Hox Control of Morphogenesis via Co-Evolution of Numbers and Affinities of Hox Binding Sites with Transcription Factor Concentrations

Hox转录因子家族(Tf)通过高水平的转录特异性驱动体节沿前后(A/P)轴的形态多样化。体内沿A/P轴的形态特征的精确分配与Hox TF在体外较差的DNA结合严格性形成对比，因为它们识别经常遇到的DNA序列。显然，其他调节蛋白和结合序列的差异，特别是低亲和力位点，在赋予HOX蛋白特异性方面起着关键作用。尽管我们对HOX在体内的功能有了更多的了解，但HOX低亲和力结合位点的出现也带来了新的问题。具体地说，高亲和力和低亲和力结合位点对HOX靶基因调控和形态发生的贡献尚不清楚，也不清楚其他同源结构域TF是否使用相同的机制，通过降低亲和力来增加特异性，从而促进不同类型HOX结合位点的进化。为了回答这些问题，我们建立了一个模型，即HOX TF变形(DFD)对AP-2基因的调控。这种调节相互作用依赖于低亲和力和高亲和力DFD结合位点的组合，控制着一个形态特征，即上颌骨环，并发生在表达其他同源结构域TF的细胞中。我们将剖析Hox Tf DFD如何通过干扰Hox位点的数量和亲和力来读取AP-2增强子中编码的调控信息，并在基于报告的分析中调节DFD的区域表达。我们将使用特定区域的过度表达和击倒来测试与DFD相似的共识序列相互作用的同源结构域TF对AP-2转录的贡献。我们将通过在UAS-GAL4救援环境中测试它们的修改对上颌环状结构形成的影响，以及使用CRISPR/CAS9设计内源性基因座来分析这些特征对形态发生的贡献。最后，我们将分析帮助DFD控制AP-2表达的调节输入，通过使用一种称为增强蛋白质组的增强子特异性蛋白质组学方法确定所有与AP-2 CRM相互作用的蛋白质。未来对单个DFD共调控因子的分析将使我们能够阐明它们如何与DFD一起对上颌节段AP-2的表达做出贡献。这项研究将为Hox TF如何控制其靶基因从而控制形态发生提供关键的见解，因为它考虑了低亲和力和高亲和力结合位点以及它们的聚集，并将测试它们对有机体适应性的重要性。这项研究还将对亲和力-特异性权衡模型做出基础性贡献，并将其扩展到同源结构域TF的整个类别。最后，它将建立一个通用的工具来分离与特定增强子片段相互作用的完整蛋白质，这将在转录调控领域开辟新的途径。

The Hox family of transcription factors (TFs) drive morphological diversification of body segments along the anterior-posterior (A/P) axis through a high level of transcriptional specificity. The precise assignment of morphological features along the A/P axis in vivo contrasts with the poor DNA binding stringency of Hox TFs in vitro, as they recognize frequently encountered DNA sequences. It is evident that other regulatory proteins and differences in binding sequences, in particular low-affinity sites, play a critical role in conferring specificity to Hox proteins. Despite increasing our understanding of Hox in vivo function, the emergence of Hox low-affinity binding sites also opened new questions. Specifically, the contribution of high- and low-affinity binging sites to Hox target gene regulation and morphogenesis is unclear and whether other homeodomain TFs use the same mechanism of increasing specificity by decreasing affinity and thereby contribute to the evolution of different types of Hox binding sites.To answer these questions we have established a model, the regulation of the AP-2 gene by the Hox TF Deformed (Dfd). This regulatory interaction depends on a combination of low- and high-affinity Dfd binding sites, controls a morphological feature, the maxillary cirri, and occurs in cells expressing other homeodomain TFs. We will dissect how the Hox TF Dfd reads the regulatory information encoded in the AP-2 enhancer by interfering with the number and affinity of Hox sites and modulate the regional expression of Dfd in reporter-based assays. We will test the contribution of homeodomain TFs interacting with similar consensus sequences as Dfd to AP-2 transcription using domain-specific over-expression and knock-downs. We will analyse the contribution of these features to morphogenesis by testing the effect of their modifications on maxillary cirri formation in a UAS-GAL4 rescue setting and by engineering the endogenous locus using the CRISPR/Cas9. Finally, we will dissect the regulatory input assisting Dfd in controlling AP-2 expression by identifying all proteins interacting with the AP-2 CRM using a enhancer-specific proteomics approach, termed EnhanceProteome. Analysis of individual Dfd co-regulators in future will allow us to elucidate how they contribute in combination with Dfd to AP-2 expression in the maxillary segment.This study will provide critical insights into how Hox TFs control their target genes and thus morphogenesis, as it takes low- and high-affinity binding sites as well as their clustering into consideration and will test their importance for organismal fitness. This study will also make fundamental contributions to the affinity-specificity trade-off model and extend it to the whole class of homeodomain TFs. Finally, it will establish a versatile tool to isolate the full complement of proteins interacting with a specific enhancer fragment, which will open new avenues in the field of transcriptional regulation.