Medina R, Gutiérrez J, Puchi M, Imschenetzky M, Montecino M
Departamento de Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepcion, Concepcion, Chile.
J Cell Biochem. 2001;83(4):554-62. doi: 10.1002/jcb.1250.
After fertilization the sea urchin sperm nucleus transforms into the male pronucleus which later fuses with the female pronucleus re-establishing the diploid genome of the embryo. This process requires remodeling of the sperm chromatin structure including the replacement of the sperm histones by maternally derived cleavage stage histone variants. In recent years, a group of protein complexes that promote chromatin-remodeling in an ATP-dependent manner have been described. To gain understanding into the molecular mechanisms operating during sea urchin male pronuclei formation, we analyzed whether chromatin-remodeling activity was present in unfertilized eggs as well as during early embryogenesis. We report that in the sea urchin Tetrapygus niger, protein extracts from the cytoplasm but not from the nucleus, of unfertilized eggs exhibit ATP-dependent nucleosome remodeling activity. This cytosolic activity was not found at early stages of sea urchin embryogenesis. In addition, by using polyclonal antibodies in Western blot analyses, we found that an ISWI-related protein is primarily localized in the cytoplasm of the sea urchin eggs. Interestingly, SWI2/SNF2-related proteins were not detected neither in the nucleus nor in the cytoplasm of unfertilized eggs. During embryogenesis, as transcriptional activity is increased an ISWI-related protein is found principally in the nuclear fraction. Together, our results indicate that the cytoplasm in sea urchin eggs contains an ATP-dependent chromatin-remodeling activity, which may include ISWI as a catalytic subunit.
受精后,海胆精子核转变为雄原核,随后与雌原核融合,重新建立胚胎的二倍体基因组。这一过程需要重塑精子染色质结构,包括用母源的卵裂期组蛋白变体取代精子组蛋白。近年来,已经描述了一组以ATP依赖方式促进染色质重塑的蛋白质复合物。为了深入了解海胆雄原核形成过程中起作用的分子机制,我们分析了未受精卵以及早期胚胎发育过程中是否存在染色质重塑活性。我们报告,在黑四角海胆中,未受精卵细胞质而非细胞核的蛋白质提取物表现出ATP依赖的核小体重塑活性。在海胆胚胎发育早期未发现这种胞质活性。此外,通过在蛋白质印迹分析中使用多克隆抗体,我们发现一种与ISWI相关的蛋白质主要定位于海胆卵的细胞质中。有趣的是,在未受精卵的细胞核和细胞质中均未检测到与SWI2/SNF2相关的蛋白质。在胚胎发育过程中,随着转录活性的增加,一种与ISWI相关的蛋白质主要存在于细胞核部分。总之,我们的结果表明,海胆卵的细胞质中含有一种ATP依赖的染色质重塑活性,其中可能包括ISWI作为催化亚基。