MODULATOR FACTOR-BINDING SEQUENCE OF THE SEA-URCHIN EARLY HISTONE H2APROMOTER ACTS AS AN ENHANCER ELEMENT

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.