以小白鼠為模式探討哺乳類精子轉錄因子CTCF：獲能效應引發CTCF酪胺酸基磷酸化會強化對甲基化標的核酸的親和力

Abstract

CTCF 是一個分佈廣且高度保守的轉錄因子，其分子量約82 kDa。先 前證實獲能的小白鼠精子之CTCF 會在酪胺酸基磷酸化。本研究探討這一 個巨大蛋白分子的酪胺酸磷酸化位置，並評估該化學修飾對標的核酸親和 力的影響。我們發現頂體反應不會導致存於頂體內CTCF 的釋放。運用基 因重組技術，融合GST 於CTCF 三個區域（domain），包括鋅指部位 （Zinc-finger domain，ZD/ residues 266-573），ZD 的N-端區域（ND/ residues 1-265）和ZD 的C-端區域（CD/ residues 574-736）。GST-ND 可被獲能精 子的酪胺酸激酶活性磷酸化，但GST-ZD 和GST-CD 卻不會。進一步將ND 的Y25, Y138, Y197, Y214, or Y226 突變成苯丙胺酸（Phenylalanine）而製備了突變 蛋白。相對原始蛋白（wild-type GST-ND），Y25F、Y138F 和Y214F 具有 相同的激酶基質活化，但Y197F 和Y226F 的基質活性則顯著降低，提示 Y197F 和Y226F 是兩個主要的磷酸化位置。進一步發現EGFR 的抑制劑 AG1478 可降低激酶對GST-ND 的磷酸化。配合可被磷酸化酪胺酸鄰近胺 基酸順序預測Y197 可被EGFR 磷酸化。運用核酸電泳動移動分析法 （electrophoretic mobility shift assay）量測CTCF 對β-APP、FpV 和c-Myc 啟 動子的親和力。相對於尚未獲能精子的CTCF，獲能精子的CTCF 對三個 啟動子的親和力較弱，但對甲基化的啟動子則親和力較強。

The CCCTC-binding nuclear factor (CTCF) is a widely expressed and highly conserved 82-KDa protein. Using mice as experimental animals, work of our previous study identified the tyrosine-phosphorylated form of CTCF in the capacitated sperm. This work was conducted to determine the tyrosine-phosphorylated sites in the CTCF molecule and to assess the impact of such a phosphorylation modification on the affinity of this nuclear factor to its target DNAs. We found that acrosomal exocytosis did not result in the release of CTCF residing in spermatozoal acrosome region. We made recombinant polypeptides of GST in frame with the N-terminal (ND/residues 1-265), zinc-finger domain (ZD/residues 266-573) and C-terminal domain (CD/residues 574-736) in CTCF. Neither GST-ZD nor GST-CD but GST-ND could be phosphorylated by the tyrosine kinase activity in the capacitated sperm. Further, Y25, Y138, Y197, Y214, or Y226 in ND of GST-ND was mutated to phenylalanine. Mutants Y25F, Y138F, and Y214F showed virtually the same substrate activity as the wild type GST-ND for the capacitation-related tyrosine phosphorylation, whereas the mutants Y197F and Y226F showed weaker substrate activity, manifesting Y197 and Y226 as the two major phosphorylation sites in which the modification of Y197 could be suppressed by an EGFR inhibitor AG1478. The electrophoretic mobility shift assay was applied to measure the affinity of spermatozoal CTCF to its DNA target sequences found in the promotors of amyloid β-protein precursor (β-APP), FpV and c-Myc. Relative to CTCF in the incapacitated sperm, the tyrosine-phosphorylated protein in the BSA-treated sperm gave weaker affinity to each target DNA, whereas it showed stronger affinity to the methylated forms of these target DNAs.