Constitutive Promoter Occupancy by the MBF-1 Activator and Chromatin Modification of the Developmental Regulated Sea Urchin alpha-H2A Histone Gene

The tandemly repeated sea urchin α-histone genes are developmentallyregulated. These genes are transcribed up to the early blastula stage andpermanently silenced as the embryos approach gastrulation. As previouslydescribed, expression of the α-H2A gene depends on the binding of theMBF-1 activator to the 5′ enhancer, while down-regulation relies on thefunctional interaction between the 3′ sns 5 insulator and the GA repeatslocated upstream of the enhancer. As persistent MBF-1 binding andenhancer activity are detected in gastrula embryos, we have studied themolecular mechanisms that prevent the bound MBF-1 from trans-activatingthe H2A promoter at this stage of development. Here we used chromatinimmunoprecipitation to demonstrate that MBF-1 occupies its site regardlessof the transcriptional state of the H2A gene. In addition, we have mappedtwo nucleosomes specifically positioned on the enhancer and promoterregions of the repressed H2A gene. Interestingly, insertion of a 26 bpoligonucleotide between the enhancer and the TATA box, led to upregulationof the H2A gene at gastrula stage, possibly by changing theposition of the TATA nucleosome. Finally, we found association of histonede-acetylase and de-acetylation and methylation of K9 of histone H3 on thepromoter and insulator of the repressed H2A chromatin. These data arguefor a role of a defined positioned nucleosome in the promoter and histonetail post-translational modifications, in the 3′ insulator and 5′ regulatoryregions, in the repression of the α-H2A gene despite the presence of theMBF-1 activator bound to the enhancer