Ficzycz A, Kaludov N K, Lele Z, Hurt M M, Ovsenek N
Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, Saskatoon, Canada.
Dev Biol. 1997 Feb 1;182(1):21-32. doi: 10.1006/dbio.1996.8459.
Replication-dependent histone genes in the mouse and Xenopus share a common regulatory element within the protein-encoding sequence called the CRAS alpha element (coding region activating sequence alpha) which has been shown to mediate normal expression in vivo and to interact with nuclear factors in vitro in a cell cycle-dependent manner. Thus far, the alpha element has only been studied in rodent cells in culture, and its effect on histone gene expression during development has not been determined. Here we examine the role of the alpha element in histone gene expression during Xenopus development which features a switch in histone gene expression from a replication-independent mode in oocytes to a replication-dependent mode in embryos after midblastula stage. In vivo expression experiments involving wild-type or alpha-mutant mouse H3.2 genes show that mutation of the CRAS alpha element results in a fourfold decline of expression in embryos, but does not affect expression in oocytes. Two distinct alpha sequence-specific binding activities were detected in both oocyte and embryonic extracts. A slowly migrating DNA-binding complex was present at relatively constant levels throughout development from the earliest stages of oogenesis through larval stages. In contrast, levels of a rapidly migrating complex were high in stage I and II oocytes, declined in stage II-VI oocytes, remained low in unfertilized eggs and cleavage stage embryos, and rose dramatically after the midblastula transition. The molecular masses of the factors forming the slow and rapidly migrating complexes were estimated to be approximately 110 and 85 kDa, respectively. DNA-binding activity of the 85 kDa alpha-binding factor was affected by phosphorylation, binding with higher affinity in the dephosphorylated state. The abrupt increase in DNA-binding activity of the 85-kDa alpha-binding factor at late blastula coincides with the switch to the replication-dependent mode of histone gene expression. We propose that the conserved alpha element present in the coding sequence of mouse and Xenopus core histone genes is required for normal replication-dependent histone expression in the developing Xenopus embryo.
小鼠和非洲爪蟾中依赖复制的组蛋白基因在蛋白质编码序列内共享一个称为CRASα元件（编码区激活序列α）的共同调控元件，该元件已被证明可介导体内正常表达，并在体外以细胞周期依赖的方式与核因子相互作用。到目前为止，α元件仅在培养的啮齿动物细胞中进行了研究，其在发育过程中对组蛋白基因表达的影响尚未确定。在这里，我们研究α元件在非洲爪蟾发育过程中组蛋白基因表达中的作用，非洲爪蟾发育的特点是组蛋白基因表达在囊胚中期后从卵母细胞中的非依赖复制模式转变为胚胎中的依赖复制模式。涉及野生型或α突变型小鼠H3.2基因的体内表达实验表明，CRASα元件的突变导致胚胎中表达下降四倍，但不影响卵母细胞中的表达。在卵母细胞和胚胎提取物中均检测到两种不同的α序列特异性结合活性。从最早的卵子发生阶段到幼虫阶段，一种迁移缓慢的DNA结合复合物在整个发育过程中以相对恒定的水平存在。相比之下，一种迁移迅速的复合物的水平在I期和II期卵母细胞中较高，在II-VI期卵母细胞中下降，在未受精卵和卵裂期胚胎中保持较低水平，并在囊胚中期转变后急剧上升。形成缓慢和迅速迁移复合物的因子的分子量估计分别约为110 kDa和85 kDa。85 kDaα结合因子的DNA结合活性受磷酸化影响，在去磷酸化状态下具有更高的亲和力。85 kDaα结合因子在囊胚后期DNA结合活性的突然增加与组蛋白基因表达向依赖复制模式的转变相吻合。我们提出，小鼠和非洲爪蟾核心组蛋白基因编码序列中存在的保守α元件是非洲爪蟾胚胎发育过程中正常依赖复制的组蛋白表达所必需的。
