Main author: Sigríður Stefanía Hlynsdóttir
Institution or Company: University of Iceland, The Science Institute
Co-Author, Institution or Company:
Pétur Orri Heiðarsson, University of Iceland, The Science Institute.
Introduction: PU.1 is a pioneer transcription factor (pTF) in the hematopoietic lineage which has been implicated in various blood disorders and diseases. As a pTF, PU.1 targets and binds to condensed chromatin and initiates cell fate change. PU.1 has two intrinsically disordered regions (IDRs) that have been demonstrated to be important for its role as a pTF, through an unknown mechanism. The role of IDRs in pTF activity is generally poorly understood but these regions may be important to recruit other TFs and/or interact with core histones or nucleosomal DNA. We intend to unravel the interactions of PU.1 and nucleosomes, including the role of the IDRs.
Methods: Single-molecule spectroscopy in combination with FRET (smFRET) is a powerful method to study the dynamics of disordered proteins. Site-specific labeling enables probing of distances within a disordered region, interactions between two molecules, and dynamical events. By labeling PU.1 and nucleosomes in various positions, we can identify important regions for PU.1´s function.
Results: We used bacterial expression systems to produce an N-terminally SUMO-tagged PU.1. After cleaving the SUMO-tag, we then optimized a series of chromatography steps to purify the wild-type protein in high yields. Finally, we labeled the two native Cysteine residues in PU.1 with fluorophores for smFRET experiments.
Conclusions: Although PU.1 has been identified as a potential therapeutic target, little is known about the biochemical and biophysical properties of the protein. By characterizing the physical principles behind the function of PU.1, we may unlock a useful tool in research of the hematopoietic system.
