DNA recognition-element binding by GRHL/CP2-family and NKX2-1 transcription factors

转录基因调控依赖于转录因子(转录因子)对启动子或增强子中DNA靶序列的识别能力。这些分子识别过程通常容忍目标序列中的微小变异，结合亲和力只有适度的损失。与关于转录因子与其共同靶序列的结合几何结构的丰富结构数据形成鲜明对比的是，人们对这些转录因子与其结合位点的序列变体之间的结构和能量相互作用知之甚少。这类数据对于评估全基因组的转铁蛋白DNA结合模式和利用生物信息学工具预测突变事件的后果具有相当大的价值。我们建议研究两种不同类型的转录因子：类颗粒蛋白GRHL1和GRHL2以及甲状腺特异因子NKX2-1，我们已经确定了它们的初始结合数据和晶体结构。GRHL1/2和NKX2-1是具有代表性的转录因子，因为它们采用了不同的DNA序列识别和结合模式，有望对依赖序列变体的DNA结合过程产生互补的见解。此外，GRHL2和NKX2-1在上皮组织发育中起协同作用，最近的数据表明GRHL2具有开创性的转铁蛋白作用。我们将确定GRHL1/2和NKX2-1与全基因组作图研究中确定的数百个DNA靶序列的结合参数，并分析这些蛋白质与数十个靶序列结合的晶体结构。我们将使用体内系统验证DNA序列变异对TF-DNA结合的影响。从我们的结构和生物物理数据中，我们的目标是推导出TF与增强子和启动子结合的通用规则，这将有助于RU的生物信息学家改进他们的算法，以识别和预测非编码基因组中的致病变体。这反过来将使临床科学家能够确定更多导致患者罕见疾病的潜在遗传原因。

Transcriptional gene regulation depends on the transcription factors’ (TFs) ability to faithfully recognize DNA target sequences in promoters or enhancers. These molecular recognition pro-cesses usually tolerate small variations within the target sequence with only moderate loss in binding affinity. In stark contrast to the wealth of structural data available on the binding geometries of TFs to their consensus target sequences, comparatively little is known about the structural and energetic interactions these TFs have with sequence variants of their binding sites. Data of this kind have considerable value for assessing genome-wide TF DNA binding patterns and for predicting consequences of mutational events by bioinformatics tools. We propose to study two distinct types of TFs: the Grainyhead-like proteins GRHL1 and GRHL2 and the thyroid-specific factor NKX2-1, for which we have determined initial binding data and crystal structures. GRHL1/2 and NKX2-1 are representative TFs because they employ distinct modes of DNA sequence recognition and binding, promising to yield complementary insight into the sequence variant-dependent DNA binding process. In addition, GRHL2 and NKX2-1 act cooperatively in epithelial tissue development, with recent data suggesting a pioneering TF role for GRHL2. We will determine the binding parameters of GRHL1/2 and NKX2-1 to hundreds of DNA target sequences identified in genome-wide mapping studies and analyze the crystal structures of these proteins bound to tens of target sequences. We will validate the impact of DNA sequence variation on TF-DNA binding using in vivo systems. From our structural and biophysical data we aim to derive generalizable rules for TF binding to enhancers and promoters that will help the RU’s bioinformaticians to improve their algorithms for identification and prediction of disease-causing variants in the non-coding genome. This in turn will enable clinical scientists to identify a larger number of underlying genetic causes of the rare diseases seen in their patients.