Systematic Analysis of Drosophila transcription factor binding specificities

描述（由申请人提供）：生物体基因组的完整注释需要系统识别顺式调节序列和结合它们的反式作用因子。对于所有生物体，这一目标的一个重要障碍是具有良好特征的DNA结合特异性的转录因子（TF）数量有限。我们已经开发了一种细菌单杂交系统，提供了一种快速的方法来表征DNA结合特异性的TF。利用这种技术，我们已经确定了15%（108/~750）的所有预测的序列特异性转录因子在果蝇中的特异性。该特异性目录包括代表12种不同类型的DNA结合结构域和所有84个独立同源结构域家族成员的蛋白质。为了补充这一数据集，我们已经开发了计算工具，映射TF结合位点频率的基因组分布，并使用这些信息来确定推定的顺式调节模块（CRM）的任何组合的TF在我们的数据集。一个基于网络的界面允许用户执行全基因组搜索的标准物质或显示结合位点频率的TF或TF的组合作为轨道内流行的Gbrowse界面。 我们现在建议表征所有剩余D的DNA结合特异性。黑腹动物TF，包括所有单体和同源寡聚TF以及来自碱性亮氨酸拉链和碱性螺旋-环-螺旋家族的所有功能性异二聚体组合。我们还将改进我们的计算工具，以提高它们在基因组内区分CRM的能力，我们将整合其他数据源（例如ChIP芯片数据集），以提高预测CRM的能力。这一努力将最终在网络访问的数据库和搜索工具，使科学界能够计算确定由任何感兴趣的因素的组合调节推定的标准物质的发展。我们的分析的结果将是全基因组注释的标准物质的子集的因素，在已知的转录调控网络的功能。 迄今为止，尚未在任何生物体中获得TF特异性的完整描述。结合改进的计算工具和广泛和不断增长的实验研究，D。黑腹转录，TF特异性的目录将允许在其整个基因组中对CRM进行系统注释。一旦开发出来，这些数据库和工具应该直接适用于其他生物体（包括人类）的标准物质注释。公共卫生相关性：尽管基因组计划已经广泛地绘制了人类和其他生物体中编码基因的DNA序列，但绘制基因开启和关闭的调控区域被证明要困难得多。我们将使用新开发的实验和计算工具来系统地绘制整个基因组中的这些控制元件。这一新的基因组“图谱”将帮助研究人员了解这些元素在正常细胞中的功能，以及这些元素的突变如何导致疾病。

DESCRIPTION (provided by applicant): The full annotation of an organism's genome requires the systematic identification of cis-regulatory sequences and the trans-acting factors that bind them. For all organisms, a significant remaining impediment to this goal is the limited number of transcription factors (TFs) with well-characterized DNA-binding specificities. We have developed a bacterial one-hybrid system that provides a rapid method to characterize the DNA-binding specificities of TFs. Using this technology, we have determined the specificity of 15% (108/~750) of all of the predicted sequence-specific transcription factors in Drosophila melanogaster. This catalog of specificities includes proteins representing 12 different types of DNA-binding domains and all 84 independent homeodomain family members. To complement this dataset we have developed computational tools that map the genomic distribution of TF binding site frequencies and use this information to identify putative cis-regulatory modules (CRMs) for any combination of TFs in our dataset. A web-based interface allows users to perform genome-wide searches for CRMs or to display binding site frequencies for TFs or combinations of TFs as tracks within the popular Gbrowse interface. We now propose to characterize the DNA-binding specificity of all remaining D. melanogaster TFs, including all monomeric and homo-oligomeric TFs as well as all functional heterodimeric combinations from the basic leucine zipper and basic helix-loop-helix families. We will also refine our computational tools to improve their ability to distinguish CRMs within the genome and we will integrate other data sources (e.g. ChIP-chip datasets) to enhance the ability to predict CRMs. This effort will culminate in the development of web-accessible database and search tools that will allow the scientific community to computationally identify putative CRMs that are regulated by any combination of factors of interest. An outgrowth of our analysis will be genome-wide annotations of CRMs for subsets of factors that function in known transcriptional regulatory networks. To date, a complete description of TF specificities has not been obtained in any organism. Combined with improved computational tools and the extensive and growing body of experimental studies on D. melanogaster transcription, a catalog of TF specificities will allow the systematic annotation of CRMs throughout its genome. Once developed, these databases and tools should be directly applicable to the annotation of CRMs in other organisms, including humans. PUBLIC HEALTH RELEVANCE: Although the genome project has extensively mapped which DNA sequences in humans and other organisms encode genes, mapping the regulatory regions that turn genes on and off has proven to be much more difficult. We will use newly developed experimental and computational tools to systematically map these control elements in an entire genome. This new genome "map" will help researchers understand how these elements function in normal cells and how mutations in these elements can lead to disease.