Molecular basis of pioneer transcription factor function in flower development

在多细胞生物中，新的发育程序的启动需要基因表达模式和染色质结构的重大变化。能够触发这些重编程事件的主要调控转录因子(TF)通常作为先驱因子。这些因子识别它们在染色质封闭区域中的同源DNA结合部位，并改变下游转录事件的染色质结构。在植物中，主要调控因子控制花发育的开始，这是一个已知的需要激活封闭染色质区域的关键发育开关。孤儿Tf LEAFY(LFY)在这种转换和花分生组织的分化中起着中心作用。使用结构方法，我们获得了LFY具有寡聚结构域的证据，该结构域允许LFY与封闭的染色质区域结合(在自然通信的修订中，合作伙伴1)。来自MADS家族的两个转录因子APETALA1和SEPALLATA3形成四聚体复合体，是花器官发生和分化的关键导向物。最近公布的数据(伙伴2)强烈表明，这两个因素能够在体内DNA结合部位局部增加染色质的可及性。研究结果表明，这些转移因子是先锋因素。我们将这些转录因子统称为植物先锋转录因子(PPTF)。然而，PPTF作用的分子机制在很大程度上是未知的。利用体外和体内的综合方法，拟议的项目将解决PPTF作用的分子机制，包括跨越分子到生物体水平的实验。生化、结晶学和原子力显微镜实验的结合将为PPTF与染色质重构体和核小体的相互作用以及它们在简化的体外系统中的动力学提供基础。基于结构知识，将产生具有改变寡聚或与重构体相互作用能力的PPTF突变体，以破译体内对PPTF功能的分子需求。通过在表达野生型或突变型PPTF的植物中进行的一系列全基因组实验，将确定PPTF如何与染色质相互作用和修饰染色质。芯片序列实验将确定PPTF结合的区域，DNaseI和MNase足迹将确定PPTF结合是否改变这些区域的染色质结构。最后，PPTF是否可以改变染色质的3D结构，以及这如何依赖于它们形成更大络合物的能力，将使用高分辨率染色质构象捕获技术进行探索。这个项目提供了一个独特的机会来连接转录因子的原子分辨率特性和它们在花切换过程中的全基因组作用。作为一个具有相当大价值的更广泛的影响，它将为研究任何生物体中主调节器的分子作用机制提供一个框架和工具包。

In multicellular organisms, the initiation of new developmental programs requires major alterations in gene expression patterns and chromatin structure. Master regulatory transcription factors (TFs) that are able to trigger these reprogramming events often act as pioneer factors. These factors recognise their cognate DNA binding sites in closed regions of chromatin and alter the chromatin structure for downstream transcriptional events. In plants, master regulatory TFs control the onset of flower development, a key developmental switch known to require the activation of closed chromatin regions. The orphan TF LEAFY (LFY) plays a central role in this switch and in the differentiation of the floral meristem. Using a structural approach, we have obtained evidence that LFY possesses an oligomerisation domain that allows LFY to bind to closed chromatin regions (in revision at Nature Communications, Partner 1). Two TFs from the MADS family, APETALA1 and SEPALLATA3, form tetrameric complexes and are key directors in floral initiation and differentiation of the floral organs. Recently published data (Partner 2) strongly suggest that these two factors are able to locally increase chromatin accessibility at their in vivo DNA-binding sites. The findings suggest that these TFs act as pioneer factors. Collectively, we term these TFs Plant Pioneer Transcription Factors (PPTFs). However, the molecular mechanisms of PPTF action are largely unknown. Using an integrated in vitro and in vivo approach, the proposed project will address the molecular mechanisms of PPTF action, incorporating experiments spanning the molecular to the organismal level. The combination of biochemical, crystallographic and atomic force microscopy experiments will provide the foundation for PPTF interactions with chromatin remodellers and nucleosomes, and their dynamics, in a simplified in vitro system. Based on structural knowledge, PPTF mutants with altered capacity to oligomerise or interact with remodellers will be generated to decipher the molecular requirements for the PPTFs in vivo function. How PPTFs interact with and modify chromatin will be determined through a series of genome wide experiments in plants expressing wild-type or mutant PPTFs. ChIP-seq experiments will identify regions bound by PPTFs, and DNaseI and MNase footprinting will determine whether PPTF binding alters the structure of chromatin in those regions. Finally, whether PPTFs can modify the 3D structure of chromatin and how this depends on their capacity to form larger complexes will be explored using high-resolution chromatin conformation capture techniques. This project provides a unique opportunity to bridge atomic resolution properties of transcription factors and their genome wide action during the floral switch. As a broader impact of considerable merit, it will provide a framework and toolkit for studying the molecular mechanisms of action of master regulators in any organism.