F. Palla et al., MODULATOR FACTOR-BINDING SEQUENCE OF THE SEA-URCHIN EARLY HISTONE H2APROMOTER ACTS AS AN ENHANCER ELEMENT, Proceedings of the National Academy of Sciences of the United Statesof America, 91(25), 1994, pp. 12322-12326
The sea urchin early H2A histone gene, like the other four members of
the repeating units, is transiently expressed during very early develo
pment. To investigate the mechanisms underlying the faithful expressio
n of the early H2A gene, we focused our attention on the modulator ele
ment. We showed by DNase I cleavage protection patterns that the modul
ator includes the upstream sequence element 1 (USE1) and mapped at nuc
leotides -137 to -108 in the early H2A gene promoter. Functional tests
conducted by microinjection into sea urchin embryos then showed that
the modulator element binds the transcriptional factor called modulato
r-binding factor 1 (MBF-1). We found in fact that coinjection of an ex
cess of the MBF-1-binding site, either as the modulator or as the USE1
, efficiently impaired the activity of the H2A promoter. An unexpected
finding was the expression of the reporter gene from the early H2A pr
omoter at the gastrula stage of embryonic development, when the early
histone genes are transcriptionally silent. In addition, we also found
that the modulator element was active at the gastrula stage. The pote
ntial enhancer activity of the modulator was tested by microinjecting
several constructs containing single or multiple copies of the modulat
or element placed 5' or 3' to a thymidine kinase gene (tk) promoter in
both sea urchin embryos and Xenopus laevis oocytes and determining th
e expression of a reporter chloramphenicol acetyltransferase gene unde
r the control of the linked tk promoter. We found that an oligonucleot
ide bearing the MBF-1-binding site activates the expression of the rep
orter gene independently of the position and orientation. We conclude
that the modulator binds the MBF-1 activator and that it is a transcri
ptional enhancer of the early H2A histone gene.