Selection of ten base-pair sequences which enhance the response of the Gal1 minimal promoter to murine Hoxa-7 in yeast.
The mouse homeodomain protein Hoxa-7, expressed under the control of an inducible promoter, was able to inducibly activate reporter genes containing 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 binding site insertion. However, further characterisation revealed that the selected elements lacked homeodomain (HD) binding core motifs and were not bound by a purified Hoxa-7/beta-galactosidase fusion protein capable of binding known sites. The minimal promoter context contains 16 HD core motifs in 410 bp. Careful re-examination of basal levels revealed a low residual response of the gal1 minimal promoter to Hoxa-7. The 11 characterised 10 bp inserts amplified Hoxa-7 responsiveness in a manner correlated to increases in basal reporter activity. Thus, a quantitative range of Hox-responsiveness was produced by slight sequence alterations that did not change HD binding sites of their relative spacing in the promoter. These data suggest how, without altering resident 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 elements were pyrimidine rich on the sense strand, containing (T)nC motifs, strikingly similar to sequences which enhance Hoxa-7 binding and activation from outside the HD contact zone. A search of defined sequence databases demonstrated that these elements were over-represented in promoters. Two elements altered the mobility shift patterns produced by cell extracts on minimal promoter fragments.
Gross
M K
MK
Abteilung für Molekulare Zellbiologie, Max-Plank-Institute für Biophysikalische Chemie, Göttingen, Germany.
Gruss
P
P
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D012641:Selection, Genetic; D013997:Time Factors; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000235:genetics*
D018398:Homeodomain Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase