Llewellyn Katrina J, Cary Peter D, McClellan James A, Guille Matthew J, Scarlett Garry P
Biophysics laboratories, School of Biological Sciences, IBBS, University of Portsmouth, Portsmouth, PO1 2DT, UK.
Biochim Biophys Acta. 2009 Nov-Dec;1789(11-12):675-80. doi: 10.1016/j.bbagrm.2009.07.007. Epub 2009 Aug 7.
We have previously shown that a critical region of the gata2 promoter contains an inverted CCAAT box and adopts a partial A-form DNA structure in vitro. At gastrula stages of development transcription requires binding of CBTF (CCAAT box transcription factor), a multi-subunit transcription factor, to this region. Xilf3 is one component of CBTF and the double stranded RNA binding domains (dsRBDs) of Xilf3 must be active for both binding to, and transcription from, this promoter. Here we determine the contribution of DNA sequence and structure at the gata2 promoter to transcriptional activity. In all the constructs we tested a CCAAT box was a requirement for full activity. However, base substitutions that increase B-form structure propensity in the sequences flanking the CCAAT box are equally able to decrease activity even if a CCAAT box is present. In contrast, mutations that maintain A-form propensity in these regions also maintain, or increase, transcription factor binding and transcriptional activity. We propose a two-component model for the interaction of CBTF with the gata2 promoter, requiring both a CCAAT sequence and flanking A-form DNA structures. These results support a novel role for dsRBDs in transcriptional regulation and suggest a function for A-form DNA in vivo.
我们之前已经表明，gata2启动子的一个关键区域包含一个反向CCAAT框，并且在体外呈现部分A-DNA结构。在原肠胚发育阶段，转录需要多亚基转录因子CBTF（CCAAT框转录因子）与该区域结合。Xilf3是CBTF的一个组分，并且Xilf3的双链RNA结合结构域（dsRBDs）对于与该启动子的结合以及从该启动子进行转录都必须是活性的。在此我们确定gata2启动子处的DNA序列和结构对转录活性的贡献。在我们测试的所有构建体中，一个CCAAT框是充分活性所必需的。然而，在CCAAT框侧翼序列中增加B-DNA结构倾向的碱基替换即使存在CCAAT框也同样能够降低活性。相反，在这些区域维持A-DNA结构倾向的突变也能维持或增加转录因子结合及转录活性。我们提出了一个CBTF与gata2启动子相互作用的双组分模型，既需要一个CCAAT序列又需要侧翼A-DNA结构。这些结果支持了dsRBDs在转录调控中的新作用，并暗示了A-DNA在体内的一种功能。
