Sox3: A transcription factor for Cyp19 expression in the frog Rana rugosa
Sox3: A transcription factor for Cyp19 expression in the frog Rana rugosa
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DOI：
10.1016/j.gene.2009.05.011
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发表时间：
2009-09-15
期刊：
影响因子：
3.5
通讯作者：
Nakamura, Masahisa
中科院分区：
文献类型：
--
作者：
Oshima, Yuki;Naruse, Kiyoshi;Nakamura, Masahisa
Cyp19在青蛙Rana rugosa的雌性蝌蚪的性腺中在性别决定期间以高水平表达。为了鉴定对Cyp19表达重要的序列元件，我们分离了携带R. rugosa Cyp19的5 ′-侧翼区的核苷酸序列，以寻找潜在的转录因子结合位点。Sox（SRY相关HIVIG盒）蛋白和Sf1结合位点被发现在Cyp19的卵巢特异性启动子区。由于Sox 3位于R. rugosa，我们使用启动子区域在非洲爪蟾A6细胞中进行了荧光素酶报告基因测定。Sox 3在细胞中驱动报告基因。但Sf 1不这样。当进行2.7kbp的Cyp 19启动子区的顺序缺失时，跨越核苷酸-191至+48的片段足以驱动报告基因的转录。在定点突变中，该区域中-57处的结合位点对于Sox 3反应性是关键的。缺少HMG box的Sox 3不能促进Cyp19的转录。此外，染色质免疫沉淀（ChIP）试验表明，DNA片段富集8倍，如通过实时PCR确定的，当染色质与抗His抗体对His标记的Sox3免疫沉淀。这些结果表明，Sox 3通过直接结合Cyp19启动子区的结合位点来激活Cyp19的转录。Sox 3似乎是一个指导无关性腺发育成卵巢的因子。皱纹。(C)2009 Elsevier B.V.保留所有权利。
Cyp19 is expressed at a high level in the gonad of the female tadpole of the frog Rana rugosa during sex determination. To identify sequence elements important for expression of Cyp19, we isolated a genomic clone (similar to 40 kbp) carrying R. rugosa Cyp19 and analyzed the nucleotide sequence of the 5'-flanking region to search for potential transcription factor binding sites. Sox (SRY-related HIVIG box) protein and Sf1 binding sites were found in the ovary-specific promoter region of Cyp19. Because Sox3 is located on the sex chromosome in R. rugosa, we conducted the luciferase reporter assay in Xenopus A6 cells using the promoter region. Sox3 drove the reporter gene in the cells. but Sf1 did not When sequential deletion of the 2.7 kbp Cyp 19-promoter region was undertaken, a fragment spanning nucleotides -191 to +48 was sufficient to drive the transcription of the reporter gene. In site-directed mutagenesis, the binding site at -57 in the region was critical for Sox3 responsiveness. Sox3 lacking the HMG box had no ability to promote Cyp19 transcription. In addition, a chromatin immunoprecipitation (ChIP) assay showed that DNA fragments were enriched 8-fold, as determined by real-time PCR, when chromatin was immunoprecipitated with the anti-His antibody against His-tagged Sox3. The results, taken together, suggest that Sox3 activates Cyp19 transcription by its direct binding to the binding site of the Cyp19 promoter region. Sox3 appears to be a factor that directs indifferent gonads to develop into an ovary in R. rugosa. (C) 2009 Elsevier B.V. All rights reserved.