BindDB: Oct4 Case Study

Epigenomic Profiling of Oct4 Promoter Due to the flexibility of the BindDB platform, more general questions related to epigenetic regulation in the ESC context can be asked. For example, how are the alternative promoters of the stem cell specific transcription factor OCT4 regulated? In both mESCs and hESCs, two main isoforms of Oct4 exist (Oct4A, Oct4B), which differ by approximately 3kb in their 5' regions and have the potential to be regulated by two distinct promoter regions (Wang and Dai, 2010). Interestingly, Oct4A and Oct4B display different temporal and spatial expression patterns (Cauffman et al., 2006). It has been hypothesized that the Oct4A is the pluripotent promoter, whereas, Oct4B is related to stress responses (Wang et al., 2009). To study these promoters in greater depth, we queried ‘Pou5f1’ in BindDB (Figure 2A). The tool automatically discerns the presence of two alternative promoters while searching for the gene's genomic location and directs the user to a heatmap of per-promoter results. From the heatmap, it is apparent that both promoters share some of their epigenetic signature including H3K4me3, H3K27ac and H3K9ac active chromatin marks (Figure 2B). Also, the OCT4 protein itself binds both of its promoters, along with other pluripotent factors such as UTX and KLF4. Beyond this common epigenetic core, the two promoters differ widely: The Oct4A promoter of the longer isoform displays binding for a plethora of factors and evidence of RNAPII suggests that Oct4 is actively transcribed in ESCs. In general, this type of large-scale epigenetic profile is characteristic of active gene regulation and goes far beyond the simple histone modification code. On the other hand, the Oct4B promoter exhibits binding for an entirely different set of factors including SMC1, SMC3, SA1, SA2, RAD21, and CTCF, the main components of the structural Cohesin complex. It is also bound by ZC3H11A, which we found to cluster globally with the Cohesin complex (data not shown). Cohesin binding to the Oct4B promoter suggests that a specific enhancer may be driving the expression of this second isoform in ESCs. On the other hand, the mediator proteins MED1 and MED12 bind the first promoter and do not cluster with the Cohesin complex as they are proposed to do. These findings may be particularly insightful into discovering the molecular mechanisms governing alternative promoter regulation of Oct4 and genes in general.Oct4启动子的表观基因组谱分析（Epigenomic Profiling of Oct4 Promoter）：鉴于BindDB平台的灵活性，我们可针对胚胎干细胞（Embryonic Stem Cell, ESC）背景下的表观遗传调控提出更多一般性研究问题。例如，干细胞特异性转录因子OCT4的可变启动子是如何被调控的？在小鼠胚胎干细胞（mouse Embryonic Stem Cell, mESCs）与人胚胎干细胞（human Embryonic Stem Cell, hESCs）中，Oct4存在两种主要同工型（Oct4A、Oct4B），二者的5'端区域差异约3kb，且理论上可被两个不同的启动子区域调控（Wang与Dai, 2010）。有趣的是，Oct4A与Oct4B展现出不同的时空表达模式（Cauffman等, 2006）。已有研究假说指出，Oct4A为多能性启动子，而Oct4B则与应激反应相关（Wang等, 2009）。为更深入地探究这两类启动子，我们在BindDB平台中检索了基因“Pou5f1”（图2A）。该工具在检索目标基因的基因组位置时，可自动识别两个可变启动子的存在，并引导用户查看对应各启动子的结果热图。从热图结果可见，两个启动子共享部分表观遗传特征，包括H3K4me3、H3K27ac及H3K9ac等活性染色质标记（图2B）。此外，OCT4蛋白自身可结合这两个启动子，同时结合的还有UTX、KLF4等其他多能性调控因子。除上述共同的表观遗传核心特征外，两类启动子还存在显著差异：较长同工型对应的Oct4A启动子可结合大量调控因子，且RNA聚合酶II（RNA Polymerase II, RNAPII）的结合证据表明，Oct4在ESC中处于活跃转录状态。一般而言，这类大规模表观基因组谱是活跃基因调控的典型特征，其调控机制远超简单的组蛋白修饰编码范畴。与之相反，Oct4B启动子结合的则是完全不同的一类因子，包括黏连蛋白复合物（Cohesin complex）的核心组分SMC1、SMC3、SA1、SA2、RAD21及CTCF。该启动子还可结合ZC3H11A，我们的研究发现该蛋白可与黏连蛋白复合物在全基因组范围内共聚类（数据未展示）。黏连蛋白结合Oct4B启动子这一现象提示，存在特定增强子驱动该第二同工型在ESC中的表达。另一方面，中介体蛋白MED1与MED12可结合Oct4A启动子，且并不如预期般与黏连蛋白复合物共聚类。上述研究结果或可为揭示调控Oct4乃至其他基因可变启动子的分子机制提供重要见解。