Hume David A, Sasmono Tedjo, Himes S Roy, Sharma Sudarshana M, Bronisz Agnieszka, Constantin Myrna, Ostrowski Michael C, Ross Ian L
Australian Research Council Special Research Centre for Functional and Applied Genomics and the Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland, Australia.
J Immunol. 2008 May 15;180(10):6733-42. doi: 10.4049/jimmunol.180.10.6733.
Many macrophage-specific promoters lack classical transcriptional start site elements such as TATA boxes and Sp1 sites. One example is the CSF-1 receptor (CSF-1R, CD115, c-fms), which is used as a model of the transcriptional regulation of macrophage genes. To understand the molecular basis of start site recognition in this gene, we identified cellular proteins binding specifically to the transcriptional start site (TSS) region. The mouse and human csf1r TSS were identified using cap analysis gene expression (CAGE) data. Conserved elements flanking the TSS cluster were analyzed using EMSAs to identify discrete DNA-binding factors in primary bone marrow macrophages as candidate transcriptional regulators. Two complexes were identified that bind in a highly sequence-specific manner to the mouse and human TSS proximal region and also to high-affinity sites recognized by myeloid zinc finger protein 1 (Mzf1). The murine proteins were purified by DNA affinity isolation from the RAW264.7 macrophage cell line and identified by mass spectrometry as EWS and FUS/TLS, closely related DNA and RNA-binding proteins. Chromatin immunoprecipitation experiments in bone marrow macrophages confirmed that EWS, but not FUS/TLS, was present in vivo on the CSF-1R proximal promoter in unstimulated primary macrophages. Transfection assays suggest that EWS does not act as a conventional transcriptional activator or repressor. We hypothesize that EWS contributes to start site recognition in TATA-less mammalian promoters.
许多巨噬细胞特异性启动子缺乏经典的转录起始位点元件，如TATA盒和Sp1位点。一个例子是集落刺激因子1受体（CSF-1R，CD115，c-fms），它被用作巨噬细胞基因转录调控的模型。为了了解该基因起始位点识别的分子基础，我们鉴定了与转录起始位点（TSS）区域特异性结合的细胞蛋白。利用帽分析基因表达（CAGE）数据鉴定了小鼠和人类csf1r的TSS。使用电泳迁移率变动分析（EMSA）分析TSS簇两侧的保守元件，以鉴定原代骨髓巨噬细胞中离散的DNA结合因子作为候选转录调节因子。鉴定出两种复合物，它们以高度序列特异性的方式与小鼠和人类TSS近端区域结合，也与髓系锌指蛋白1（Mzf1）识别的高亲和力位点结合。通过DNA亲和分离从RAW264.7巨噬细胞系中纯化出小鼠蛋白，并通过质谱鉴定为EWS和FUS/TLS，它们是密切相关的DNA和RNA结合蛋白。骨髓巨噬细胞中的染色质免疫沉淀实验证实，在未刺激的原代巨噬细胞中，CSF-1R近端启动子在体内存在EWS，但不存在FUS/TLS。转染实验表明，EWS不作为传统的转录激活剂或抑制剂。我们假设EWS有助于无TATA的哺乳动物启动子中的起始位点识别。
