Systematic Analysis of Drosophila transcription factor binding specificities

描述(由申请人提供)：有机体基因组的完整注释需要系统地识别顺式调控序列和结合它们的反式作用因子。对于所有生物体来说，实现这一目标的一个重要障碍是转录因子(TF)的数量有限，这些转录因子具有良好的DNA结合特异性。我们已经开发了一种细菌单杂交系统，它提供了一种快速表征TF与DNA结合的特异性的方法。利用这项技术，我们已经确定了果蝇所有预测的序列特异性转录因子中15%(108/~750)的特异性。这个特异性目录包括代表12种不同类型的DNA结合域的蛋白质和所有84个独立的同源结构域家族成员。为了补充这个数据集，我们开发了计算工具来绘制TF结合位点频率的基因组分布图，并使用这些信息来识别我们数据集中任何TF组合的假定顺式调节模块(CRM)。基于网络的界面允许用户执行全基因组范围的CRM搜索，或者将TF或TF的组合的结合位点频率显示为流行的GBROWSE界面内的轨道。 我们现在建议表征所有剩余的黑腹果蝇转录因子的DNA结合特异性，包括所有单体和同源低聚转录因子，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中区分CRM的能力，并将整合其他数据源(如芯片数据集)，以增强预测CRM的能力。这一努力的最终结果将是开发可上网的数据库和搜索工具，使科学界能够通过计算确定由感兴趣的因素的任何组合管理的假定的标准对照。我们分析的结果将是针对在已知转录调控网络中起作用的因子子集的全基因组CRM注释。 到目前为止，还没有在任何生物体中获得对转铁蛋白特异性的完整描述。结合改进的计算工具和对黑腹果蝇转录的广泛和不断增长的实验研究，转铁蛋白特异性的目录将允许对整个基因组中的CRM进行系统的注释。一旦开发出来，这些数据库和工具应该直接适用于对包括人类在内的其他生物体中的CRM进行注释。与公共卫生相关：尽管基因组计划已经广泛地绘制了人类和其他生物中哪些DNA序列编码基因，但事实证明，绘制开启和关闭基因的调节区要困难得多。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。这张新的基因组图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.