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

Hox 通过 Hox 结合位点的数量和亲和力与转录因子浓度的共同进化来控制形态发生

• 批准号: 158148543
• 负责人: Professorin Dr. Ingrid Lohmann
• 金额: --
• 依托单位: Centre for Organismal Studies (COS)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/158148543?language=en
• 关键词: Hox; Control; Morphogenesis; via; Co

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.

Hox家族的转录因子（TF）通过高水平的转录特异性沿着前-后（A/P）轴驱动身体节段的形态多样化。体内形态学特征沿着A/P轴的精确分配与体外Hox TF的差的DNA结合严格性形成对比，因为它们识别经常遇到的DNA序列。很明显，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。尽管增加了我们对Hox体内功能的理解，但Hox低亲和力结合位点的出现也带来了新的问题。具体来说，高和低亲和力结合位点的Hox靶基因调控和形态发生的贡献是不清楚的，是否其他同源域TF使用相同的机制，增加特异性，通过降低亲和力，从而有助于不同类型的Hox结合sites.To回答这些问题，我们已经建立了一个模型，由Hox TF变形（Dfd）的AP-2基因的调节。这种调节相互作用依赖于低和高亲和力Dfd结合位点的组合，控制形态学特征，上颌纤毛，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PCR的检测中调节Dfd的区域表达。我们将使用结构域特异性过表达和敲低来测试同源结构域TF与类似的共有序列Dfd相互作用对AP-2转录的贡献。我们将分析这些特征对形态发生的贡献，方法是在UAS-GAL 4拯救环境中测试它们的修饰对上颌纤毛形成的影响，并使用CRISPR/Cas9工程化内源性基因座。最后，我们将解剖的监管输入协助Dfd控制AP-2的表达，通过识别所有的蛋白质与AP-2 CRM使用增强子特异性蛋白质组学的方法，称为增强蛋白质组。未来对单个Dfd辅调节因子的分析将使我们能够阐明它们如何与Dfd结合对上颌骨段AP-2表达做出贡献。这项研究将为Hox TF如何控制其靶基因以及形态发生提供重要见解，因为它需要考虑低亲和力和高亲和力结合位点及其聚集性，并将测试它们对生物体适应性的重要性。本研究也将为亲和性-特异性权衡模型做出基础性贡献，并将其扩展到整个同源结构域TF类。最后，它将建立一个通用的工具，以分离与特定增强子片段相互作用的蛋白质的完整互补，这将在转录调控领域开辟新的途径。