Multiple SSAP binding sites constitute the stage-specific enhancer of the sea urchin late H1beta gene.

The sea urchin late histone H1 genes are expressed at low levels up until mid-blastula stage of development when an enhancer element activates transcription to higher levels. Stage-specific activator protein (SSAP) was previously identified as the transcription factor that binds to a sequence motif within the late H1-specific enhancer, USE IV, and mediates this stage-specific activation. However, another conserved late H1-specific element, USE III, was also shown to contribute to the activated expression of the late H1 genes. To attain a better understanding of the mechanism of blastula stage activation an extended analysis of the late H1-specific DNA sequences of the SpH1beta gene was performed. Our findings indicate that this region, located between positions -320 and -200, consists of three SSAP binding sites, USE IV, USE III, and another site located between the two, termed Site 2. Although SSAP binds to USE IV in vitro with 10-15-fold higher affinity than to either of the other two sites, multiple sites are necessary for activation. Multimers of either USE IV or USE III activate mid-blastula stage transcription to similar levels in the context of a functional H1beta basal promoter, but not with a TATA box alone. In addition, multimers of USE IV activate expression of a reporter construct containing an early histone H1 promoter at an embryonic stage when it is normally repressed. We propose a mechanism for mid-blastula activation of the late histone H1 genes where SSAP binding sites activate expression, but require the presence of the cis sequences of the basal promoter to function.