Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene.

H Pöpperl,M S Featherstone

doi:10.1128/mcb.13.1.257

Abstract

Hox genes play an important role in the process of vertebrate pattern formation, and their expression is intricately regulated both temporally and spatially. All-trans-retinoic acid (RA), a physiologically active metabolite of vitamin A, affects the expression of a large number of Hox genes in vitro and in vivo. However, the regulatory mechanisms underlying the RA response of these genes have not been extensively studied, and no response element for RA receptors (RARs) has been characterized in a Hox regulatory region. The expression of murine Hox-4.2 and its human homolog, HOX4B, is increased in embryonal carcinoma (EC) cell lines upon RA treatment (M. S. Featherstone, A. Baron, S. J. Gaunt, M.-G. Mattei, and D. Duboule, Proc. Natl. Acad. Sci. USA 85:4760-4764, 1988; A. Simeone, D. Acampora, V. Nigro, A. Faiella, M. D'Esposito, A. Stornaiuolo, F. Mavilio, and E. Boncinelli, Mech. Dev. 33:215-228, 1991). Using transient expression assays, we showed that luciferase reporter gene constructs carrying genomic sequences located upstream of Hox-4.2 responded to RA in murine P19 EC cells. A 402-bp NcoI fragment was necessary for the RA responsiveness of reporter constructs. This fragment contained a regulatory element, 5'-AGGTGA(N)5AGGTCA-3', that closely resembles the consensus sequence for an RA response element. The Hox-4.2 RA response element was critical for the RA induction and specifically bound RARs. In addition, the response to RA could be inhibited by expressing a dominant negative form of RAR alpha in transfected P19 EC cells. These results suggested that Hox-4.2 is a target for RAR-mediated regulation by RA.

Highlights

McGill Cancer Centre '2 and Departments of Oncology2 and Medicine, 1,2 Division of Experimental Medicine, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G 1Y6
We tested whether a related sequence in the NcoI fragment could function as a vitamin D3 response element (VDRE) in a similar assay that led to the identification of the Hox-4.2 RA response element (RARE)
The mechanistic basis for the response in embryonal carcinoma (EC) cells or embryonic stem cells may include a direct action at the transcriptional level [31, 43, 47, 57], an indirect effect on transcription mediated by transcriptional regulators that are themselves induced by retinoic acid (RA) [47], and posttranscriptional mechanisms [6, 47]

Summary

MATERIALS AND METHODS

Mobility shift assays with nuclear extracts from P19 EC cells contained, in a volume of 20 pl, 10 ,g of protein, 300 ,ug of bovine serum albumin per ml, 8 mM Tris-HCl (pH 7.9), 12 mM HEPESKOH (pH 7.9), 80 mM KCl, 3 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 12% (vol/vol) glycerol, 2 ,g of poly(dI-dC), 4 x 104 to 5 x 104 cpm of double-stranded oligonucleotide For gel shift assays with whole cell extracts, the binding reaction was carried out in 25 pl containing 10 ,g of protein from P19 EC cells (nuclear or whole cell extracts) or 2.5 to 3 p,g of Cos cell protein, 280 p,g of bovine serum albumin per ml, 20 mM Tris-HCl (pH 7.9), 100 mM KCl, 3 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 12% (vol/vol) glycerol, 2.5 ,ug of poly(dI-dC), and double-stranded oligonucleotides as described above. The sequence of the NcoI fragment containing the Hox-4.2 RARE has been deposited in the EMBL data library under accession number X65950

RESULTS

DISCUSSION

RARE could affect the RA response shows that the