SELECTION OF 10 BASE-PAIR SEQUENCES WHICH ENHANCE THE RESPONSE OF THEGAL1 MINIMAL PROMOTER TO MURINE HOXA-7 IN YEAST

The mouse homeodomain protein Hoxa-7, expressed under the control of a n inducible promoter, was able to inducibly activate reporter genes co ntaining multimerized Hoxa-7 binding sites in Saccharomyces cerevisiae . This tight regulation was exploited in an attempt to screen for Hoxa -7 responsive elements. A reporter library consisting of a randomised 10 bp element inserted into the minimal gal1 promoter was constructed. In a surprisingly small screen, 24 reporters were isolated which had all of the transactivation characteristics expected for a Hoxa-7 bindi ng site insertion. However, further characterisation revealed that the selected elements lacked homeodomain (HD) binding core motifs and wer e not bound by a purified Hoxa-7/beta-galactosidase fusion protein cap able of binding known sites. The minimal promoter context contains 16 HD core motifs in 410 bp. Careful re-examination of basal levels revea led a low residual response of the gal1 minimal promoter to Hoxa-7. Th e 11 characterised 10 bp inserts amplified Hoxa-7 responsiveness in a manner correlated to increases in basal reporter activity. Thus, a qua ntitative range of Hox-responsiveness was produced by slight sequence alterations that did not change HD binding sites of their relative spa cing in the promoter. These data suggest how, without altering residen t HD base contact zones, mouse promoters could be optimised by natural selection to give appropriate quantitative outputs in each anatomical region defined by an assortment of Hox proteins. The selected element s were pyrimidine rich on the sense strand, containing (T)(n)C motifs, strikingly similar to sequences which enhance Hoxa-7 binding and acti vation from outside the HD contact zone. A search of defined sequence databases demonstrated that these elements were over-represented in pr omoters. Two elements altered the mobility shift patterns produced by cell extracts on minimal promoter fragments.