Otsuki Akihito, Suzuki Mikiko, Katsuoka Fumiki, Tsuchida Kouhei, Suda Hiromi, Morita Masanobu, Shimizu Ritsuko, Yamamoto Masayuki
Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan.
Center for Radioisotope Sciences, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan.
Free Radic Biol Med. 2016 Feb;91:45-57. doi: 10.1016/j.freeradbiomed.2015.12.005. Epub 2015 Dec 8.
Nrf2-small Maf (sMaf) heterodimer is essential for the inducible expression of cytoprotective genes upon exposure to oxidative and xenobiotic stresses. While the Nrf2-sMaf heterodimer recognizes DNA sequences referred to as the antioxidant/electrophile responsive element (ARE/EpRE), we here define these DNA sequences collectively as CNC-sMaf binding element (CsMBE). In contrast, large and small Maf proteins are able to form homodimers that recognize the Maf recognition element (MARE). CsMBE and MARE share a conserved core sequence but they differ in the 5'-adjacent nucleotide neighboring the core. Because of the high similarity between the CsMBE and MARE sequences, it has been unclear how many target binding sites and target genes are shared by the Nrf2-sMaf heterodimers and Maf homodimers. To address this issue, we introduced a substitution mutation of alanine to tyrosine at position 502 in Nrf2, which rendered the DNA-binding domain structure of Nrf2 similar to Maf, and generated knock-in mice expressing the Nrf2(A502Y) mutant. Our chromatin immunoprecipitation-sequencing analyses showed that binding sites of Nrf2(A502Y)-sMaf were dramatically changed from CsMBE to MARE in vivo. Intriguingly, however, one-quarter of the Nrf2(A502Y)-sMaf binding sites also bound Nrf2-sMaf commonly and vice versa. RNA-sequencing analyses revealed that Nrf2(A502Y)-sMaf failed to induce expression of major cytoprotective genes upon stress stimulation, which increased the sensitivity of Nrf2(A502Y) mutant mice to acute acetaminophen toxicity. These results demonstrate that the unique cistrome defined as CsMBE is strictly required for the Nrf2-sMaf heterodimer function in cytoprotection and that the roles played by CsMBE differ sharply from those of MARE.
Nrf2-小Maf（sMaf）异二聚体对于细胞在暴露于氧化应激和外源性应激时诱导细胞保护基因的表达至关重要。虽然Nrf2-sMaf异二聚体识别被称为抗氧化剂/亲电反应元件（ARE/EpRE）的DNA序列，但我们在此将这些DNA序列统称为CNC-sMaf结合元件（CsMBE）。相比之下，大Maf蛋白和小Maf蛋白能够形成识别Maf识别元件（MARE）的同二聚体。CsMBE和MARE共享一个保守的核心序列，但它们在核心相邻的5'侧翼核苷酸上有所不同。由于CsMBE和MARE序列之间的高度相似性，目前尚不清楚Nrf2-sMaf异二聚体和Maf同二聚体共享多少靶结合位点和靶基因。为了解决这个问题，我们在Nrf2的第502位引入了丙氨酸到酪氨酸的替换突变，这使得Nrf2的DNA结合结构域结构类似于Maf，并生成了表达Nrf2（A502Y）突变体的敲入小鼠。我们的染色质免疫沉淀测序分析表明，在体内，Nrf2（A502Y）-sMaf的结合位点从CsMBE显著转变为MARE。然而，有趣的是，四分之一的Nrf2（A502Y）-sMaf结合位点也通常与Nrf2-sMaf结合，反之亦然。RNA测序分析显示，在应激刺激下，Nrf2（A502Y）-sMaf未能诱导主要细胞保护基因的表达，这增加了Nrf2（A502Y）突变小鼠对急性对乙酰氨基酚毒性的敏感性。这些结果表明，定义为CsMBE的独特顺反组对于Nrf2-sMaf异二聚体在细胞保护中的功能是严格必需的，并且CsMBE所起的作用与MARE的作用截然不同。
