Molecular basis of pioneer transcription factor function in flower development

• Applicant: Professorin Dr. Kerstin Kaufmann
• Subject Area: Plant Cell and Developmental Biology
• Term: from 2016 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 316736798

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.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partners: Dr. Francois Parcy; Privatdozentin Dr. Chloe Zubieta