THE INTERPLAY OF HALF-SITE SEQUENCE AND SPACING ON THE ACTIVITY OF DIRECT REPEAT THYROID-HORMONE RESPONSE ELEMENTS

Direct repeats of the hexamer AGGTCA can serve as response elements fo r vitamin D, thyroid hormone, or retinoic acid. The specificity of the response appears to reside in the spacing between the hexamers, with response elements for vitamin D restricted to direct repeats separated by a 3-base pair (bp) spacer, thyroid hormone a 4-bp spacer, and reti noic acid a 5-bp spacer (3-4-5 rule). Recently we have shown that the optimum thyroid hormone receptor binding site consists of an 8-bp sequ ence (TAAGGTCA), not a hexamer. Therefore we tested whether the 3-4-5 rule is valid for octamer sequence direct repeats. In transfection exp eriments octamer direct repeats with 3-, 4-, or 5-bp spacers conferred equivalently strong thyroid hormone responses, although a repeat with a 9-bp spacer was substantially weaker. For the 4 and 5-bp spacer con structs, the 5' half-site octamer had as strong an influence on thyroi d hormone induction as did the 3' half-site octamer, although for the 3-bp spacer construct the 5' octamer was marginally less potent than t he 3' octamer. Transfection and gel shift experiments did not suggest a simple correlation between the binding of thyroid hormone receptor-r etinoid X receptor heterodimers and thyroid hormone induction from the se response elements. We conclude that half site sequence can override the effect of spacing in determining the hormone responsiveness of a direct repeat response element. In addition, the thyroid hormone respo nse may not be due simply to the binding of thyroid hormone receptor-r etinoid X receptor heterodimers to the DNA.