Shago M, Giguére V
Molecular Oncology Group, Royal Victoria Hospital, Montréal, Québec, Canada.
Mol Cell Biol. 1996 Aug;16(8):4337-48. doi: 10.1128/MCB.16.8.4337.
One of the primary goals in transcription factor research is the elucidation of the genetic networks controlled by a factor or by members of a family of closely related factors. The pleiotropic effects of retinoic acid (PA) in the developing and adult animal are mediated by ligand-inducible transcription factors (RA receptors [RARs] and retinoid X receptors [RXRs]) that belong to the superfamily of nuclear receptors. Regulatory regions of PA effector genes contain RAR and RXR binding sites (RAR elements [RAREs] and RXR elements [RXREs]) that generally consist of direct or everted repeats of the core half-site motif, (A/G)G(G/T)TCA. In order to identify novel genes regulated by RA, we devised a selection strategy based on the premise that regulatory regions of a large number of housekeeping and tissue-specific genes are embodied within CpG island DNA. In this method, referred to as CpG-selected and amplified binding, fragments derived from the CpG island fraction of the murine genome are selected by a gel mobility shift assay using in vitro-transcribed and -translated RXR-RAR. Multiple rounds of selection coupled with amplification of the fragments by PCR enabled us to clone a population of CG-rich fragments of which approximately one-fifth contained consensus RAREs or RXREs. Twelve genomic fragments containing novel response elements are described, and the transcription unit associated with one of them, NN-84AG, was characterized in detail. The mouse NN-84AG transcript is upregulated by RA in F9 embryonal carcinoma cells and is homologous to an expressed sequence tag (EST41159) derived from a human infant brain cDNA library. Cloning of the murine NN8-4AG genomic sequence places the RXRE in the proximity of the transcription initiation sites of the gene. Although sequence analysis indicates that the EST41159 gene product is novel, a region of amino acid identity with sequences of a yeast polypeptide of, as yet, unknown function and the Drosophila trithorax protein suggests the presence of an evolutionarily and functionally conserved domain. Our study demonstrates that transcription factor binding sites and corresponding regulated genes can be identified by selecting fragments derived from the CpG island fraction of the genome.
转录因子研究的主要目标之一是阐明由一个因子或密切相关因子家族的成员所控制的基因网络。视黄酸（RA）在发育中和成年动物中的多效性作用是由属于核受体超家族的配体诱导型转录因子（RA受体[RARs]和视黄酸X受体[RXRs]）介导的。RA效应基因的调控区域包含RAR和RXR结合位点（RAR元件[RAREs]和RXR元件[RXREs]），它们通常由核心半位点基序(A/G)G(G/T)TCA的直接或反向重复组成。为了鉴定受RA调控的新基因，我们基于大量管家基因和组织特异性基因的调控区域存在于CpG岛DNA中的前提设计了一种筛选策略。在这种称为CpG选择和扩增结合的方法中，使用体外转录和翻译的RXR - RAR通过凝胶迁移率变动分析从鼠基因组的CpG岛部分衍生的片段。多轮选择以及通过PCR对片段进行扩增使我们能够克隆一群富含CG的片段，其中约五分之一包含共有RAREs或RXREs。描述了12个含有新反应元件的基因组片段，并详细表征了与其中一个片段NN - 84AG相关的转录单元。小鼠NN - 84AG转录本在F9胚胎癌细胞中被RA上调，并且与源自人类婴儿脑cDNA文库的一个表达序列标签（EST41159）同源。鼠NN8 - 4AG基因组序列的克隆将RXRE置于该基因转录起始位点附近。尽管序列分析表明EST41159基因产物是新的，但与功能未知的酵母多肽和果蝇三体胸蛋白的序列存在氨基酸同一性区域，这表明存在进化上和功能上保守的结构域。我们的研究表明，通过选择源自基因组CpG岛部分的片段可以鉴定转录因子结合位点和相应的调控基因。
