Brown R Clark, Pattison Scott, van Ree Janine, Coghill Elise, Perkins Andrew, Jane Stephen M, Cunningham John M
Division of Experimental Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Mol Cell Biol. 2002 Jan;22(1):161-70. doi: 10.1128/MCB.22.1.161-170.2002.
Characterization of the mechanism(s) of action of trans-acting factors in higher eukaryotes requires the establishment of cellular models that test their function at endogenous target gene regulatory elements. Erythroid Krüppel-like factor (EKLF) is essential for beta-globin gene transcription. To elucidate the in vivo determinants leading to transcription of the adult beta-globin gene, functional domains of EKLF were examined in the context of chromatin remodeling and transcriptional activation at the endogenous locus. Human EKLF (hEKLF) sequences, linked to an estrogen-responsive domain, were studied with an erythroblast cell line lacking endogenous EKLF expression (J2eDeltaeklf). J2eDeltaeklf cells transduced with hEKLF demonstrated a dose-dependent rescue of beta-globin transcription in the presence of inducing ligand. Further analysis using a series of amino-terminal truncation mutants of hEKLF identified a distinct internal domain, which is sufficient for transactivation. Interestingly, studies of the chromatin structure of the beta-promoter revealed that a smaller carboxy-terminal domain generated an open promoter configuration. In vitro and in vivo binding studies demonstrated that this region interacted with BRG1, a component of the SWI/SNF chromatin remodeling complex. However, further study revealed that BRG1 interacted with an even smaller domain of EKLF, suggesting that additional protein interactions are required for chromatin remodeling at the endogenous beta-promoter. Taken together, our findings support a stepwise process of chromatin remodeling and coactivator recruitment to the beta-globin promoter in vivo. The J2eDeltaeklf inducible hEKLF system will be a valuable tool for further characterizing the temporal series of events required for endogenous beta-globin gene transcription.
对高等真核生物中转录因子作用机制的表征需要建立细胞模型，以在内源靶基因调控元件处测试其功能。红细胞Krüppel样因子（EKLF）对β-珠蛋白基因转录至关重要。为了阐明导致成人β-珠蛋白基因转录的体内决定因素，在内源基因座的染色质重塑和转录激活的背景下研究了EKLF的功能域。将与雌激素反应域相连的人EKLF（hEKLF）序列，用缺乏内源性EKLF表达的成红细胞系（J2eDeltaeklf）进行研究。用hEKLF转导的J2eDeltaeklf细胞在存在诱导配体的情况下表现出β-珠蛋白转录的剂量依赖性挽救。使用一系列hEKLF的氨基末端截短突变体进行的进一步分析确定了一个独特的内部结构域，该结构域足以进行反式激活。有趣的是，对β-启动子染色质结构的研究表明，一个较小的羧基末端结构域产生了开放的启动子构型。体外和体内结合研究表明，该区域与SWI/SNF染色质重塑复合物的一个组分BRG1相互作用。然而，进一步的研究表明，BRG1与EKLF的一个甚至更小的结构域相互作用，这表明在内源β-启动子处进行染色质重塑还需要其他蛋白质相互作用。综上所述，我们的研究结果支持了体内染色质重塑和共激活因子募集到β-珠蛋白启动子的逐步过程。J2eDeltaeklf诱导型hEKLF系统将是进一步表征内源性β-珠蛋白基因转录所需的时间序列事件的有价值工具。
