ENHANCER BLOCKING ACTIVITY LOCATED NEAR THE 3' END OF THE SEA-URCHIN EARLY H2A HISTONE GENE

The sea urchin early histone repeating unit contains one copy of each of the five histone genes whose coordinate expression during developme nt is regulated by gene-specific elements, To learn how within the his tone repeating unit a gene-specific activator can be prevented to comm unicate with the heterologous promoters, we searched for domain bounda ries by using the enhancer blocking assay, We focused on the region ne ar the 3' end of the H2A gene where stage-specific nuclease cleavage s ites appear upon silencing of the early histone genes, We demonstrated that a DNA fragment of 265 bp in length, defined as sns (for silencin g nucleoprotein structure), blocked the enhancer activity of the H2A m odulator in microinjected sea urchin embryos only when placed between the enhancer elements and the promoter, We also found that sns silence d the modulator elements even when placed at 2.7 kb from the promoter, By contrast, the enhancer activity of the modulator sequences, locate d downstream to the coding region, was not affected when sns was posit ioned in close proximity to the promoter, Finally, the H2A sns fragmen t placed between the simian virus 40 regulative region and the tk prom oter repressed chloramphenicol acetyltransferase expression in transfe cted human cell lines, We conclude that 3' end of the H2A gene contain s sequence elements that behave as functional barriers of enhancer fun ction in the enhancer blocking assay, Furthermore, our results also in dicate that the enhancer blocking function of sns lacks enhancer and s pecies specificity and that it can act in transient assays.