Chen Li, Peng Zhihua, Bateman Erik
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington, VT 05405, USA.
Nucleic Acids Res. 2004 Feb 19;32(4):1251-60. doi: 10.1093/nar/gkh297. Print 2004.
Transcription of the TATA box binding protein (TBP) gene in Acanthamoeba castellanii is regulated by TATA box binding protein promoter binding factor (TPBF), which binds to an upstream TBP promoter element to stimulate transcription, and to a TATA proximal element, where it represses transcription. In order to extend these observations to the in vivo chromatin context, the TBP gene was examined by in situ footprinting and chromatin immunoprecipitation (ChIP). Acanthamoeba DNA is nucleosomal with a repeat of approximately 160 bp, and an intranucleosomal DNA periodicity of 10.5 bp. The TBP gene comprises a 220 bp micrococcal nuclease hypersensitive site corresponding to the promoter regulatory elements previously identified, flanked by protected regions of a size consistent with the presence of nucleosomes. ChIP data indicated that TPBF is associated with the TBP, TPBF and MIL gene promoters, but not to the CSP21, MIIHC, 5SrRNA or 39SrRNA promoters, or to the MIL gene C-terminal region. Binding by TPBF to the TPBF and MIL gene promoters was confirmed by in vitro assays. These results validate the in vitro model for TBP gene regulation and further suggest that TPBF may be autoregulated and may participate in the regulation of the MIL gene.
卡氏棘阿米巴中TATA框结合蛋白（TBP）基因的转录受TATA框结合蛋白启动子结合因子（TPBF）调控，该因子与上游TBP启动子元件结合以刺激转录，并与TATA近端元件结合，在该元件处它抑制转录。为了将这些观察结果扩展到体内染色质环境，通过原位足迹法和染色质免疫沉淀（ChIP）对TBP基因进行了检测。卡氏棘阿米巴DNA是核小体形式的，重复单元约为160 bp，核小体内DNA周期性为10.5 bp。TBP基因包含一个220 bp的微球菌核酸酶超敏位点，对应于先前鉴定的启动子调控元件，两侧是与核小体存在一致大小的保护区域。ChIP数据表明，TPBF与TBP、TPBF和MIL基因启动子相关，但与CSP21、MIIHC、5SrRNA或39SrRNA启动子或MIL基因C末端区域无关。通过体外试验证实了TPBF与TPBF和MIL基因启动子的结合。这些结果验证了TBP基因调控的体外模型，并进一步表明TPBF可能是自我调控的，且可能参与MIL基因的调控。
