Ling Guoyu, Hauer Charles R, Gronostajski Richard M, Pentecost Brian T, Ding Xinxin
New York State Department of Health, and School of Public Health, Wadsworth Center, State University of New York, Empire State Plaza, Albany, NY 12201, USA.
J Biol Chem. 2004 Jul 2;279(27):27888-95. doi: 10.1074/jbc.M403705200. Epub 2004 Apr 28.
Rat CYP2A3 and its mouse and human orthologs are expressed preferentially in the olfactory mucosa. We found previously that an element in the proximal promoter region of CYP2A3 (the nasal predominant transcriptional activating (NPTA) element), which is similar to a nuclear factor 1 (NFI)-binding site, is critical for transcriptional activation of CYP2A3 in vitro. We proposed that this element might be important for tissue-selective CYP2A3 expression. The goals of the present study were to characterize NPTA-binding proteins and to obtain more definitive evidence for the role of NFI in the transcriptional activation of CYP2A3. The NPTA-binding proteins were isolated by DNA-affinity purification from rat olfactory mucosa. Mass spectral analysis indicated that isoforms corresponding to all four NFI genes were present in the purified NPTA-binding fraction. Further analysis of NPTA-binding proteins led to the identification of a novel NFI-A isoform, NFI-A-short, which was derived from alternative splicing of the NFI-A transcript. Transient transfection assay showed that NFI-A2, an NFI isoform previously identified in the olfactory mucosa, transactivated the CYP2A3 promoter, whereas NFI-A-short, which lacks the transactivation domain, counteracted the activation. Chromatin immunoprecipitation assays indicated that NFI proteins are associated with the CYP2A3 promoter in vivo, in rat olfactory mucosa, but essentially not in the liver where the CYP2A3 promoter is hypermethylated and CYP2A3 is not expressed. These data strongly support a role for NFI transcription factors in the transcriptional activation of CYP2A3.
大鼠CYP2A3及其小鼠和人类直系同源基因在嗅觉黏膜中优先表达。我们之前发现，CYP2A3近端启动子区域的一个元件（鼻优势转录激活（NPTA）元件），它类似于核因子1（NFI）结合位点，在体外对CYP2A3的转录激活至关重要。我们提出这个元件可能对CYP2A3的组织选择性表达很重要。本研究的目的是鉴定NPTA结合蛋白，并获得关于NFI在CYP2A3转录激活中作用的更确凿证据。通过DNA亲和纯化从大鼠嗅觉黏膜中分离出NPTA结合蛋白。质谱分析表明，纯化的NPTA结合组分中存在与所有四个NFI基因相对应的异构体。对NPTA结合蛋白的进一步分析导致鉴定出一种新的NFI-A异构体，即NFI-A-short，它源自NFI-A转录本的可变剪接。瞬时转染实验表明，之前在嗅觉黏膜中鉴定出的一种NFI异构体NFI-A2可激活CYP2A3启动子，而缺乏反式激活结构域的NFI-A-short则可抵消这种激活作用。染色质免疫沉淀实验表明，在大鼠嗅觉黏膜的体内，NFI蛋白与CYP2A3启动子相关联，但在肝脏中基本不相关，在肝脏中CYP2A3启动子发生高甲基化且CYP2A3不表达。这些数据有力地支持了NFI转录因子在CYP2A3转录激活中的作用。
