Medina R, Paredes R, Puchi M, Imschenetzky M, Montecino M
Departamento de Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepcion, Casilla 160-C, Concepcion, Chile.
Gene. 2001 Jul 11;272(1-2):237-48. doi: 10.1016/s0378-1119(01)00534-0.
During sea urchin embryogenesis the early histone genes are temporally expressed to accommodate the high demand for histone proteins during DNA replication at early cleavage stages of development. The early histone genes are transcriptionally active from the 16-cell stage, reaching a peak in expression at the 128-cell stage that gradually decreases until expression is completely inhibited at the late blastula stage. We are studying the gene regulatory mechanisms that control early histone gene expression in sea urchins to understand the interrelationships between chromatin remodeling and transcriptional activation during development. Here, we have investigated chromatin organization and transcription factor interactions by analyzing nuclease hypersensitivity and protein binding in the promoter region of the early histone H3 gene from the sea urchin Tetrapygus niger. We have found a DNase I hypersensitive domain centered at -90 in the early histone H3 gene promoter which is only detected in embryos at the 128-cell stage expressing high levels of early histone H3 mRNA. This hypersensitive site (-110 to -70) encompasses two regulatory elements (TnH3NFH3.1 and TnH3CCAAT). The -94 to -77 region of the histone H3 promoter is recognized by a transcription factor complex in nuclear extracts from 128-cell embryos. Methylation interference analysis and competition studies demonstrated a specific interaction at the CCAAT sequence. Using specific antibodies we find that the homeodomain transcription factor CDP/cut is the DNA-binding component of the complex interacting with the early histone H3 gene promoter in T. niger. Our results provide further evidence for the functional role of CDP/cut in developmental regulation of histone gene expression in phylogenetically diverse eukaryotic species.
在海胆胚胎发育过程中，早期组蛋白基因在发育的早期卵裂阶段进行DNA复制时，会随着时间的推移表达，以满足对组蛋白的高需求。早期组蛋白基因从16细胞阶段开始转录活跃，在128细胞阶段达到表达峰值，随后逐渐下降，直到在囊胚后期表达完全被抑制。我们正在研究控制海胆早期组蛋白基因表达的基因调控机制，以了解发育过程中染色质重塑与转录激活之间的相互关系。在此，我们通过分析来自黑四角海胆早期组蛋白H3基因启动子区域的核酸酶超敏感性和蛋白质结合，研究了染色质组织和转录因子相互作用。我们发现在早期组蛋白H3基因启动子中，一个以-90为中心的DNase I超敏感区域，该区域仅在表达高水平早期组蛋白H3 mRNA的128细胞阶段胚胎中检测到。这个超敏感位点（-110至-70）包含两个调控元件（TnH3NFH3.1和TnH3CCAAT）。组蛋白H3启动子的-94至-77区域被128细胞胚胎核提取物中的转录因子复合物识别。甲基化干扰分析和竞争研究表明在CCAAT序列处存在特异性相互作用。使用特异性抗体，我们发现同源域转录因子CDP/cut是与黑四角海胆早期组蛋白H3基因启动子相互作用的复合物的DNA结合成分。我们的结果为CDP/cut在系统发育上不同的真核生物物种中组蛋白基因表达的发育调控中的功能作用提供了进一步证据。
