Palla F, Melfi R, Di Gaetano L, Bonura C, Anello L, Alessandro C, Spinelli G
Istituto di Biologia dello Sviluppo del Consiglio Nazionale delle Ricerche, Palermo, Italy.
Biol Chem. 1999 Feb;380(2):159-65. doi: 10.1515/BC.1999.024.
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.
海胆早期组蛋白基因的转录在早期卵裂期间短暂发生，在桑椹胚阶段达到最大值，并在原肠胚阶段下降到检测不到的水平。为了确定负责H2A基因转录时间和水平的调控元件，我们利用核提取物中的启动子结合研究以及由早期H2A启动子驱动的CAT转基因的显微注射进行研究。我们发现，桑椹胚和原肠胚核蛋白在H2A启动子上产生了难以区分的DNase I足迹模式。检测到分别以调节子/增强子和CCAAT框为中心的两个相互作用位点。调节子的缺失或过量调节子序列的共注射严重影响了由无增强子且含调节子的H2A启动子驱动的两个转基因的表达。最后，一个包含3'编码和H2A后间隔序列的DNA片段，在沉默时先前已绘制出三个微球菌核酸酶超敏位点，在原肠胚阶段特异性抑制了由H2A启动子驱动的转基因的表达。这些结果表明，调节子对于早期H2A基因的表达至关重要，并且下调序列位于H2A基因的3'端附近。
