Palla F, Bonura C, Anello L, Di Gaetano L, Spinelli G
Dipartimento di Biologia Cellulare e dello Sviluppo, Consiglio Nazionale delle Richerche, Palermo, Italy.
Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12322-6. doi: 10.1073/pnas.91.25.12322.
The sea urchin early H2A histone gene, like the other four members of the repeating units, is transiently expressed during very early development. To investigate the mechanisms underlying the faithful expression of the early H2A gene, we focused our attention on the modulator element. We showed by DNase I cleavage protection patterns that the modulator includes the upstream sequence element 1 (USE1) and mapped at nucleotides -137 to -108 in the early H2A gene promoter. Functional tests conducted by microinjection into sea urchin embryos then showed that the modulator element binds the transcriptional factor called modulator-binding factor 1 (MBF-1). We found in fact that coinjection of an excess of the MBF-1-binding site, either as the modulator or as the USE1, efficiently impaired the activity of the H2A promoter. An unexpected finding was the expression of the reporter gene from the early H2A promoter at the gastrula stage of embryonic development, when the early histone genes are transcriptionally silent. In addition, we also found that the modulator element was active at the gastrula stage. The potential enhancer activity of the modulator was tested by microinjecting several constructs containing single or multiple copies of the modulator element placed 5' or 3' to a thymidine kinase gene (tk) promoter in both sea urchin embryos and Xenopus laevis oocytes and determining the expression of a reporter chloramphenicol acetyltransferase gene under the control of the linked tk promoter. We found that an oligonucleotide bearing the MBF-1-binding site activates the expression of the reporter gene independently of the position and orientation. We conclude that the modulator binds the MBF-1 activator and that it is a transcriptional enhancer of the early H2A histone gene.
海胆早期H2A组蛋白基因与重复单元的其他四个成员一样，在非常早期的发育过程中短暂表达。为了研究早期H2A基因忠实表达的潜在机制，我们将注意力集中在调节元件上。我们通过DNA酶I切割保护模式表明，调节子包括上游序列元件1（USE1），并定位在早期H2A基因启动子的核苷酸-137至-108处。然后通过显微注射到海胆胚胎中进行的功能测试表明，调节元件结合了称为调节子结合因子1（MBF-1）的转录因子。事实上，我们发现共注射过量的MBF-1结合位点，无论是作为调节子还是作为USE1，都能有效损害H2A启动子的活性。一个意外的发现是，在胚胎发育的原肠胚阶段，当早期组蛋白基因转录沉默时，早期H2A启动子的报告基因表达。此外，我们还发现调节元件在原肠胚阶段具有活性。通过在海胆胚胎和非洲爪蟾卵母细胞中显微注射几种构建体来测试调节子的潜在增强子活性，这些构建体包含置于胸苷激酶基因（tk）启动子5'或3'端的单个或多个调节子元件拷贝，并确定连接的tk启动子控制下的报告氯霉素乙酰转移酶基因的表达。我们发现带有MBF-1结合位点的寡核苷酸可独立于位置和方向激活报告基因的表达。我们得出结论，调节子结合MBF-1激活剂，并且它是早期H2A组蛋白基因的转录增强子。
