Regulation of the Sea Urchin Early H2A Histone Gene Expression Depends on the Modulator Element and on Sequences Located near the 3′ End

Abstract

Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the modulator or coinjection of an excess of modulator sequences severely affected the expression of two transgenes driven by the enhancer-less and modulator-containing H2A promoter. Finally, a DNA fragment containing 3' coding and post-H2A spacer sequences, where upon silencing three micrococcal nuclease hypersensitive sites were previously mapped, specifically repressed at the gastrula stage the expression of the transgene driven by the H2A promoter. These results indicate that the modulator is essential for the expression of early H2A gene and that sequences for downregulation are localized near the 3' end of the H2A gene.

Down-regulation of Early Sea Urchin Histone H2A Gene Relies on cis Regulative Sequences Located in the 5′ and 3′ Regions and Including the Enhancer Blocker sns

“…The results of the RNase protection assays carried out with RNA samples from the same number of microinjected embryos are shown in Figure 2. In agreement with previous observations, 17 the reporter transgene driven by the a-H2A enhancer/promoter fragment was not silenced at gastrula stage and it was constitutively expressed. By contrast, it followed the timing of expression of the a-H2A endogenous gene when the sns 2 sequence was included in the transgene transcripton unit.…”

“…As described, repression of the a-H2A gene at gastrula stage depends most probably on regulative sequences located in the sns 2 DNA fragment. 17 Such evidence is in contrast with the presence of negative sequence elements in the promoter region of a-H1 and a-H3 genes. 19,20 It is of great interest that sns 2 includes the sns enhancer blocker.…”

“…As mentioned above, when the a-H2A gene is silenced as the sea urchin embryo enters the gastrula stage, the enhancer remains constitutively active and the binding of the MBF-1 transcription factor persists. 17 So, the enhancer does not play a predominant role in the down-regulation of the a-H2A gene expression. As the embryo approaches gastrulation, the only major change that is detected is the structural change of the early histone genes' chromatin configuration.…”

“…Down-regulation depends on sequence elements present in the sns 2 fragment. 17 sns 2 extends further upstream and further downstream than the sns enhancer blocking element. 38 It contains the last 90 bp of the protein-coding Figure 2.…”

“…16 In addition, persistent MBF-1 binding activity and expression of a transgene driven by the enhancer can be demonstrated after silencing of the a-H2A gene at gastrula stage. 17 cis-Acting regulative sequence elements have also been characterized in the promoter of the other four genes of the early histone repeating unit of Strongylocentrotus purpuratus. Maximum expression of a-H2B relies only on the octamer element, 18 while for a-H3, the CCAAT box and a gene-specific element are all that is required for high level ontogenic expression.…”