Universal, Compact Combinatorial Microarrays for DNA Binding Site Discovery

描述(申请人提供)：转录因子(TF)和它们的DNA结合位点之间的相互作用是细胞内调控网络的组成部分。这些相互作用控制着发育和对环境压力的反应的关键步骤，在人类中，它们的功能障碍可能会导致各种疾病的进展。到目前为止，只有一小部分序列特异的转录因子被很好地表征，足以让我们知道它们可以结合的所有序列，同样重要的是，它们不能结合的序列。这种结合位点序列数据的稀疏性是非常有问题的，因为这些稀疏数据集然后被用来搜索这些位点的基因组出现，并预测了许多假阳性和假阴性结合位点。生物界最终需要的是关于所有可能的DNA序列变体的更完整的Tf结合位点数据。这些数据将使我们能够提高预测基因组序列中功能顺式调控元件的准确性。我们已经计算出了我们认为是所有可能结合位点的最紧凑的表示，它仍然允许恢复DNA结合分子的序列特异性。这项技术的优点是，所有可能的DNA序列变体都可以在DNA微阵列上以空间和成本高效的方式表示，因此只需要合成最少数量的单个DNA序列和单个DNA斑点。在这个项目中，我们将：(1)开发紧凑型组合DNA微阵列在蛋白质结合微阵列(PBM)实验中的应用，以确定序列特异性TF的所有可能的DNA结合位点；(2)使用紧凑型组合DNA微阵列确定-15酿酒酵母TF的所有可能的DNA结合位点的结合亲和力，并创建这些数据的数据库；以及(3)评估完整的结合特异性数据和结合亲和力数据对于改进体内TF结合位点预测的有效性。目前还没有其他技术可以像紧凑的组合DNA微阵列PBM技术那样高通量地确定所有候选DNA结合位点与TFS的相对结合亲和力。这些研究应该能够更好地理解真核基因组中转铁蛋白结合位点的结合亲和力的重要性。这样的数据还可以提高在高等真核基因组中预测顺式调节模块的准确性。

DESCRIPTION (provided by applicant): The interactions between transcription factors (TFs) and their DNA binding sites are an integral part of the regulatory networks within cells. These interactions control critical steps in development and responses to environmental stresses, and in humans their dysfunction can contribute to the progression of various diseases. Thus far only a small handful of sequence-specific TFs have been characterized well enough for us to know all the sequences that they can, and just as importantly, can not bind. This sparseness of this binding site sequence data is highly problematic, because these sparse datasets are then used to search for genomic occurrences of these sites, with many false positive and false negative binding sites being predicted. Ultimately what the biological community needs is much more complete TF binding site data on all possible DNA sequence variants. These data will allow us to improve the accuracy with which we can predict functional cis regulatory elements within genomic sequence. We have calculated what we believe is a maximally compact representation of all possible binding sites that still allows the sequence specificities of DNA binding molecules to be recovered. The advantage of this technology is that all possible DNA sequence variants can be represented on DNA microarrays in a space- and cost- efficient manner, so that only a minimal number of individual DNA sequences and individual DNA spots need to be synthesized. In this project, we will: (1) develop the use of compact combinatorial DNA microarrays in protein binding microarray (PBM) experiments for identifying all possible DNA binding sites of sequence-specific TFs; (2) determine the binding affinities of all possible DNA binding sites for -15 Saccharomyces cerevisiae TFs using compact combinatorial DNA microarrays and create a database of these data; and (3) evaluate the utility of complete binding specificity data and binding affinity data for improved prediction of in vivo TF binding sites. There exists no other technology for the determination of the relative binding affinities of all candidate DNA binding sites for TFs that is as high-throughput as the compact combinatorial DNA microarray PBM technology. These studies should permit a better understanding of the importance of the binding affinities of TF binding sites in eukaryotic genomes. Such data may also increase the accuracy with which cis regulatory modules can be predicted in higher eukaryotic genomes.