Huang Chiu-Jung, Wu Shinn-Chih, Choo Kong-Bung
Department of Animal Science, College of Agriculture, Chinese Culture University, Taipei 11192, Taiwan.
Biochem J. 2005 Apr 15;387(Pt 2):367-75. doi: 10.1042/BJ20041364.
Maternal-to-zygotic transition of a fertilized egg and the subsequent pre-implantation development of the embryo involve zygotic genome activation and reprogramming of gene expression. The goal of the present study is to establish a model suitable for the characterization of transcriptional modulation of mammalian pre-implantation development. Rnf35 is a mouse RING-finger protein gene that is temporally transcribed in the early embryo, but is permanently silenced before the blastocyst stage of development. We first show that the Chinese-hamster ovary-K1 cells are unique in supporting Rnf35 promoter activities in transient transfection assays. Using the permissive Chinese-hamster ovary-K1 cell line, we show that Rnf35 transcription is driven by an Inr (initiator) core promoter element in the absence of a TATA box; the Inr promoter function is confirmed by direct microinjection of mouse one-cell embryos. This is the first demonstration of the involvement of an Inr core promoter element in transcription in pre-implantation development. We show that the Rnf35 promoter is regulated by three obligatory Y-box (CCAAT-box) elements: two Y boxes (Y(I) and Y(II)) located at -81 are coupled in a palindrome and act synergistically in contributing to Rnf35 transcription; the third Y box (Y(III)) is situated at -13, just upstream of the Inr element, and may be an integral part of the Inr function. Electrophoretic mobility-shift assays and competition experiments further reveal that the Y(I) box is bound by the ubiquitous NF-Y (nuclear factor-Y)/CBF (CCAAT-binding factor) and that Y(II) is targeted by an unidentified protein(s) that acts synergistically with the NF-Y. We suggest that the NF-Y, targeting at a Y-box sequence, may function as an important activator in transcriptional regulation of the Rnf35 gene in the pre-implantation embryo.
受精卵从母源向合子的转变以及随后胚胎的植入前发育涉及合子基因组激活和基因表达重编程。本研究的目的是建立一个适合表征哺乳动物植入前发育转录调控的模型。Rnf35是一个小鼠泛素连接酶蛋白基因，在早期胚胎中暂时转录，但在发育的囊胚阶段之前永久沉默。我们首先表明，中国仓鼠卵巢-K1细胞在瞬时转染实验中支持Rnf35启动子活性方面具有独特性。使用允许的中国仓鼠卵巢-K1细胞系，我们表明在没有TATA框的情况下，Rnf35转录由Inr（起始子）核心启动子元件驱动；通过直接显微注射小鼠单细胞胚胎证实了Inr启动子功能。这是首次证明Inr核心启动子元件参与植入前发育中的转录。我们表明Rnf35启动子受三个必需的Y框（CCAAT框）元件调控：位于-81的两个Y框（Y(I)和Y(II)）以回文形式耦合，并协同作用促进Rnf35转录；第三个Y框（Y(III)）位于Inr元件上游-13处，可能是Inr功能的一个组成部分。电泳迁移率变动分析和竞争实验进一步揭示，Y(I)框与普遍存在的NF-Y（核因子-Y）/CBF（CCAAT结合因子）结合，而Y(II)被一种未鉴定的蛋白质靶向，该蛋白质与NF-Y协同作用。我们认为，靶向Y框序列的NF-Y可能在植入前胚胎中Rnf35基因的转录调控中起重要激活作用。
