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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Identification of target genes in breast cancer cells directly regulated by the SRC-3/AIB1 coactivator.
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Identification of target genes in breast cancer cells directly regulated by the SRC-3/AIB1 coactivator.

机译：在SRC-3 / AIB1共激活因子直接调控的乳腺癌细胞中鉴定靶基因。

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Steroid receptor coactivator-3 (SRC-3/AIB1) is a coactivator for nuclear receptors and other transcription factors and an oncogene that contributes to growth regulation and development of mammary and other tumor types. Because of its biological functions, it is important to identify genes regulated by SRC-3. However, because coactivators do not bind DNA directly, extensive work is required to determine whether genes identified by RNA profiling approaches are direct or indirect targets. Here, we report the use of chromatin immunoprecipitation (ChIP)-based assays that involve genomic mapping and computational analyses of immunoprecipitated DNA to identify SRC-3-binding target genes in estradiol (E2)-treated MCF-7 breast cancer cells. We identified 18 SRC-3 genomic binding sites and demonstrated estrogen receptor-alpha (ERalpha) binding to all of them. Both E2-dependent and -independent SRC-3/ERalpha-binding sites were identified. RNA polymerase II ChIP assays were used to determine the correlation between SRC-3 and ERalpha binding and recruitment of the transcriptional machinery. These assays, in conjunction with analyses of RNA obtained from E2-treated cells, lead to the identification of SRC-3/ERalpha-associated genes. The ability of SRC family coactivators to regulate the expression of one of these genes, PARD6B/Par6, was confirmed by using cells individually depleted of SRC-1, SRC-2, or SRC-3 by small interfering RNA. The method described herein can be used to identify genes regulated by non-DNA-binding factors, such as other coactivators or corepressors, as well as DNA-binding transcription factors, and provides information on their binding location that can accelerate further gene characterization.
机译：类固醇受体共激活因子3（SRC-3 / AIB1）是核受体和其他转录因子的共激活因子，也是致癌基因，可促进乳腺和其他肿瘤类型的生长调节和发育。由于其生物学功能，重要的是鉴定受SRC-3调控的基因。但是，由于共激活因子不能直接结合DNA，因此需要大量工作来确定通过RNA分析方法鉴定的基因是直接还是间接靶标。在这里，我们报告使用基于染色质免疫沉淀（ChIP）的检测方法，该方法涉及基因组定位和免疫沉淀DNA的计算分析，以鉴定雌二醇（E2）治疗的MCF-7乳腺癌细胞中的SRC-3结合靶基因。我们确定了18个SRC-3基因组结合位点，并证明了雌激素受体-α（ERalpha）绑定到所有这些。确定了E2依赖性和非依赖性的SRC-3 / ERalpha结合位点。 RNA聚合酶II ChIP分析用于确定SRC-3和ERalpha结合与转录机制募集之间的相关性。这些测定，结合从E2处理的细胞中获得的RNA分析，可鉴定出SRC-3 / ERalpha相关基因。 SRC家族共激活因子调节这些基因之一PARD6B / Par6的表达的能力通过使用小干扰RNA分别耗尽了SRC-1，SRC-2或SRC-3的细胞来证实。本文描述的方法可用于鉴定受非DNA结合因子（例如其他共激活因子或共加压因子）以及DNA结合转录因子调控的基因，并提供有关其结合位置的信息，这些信息可加速进一步的基因表征。

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