Líffræðifélag Íslands - biologia.is
Líffræðiráðstefnan 2021
Erindi/veggspjald / Talk/poster V49
Höfundar / Authors: Kinga Sofia Demény, Pétur Orri Heiðarsson
Starfsvettvangur / Affiliations: University of Iceland, The Science Institute
Kynnir / Presenter: Kinga Sofia Demény
Cell reprogramming has the potential to become a major tool in future medicine allowing the induction of pluripotent stem cells (iPSCs) or control over cell differentiation. Pioneer transcription factors (pTFs) are key players in cell reprogramming where they invade and open condensed chromatin and hence alter gene expression networks and thus cell fate. PTFs have well conserved DNA binding domains flanked by long intrinsically disordered regions, some of which contribute to chromatin opening. A powerful tool to study dynamics and kinetics of intrinsically disordered proteins is single-molecule Förster Resonance Energy Transfer (smFRET). However, to be able to decipher the physical and chemical properties that allow pTFs to access closed chromatin and modify cell fate, it is crucial to have a well-defined chromatin landscape. Here we illustrate our methodology to reconstitute chromatin constructs in vitro; from expressing and purifying human core histones, through the design of modified and natural DNA sequences, and to chromatin reconstitution. Finally, we describe and compare two different smFRET techniques using either freely diffusing or surface immobilized single molecules, to understand the conformations and dynamics of chromatin. These experiments allow us to map the conformational dynamics and kinetic mechanisms of pTFs, enabling us to decode their complex molecular mechanisms.