Thyroid and steroid hormone response elements

Evidence for direct transcriptional regulation of the gene by retinoic acid prompted the search for cognate binding sites (Rodaway et al, 1990). Retinoid receptors are members of the steroid receptor superfamily of proteins that function as ligand- dependent transcription factors, and includes steroids, thyroid hormones, and vitamin D3 (Green, 1993). Retinoic acid exerts its effects through two classes of receptor, the retinoic acid receptor (RAR), and retinoid X receptors (RXR). This classification is based on differences in primary structure, sensitivity to synthetic ligands, and ability to regulate expression of different target genes. Each receptor sub-family consists of several isoforms referred to as RAR or RXR a , p, and y, suggesting that they are involved in different processes. The RAR family are activated by all-trans and 9-cis RA, whereas the RXR family are activated exclusively by 9-cis RA (Heyman et al, 1992). Unlike oestrogen and glucocorticoid receptors which form homodimers and recognise short palindromic DNA sequences, the vitamin D receptor (VDR), thyroid hormone receptor (TR), and RAR all bind to a direct repeat of the consensus GGTCA usually as a heterodimer with RXR (Beato, 1989; Umesono et al. 1991; Yu et al. 1991; Kliewer et al. 1992). The spacing of the direct repeat determines binding preference, and although it was originally proposed that each heterodimer acted through a unique direct repeat (VDR/RXR, 3bp; TR/RXR, 4bp; RAR/RXR, 5bp), there now appears to be considerable degeneracy (Naar et al, 1991).

Four repeated motifs GGTC/GA with exactly 13 bp between each core consensus are therefore excellent candidate binding sites for members of the nuclear hormone superfamily. The arrangement o f the repeats particularly resembles response elements for RA, D3 and thyroid hormone (T3). However, either in the context of the natural promoter, or a heterologous promoter, these elements were unable to confer RA, D3 or T3 inducibility to a reporter gene in myeloid cells. Similarly, in CVl or F9 cells, co­ transfection o f RAR, RXR, or VDR receptors did not support induction of reporter gene expression. It is therefore unlikely that these motifs are RAR or VDR response elements, the implication being that the RARE lies outside the proximal 3.1 kb of the promoter. Alternatively, the interpretation o f previous run-off transcription data is incorrect, and the p47^^^^ gene transcription is not directly inducible by retinoic acid.

One particular factor making interpretation of results more complex, is that terminal differentiation of HL60 cells is induced by RA, which itself causes widespread changes of gene expression.

In the absence o f detectable induction by RA, D3 or T3, it is interesting to speculate on the function of this DNA sequence. Two features which are unique to these sites are the spacing between each presumed half site (13bp), and the consensus sequence TGGTC/GA. Almost invariably, the natural response elements for these hormones are based on the hexamer consensus PuGGTC/GA, the only example o f a hexamer half site starting with T being the chicken vitellogenin A2 oestrogen response element (cVitERE, TGGTCA), which is arranged as a palindrome relative to its complementary half site (TGACCG) (Slater et al, 1991). Other members o f the nuclear hormone receptor superfamily have now been described, many of which are termed ‘orphan’ because the ligand is unknown (Issemann and Green, 1990), and which bind as homodimers or as heterodimers with RXR. The repeated motifs in the p47^^°^ promoter may act as a binding site for one of these, or for a unique member as yet undescribed. One candidate subfamily o f orphan receptors are the chicken ovalbumin upstream promoter transcription factors (COUP-TF), which on the basis o f amino acid sequence in the stem of the first zinc finger (P box), are related to TR and RAR (Wang et al, 1991). COUP- TF s have been found to activate promoters o f a number of genes, and to down regulate hormonal induction of some genes by TR, RAR, and VDR (Cooney et al, 1991,1992,1993; Liu et al, 1993; Burbach et al. 1994). COUP-TF is relatively promiscuous for binding to diverse GGTCA repeat orientations and spacings, although binding affinity tends to diminish with increasing spacing, and one of 13bp has not been reported. Evidence for a COUP-like factor or factors binding to the p47^^'’^ promoter is based on weak enhancer activity in the context of both the natural and heterologous promoters. Furthermore, specific binding of a 38kDa protein to a 200bp fragment containing all four motifs can be competed by oligonucleotide sequences containing 2 half sites (Dr Colin Casimir, personal communication), and is reminiscent o f the low molecular weight class o f COUP-TF.

The arrangement o f the four core GGTC/GA motifs is unique. The G-rich sequences after the first and third repeat particularly, suggest that each pair constitutes a functional binding site, and that intervening sequence for each pair in some way directs the conformation o f DNA to enable specific binding to occur. The equality o f spacing between each m otif may signify a requirement for simultaneous occupancy and co- operativity at all four sites. Alternatively, these motifs may form 4 independent binding sites each with imperfect or palindromic second h alf sites, and would suggest that different receptors could bind within the same region (Fig 3.14).

[A]

[B]

Fig 3.14. Speculative arrangem ent and orientation of hormone receptor binding elements. Equal spacing betw een each half-site suggests that they m ay co-operate as tw o pairs o f binding sites [A ]. A lternatively, each half-site may have a unique partner.