Down-regulation of early sea urchin histone H2A gene relies on cis regulative sequences located in the 5’ and 3’ regions including the enhancer blocker sns

The tandem repeated sea urchin a-histone genes are developmentally regulated by gene-specific promoter elements. Coordinate transcription of the five genes begins after meiotic maturation of the oocyte, continues through cleavage, and reaches its maximum at morula stage, after which these genes are shut off and maintained in a silenced state for the life cycle of the animal. Although cis regulative sequences affecting the timing and the level of expression of these genes have been characterized, much less is known about the mechanism of their repression. Here we report the results of a functional analysis that allowed the identification of the sequence elements needed for the silencing of the a-H2A gene at gastrula stage. We found that important negative regulative sequences are located in the 462 bp sns 5 fragment located in the 30 region. Remarkably, sns 5 contains the sns enhancer blocking element and the most 30 H2A codons. In addition, we made the striking observation that inhibition of the anti-enhancer activity of sns, by titration of the binding proteins in microinjected embryos, also affected the capability of sns 5 to down-regulate transgene expression at gastrula stage. A further sequence element essential for repression of the H2A gene was identified upstream of the enhancer, in the 50 region, and contains four GAGA repeats. Altogether these findings suggest that downregulation of the a-H2A gene occurs by the functional interaction of the 50 and 30 cis sequence elements. These results demonstrate the involvement of a genomic insulator in the silencing of gene expression.