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改变。能够触发这些重编程事件的主调节转录因子（TF）通常充当先锋因子。这些因子识别染色质封闭区域中的同源DNA结合位点，并改变下游转录事件的染色质结构。在植物中，主调节转录因子控制花发育的开始，这是已知需要激活闭合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核心作用。使用结构的方法，我们已经获得的证据表明，LFY拥有一个寡聚化结构域，使LFY结合到封闭的染色质区域（在自然通讯，合作伙伴1修订）。MADS家族的两个转录因子APETALA 1和SEPALLATA 3形成四聚体复合物，是花器官的花发生和分化的关键指示剂。最近发表的数据（合作伙伴2）强烈表明，这两个因素能够局部增加染色质的可及性在其体内DNA结合位点。研究结果表明，这些TF作为先驱因素。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。 使用一个综合的体外和体内的方法，拟议的项目将解决PPTF行动的分子机制，结合实验跨越分子到生物体水平。生物化学，晶体学和原子力显微镜实验的组合将提供PPTF与染色质重塑和核小体的相互作用，以及它们的动力学，在一个简化的体外系统的基础。基于结构知识，将产生具有改变的寡聚化或与重塑物相互作用的能力的PPTF突变体，以破译PPTF体内功能的分子要求。PPTF如何与染色质相互作用和修饰染色质将通过在表达野生型或突变型PPTF的植物中进行的一系列基因组范围的实验来确定。ChIP-seq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，PPTFs是否可以修改染色质的3D结构，以及这如何取决于它们形成更大复合物的能力，将使用高分辨率染色质构象捕获技术进行探索。该项目提供了一个独特的机会，桥梁原子分辨率的转录因子和它们的基因组范围内的行动在花开关的属性。作为一个具有相当大价值的更广泛的影响，它将提供一个框架和工具包，用于研究任何生物体中主调节剂的分子作用机制。

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.