Hashimoto Hideharu, Wang Dongxue, Steves Alyse N, Jin Peng, Blumenthal Robert M, Zhang Xing, Cheng Xiaodong
Department of Biochemistry, Emory University, Atlanta, GA 30322, USA.
Genetics and Molecular Biology Graduate Program, Laney Graduate School, Emory University, Atlanta, GA 30322, USA.
Nucleic Acids Res. 2016 Dec 1;44(21):10177-10185. doi: 10.1093/nar/gkw774. Epub 2016 Sep 4.
Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay.
哺乳动物基因组甲基化的重编程至关重要，但人们对此了解甚少。Klf4是一种指导重编程的转录因子，它包含一个具有三个连续C2H2锌指的DNA结合结构域。Klf4在特定序列中识别CpG或TpG。小鼠Klf4 DNA结合结构域对甲基化的CpG或TpG具有大致相同的亲和力，而对未修饰的CpG亲和力略低。尽管已知Glu446的侧链与5-甲基胞嘧啶（5mC）或胸腺嘧啶（5-甲基尿嘧啶）的甲基接触，但这种关键偏好的结构基础尚不清楚。我们通过诱变研究了Glu446的作用。用天冬氨酸取代Glu446（E446D）导致对未修饰胞嘧啶的偏好，这是因为对5mC的亲和力降低。相反，用脯氨酸取代Glu446（E446P）使对5mC的亲和力提高了两个数量级。结构分析揭示了脯氨酸的脂肪族环状结构与嘧啶（5mC或T）的5-甲基之间的疏水相互作用。与野生型Klf4（E446）一样，446位的脯氨酸不直接与5mC的N4氮或胸腺嘧啶的O4氧相互作用。相反，未甲基化胞嘧啶的环外N4氨基（NH）及其环碳C5原子与E446D变体的天冬氨酸羧酸盐直接形成氢键。这两种相互作用都将导致对胞嘧啶的偏好超过胸腺嘧啶，后者可以解释E446D对未甲基化胞嘧啶的偏好。最后，我们在荧光素酶报告基因测定中评估了这些Klf4突变体调节甲基化和未甲基化启动子转录的能力。
