Functional characterization of a natural retinoic acid responsive element

Retinoic acid receptor (RAR) and thyroid hormone receptor (T₃R) are thought to bind as dimers to a T₃ responsive element (T₃REpal) comprised of inverted repeats of the half-site motif GGTCA. However, a RA responsive element (βRARE) was previously identified in the promoter of the RAR/β2 gene which contains two direct repeats of the motif GTTCA spaced by a six nucleotide gap. We now demonstrate the ability of RARα, β and γ to bind to and transactivate through this element and that the two direct repeats comprise the βRARE. Surprisingly, the GTTCA motifs rearranged to form a palindrome do not confer RA responsiveness to a heterologous promoter. Furthermore, no significant level of transactivation is detected by ligand-activated RAR through the Moloney murine leukaemia virus T₃RE, which comprises two direct repeats of the sequence GGTCA/C spaced by a five nucleotide gap. Similarly, T₃R does not induce gene expression through the βRARE. This study establishes the preference of T₃R to transactivate through direct repeats spaced by a five nucleotide gap as opposed to a six nucleotide gap. In contrast, RAR appears to be more flexible with respect to spacing requirements between repeats, although higher levels of transactivation are obtained through direct repeats spaced by a six nucleotide gap. Interestingly, although some elements mediate either RA or T₃ induction, changing a single nucleotide in the MoMLV T₃RE with a five nucleotide spacing creates a promiscuous RA/T₃ responsive element.