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 developmentallyregulated by gene-specific promoter elements. Coordinate transcription ofthe five genes begins after meiotic maturation of the oocyte, continuesthrough cleavage, and reaches its maximum at morula stage, after whichthese genes are shut off and maintained in a silenced state for the life cycleof the animal. Although cis regulative sequences affecting the timing andthe level of expression of these genes have been characterized, much less isknown about the mechanism of their repression. Here we report the resultsof a functional analysis that allowed the identification of the sequenceelements needed for the silencing of the a-H2A gene at gastrula stage. Wefound that important negative regulative sequences are located in the462 bp sns 5 fragment located in the 30 region. Remarkably, sns 5 containsthe sns enhancer blocking element and the most 30 H2A codons. In addition,we made the striking observation that inhibition of the anti-enhanceractivity of sns, by titration of the binding proteins in microinjected embryos,also affected the capability of sns 5 to down-regulate transgene expressionat gastrula stage. A further sequence element essential for repression of theH2A gene was identified upstream of the enhancer, in the 50 region, andcontains four GAGA repeats. Altogether these findings suggest that downregulationof the a-H2A gene occurs by the functional interaction of the 50and 30 cis sequence elements. These results demonstrate the involvement ofa genomic insulator in the silencing of gene expression.