Hilton Traci L, Wang Edith H
University of Washington, Department of Pharmacology, School of Medicine, Health Sciences Center, Box 357280, Seattle, Washington 98195-7280, USA.
J Biol Chem. 2003 Apr 11;278(15):12992-3002. doi: 10.1074/jbc.M300412200. Epub 2003 Feb 4.
Cyclin D1 is an oncogene that regulates progression through the G(1) phase of the cell cycle. A temperature-sensitive missense mutation in the transcription factor TAF1/TAF(II)250 induces the mutant ts13 cells to arrest in late G(1) by decreasing transcription of cell cycle regulators, including cyclin D1. Here we provide evidence that TAF1 serves two independent functions, one at the core promoter and one at the upstream activating Sp1 sites of the cyclin D1 gene. Using in vivo genomic footprinting, we have identified protein-DNA interactions within the cyclin D1 core promoter that are disrupted upon inactivation of TAF1 in ts13 cells. This 33-bp segment, which we termed the TAF1-dependent element 1 (TDE1), contains an initiation site that displays homology to the consensus motif and is sufficient to confer a requirement for TAF1 function. Electrophoretic mobility shift assays reveal that binding of ts13-TAF1-containing TFIID complexes to the cyclin D1 TDE1 occurs at 25 degrees C but not at 37 degrees C in vitro and involves the initiator element. Temperature-dependent DNA binding activity is also observed for TAF1-TAF2 heterodimers assembled with the ts13 mutant but not the wild-type TAF1 protein. These data suggest that a function of TAF is required for the interaction of TFIID with the cyclin D1 initiator. Our finding that recruitment of TFIID, by insertion of a TBP binding site upstream of the TDE1, restores basal but not activated transcription supports the model that TAF1 carries out two independent functions at the cyclin D1 promoter.
细胞周期蛋白D1是一种癌基因，可调节细胞周期G1期的进程。转录因子TAF1/TAF（II）250中的一个温度敏感错义突变通过降低包括细胞周期蛋白D1在内的细胞周期调节因子的转录，诱导突变体ts13细胞在G1晚期停滞。在这里，我们提供证据表明TAF1具有两种独立的功能，一种在核心启动子处，另一种在细胞周期蛋白D1基因的上游激活Sp1位点处。使用体内基因组足迹法，我们在细胞周期蛋白D1核心启动子内鉴定了蛋白质-DNA相互作用，这种相互作用在ts13细胞中TAF1失活后被破坏。这个33bp的片段，我们称之为TAF1依赖元件1（TDE1），包含一个与共有基序具有同源性的起始位点，并且足以赋予对TAF1功能的需求。电泳迁移率变动分析表明，含ts13-TAF1的TFIID复合物与细胞周期蛋白D1 TDE1的结合在体外25℃时发生，但在37℃时不发生，并且涉及起始元件。对于与ts13突变体组装但不与野生型TAF1蛋白组装的TAF1-TAF2异二聚体，也观察到温度依赖性DNA结合活性。这些数据表明，TFIID与细胞周期蛋白D1起始元件的相互作用需要TAF的一种功能。我们的发现，即通过在TDE1上游插入一个TBP结合位点来招募TFIID，可恢复基础转录但不能恢复激活转录，支持了TAF1在细胞周期蛋白D1启动子处执行两种独立功能的模型。
