EXTREME POSITION DEPENDENCE OF A CANONICAL HORMONE RESPONSE ELEMENT

Hormone response elements (HREs) are considered enhancers, activating transcription in a relatively position- and orientation-independent fa shion. Upon binding to an HRE, steroid receptors presumably contact co activators and/or proteins associated with the transcription initiatio n complex. As a receptor target site is moved further from a fixed pos ition such as the TATA box, not only will the spatial separation of th e receptor with respect to its interaction partners change, so will th e orientation due to the rotation of the DNA helix. Additional constra ints may be imposed by the assembly of DNA into chromatin. Therefore, we have endeavored to test rigorously the assertion that HRE action is position independent. We have constructed a series of 42 chlorampheni col acetyltransferase expression vectors that contain a single progest erone/glucocorticoid receptor-binding site separated from a TATA box b y 4 to 286 bp. The enhancer activity of the HRE was assessed after tra nsient transfection of progesterone receptor-expressing fibroblasts. W e find that the position of the HRE has a dramatic influence on induct ion by progestins. When closely juxtaposed to the TATA box, the HRE wa s unable to support a hormone response, perhaps due to direct steric h indrance with the transcription initiation complex. Full activity was gained by moving the HRE 10 bp further from the TATA sequence. As the HRE was moved incrementally further, activity remained near maximal ov er the next 26 bp. HRE activity then declined over the subsequent 26 b p and remained low for another 2.5 helical turns. Surprisingly, a narr ow window of HRE activity occurred at an HRE-TATA box separation of 90 -100 bp. Little or no hormone-induced transcriptional activity was obs erved when the HRE was positioned further from the TATA box. The addit ion of a second HRE or a basal (nuclear factor-1) element failed to re lieve this constraint. A similar series of experiments was carried out in a mammary carcinoma cell line that expressed high levels of both g lucocorticoid and progestin receptors. Data in these cells indicate th at glucocorticoids and progestins supported a similar HRE position-act ivity profile, but this pattern of HRE activity was quite distinct fro m that seen in fibroblasts. This may be indicative of cell type-specif ic interactions between steroid receptors and adapter/coactivator prot eins or cell type-specific activities such as acetylases or deacetylas es participating in the steroid response.