Mk. Gross et P. Gruss, SELECTION OF 10 BASE-PAIR SEQUENCES WHICH ENHANCE THE RESPONSE OF THEGAL1 MINIMAL PROMOTER TO MURINE HOXA-7 IN YEAST, Journal of Molecular Biology, 247(2), 1995, pp. 173-190
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.