, RARb mRNA was undetectable before RA-treatment but was strongly We next compared the RA-regulation of MyoD and myogenin gene expression over a time course period of RA treatment for C2-neo, C2-dnRAR1 and C2-dnRXR1 cells.…”