Midway Evaluation in Biochemistry - Sigríður Stefanía Hlynsdóttir

Student: Sigríður Stefanía Hlynsdóttir

Study programme: Biochemistry

Title of thesis:
Studying the mechanism of chromatin binding and opening by the pioneer transcription factor PU.1: The role of intrinsically disordered regions

Abstract
Pioneer transcription factors (pTFs) have recently emerged as crucial components of transcription during development and in cancer biology. Although classical TFs usually require DNA accessibility for binding, pTFs possess unique abilities beyond other TFs; they can target condensed, nucleosome-rich chromatin and initiate cell-fate changes by remodeling, exposing genes for transcription. Pioneer transcription factors are master regulators of cell fate and may hold the key to accessing the full potential of cell and gene-therapy. Mapping the molecular principles of cell reprogramming is dependent on understanding how pTFs bind and alter condensed chromatin. Purine-rich sequence DNA-binding protein 1 (PU.1) is an important pTF in the hematopoietic lineage and has a role at multiple stages of hematopoiesis. PU.1 has been implicated in various blood disorders and has been identified as a critical component in many autoimmune diseases. PU.1 consists of a structured DNA binding domain and an intrinsically disordered region (IDR), common amongst pTFs. The IDR is important for PU.1´s pioneer activity through a poorly understood mechanism but this region may be important to recruit other TFs or interact with the nucleosome, either the core histones or nucleosomal DNA. Although PU.1 has been identified as a potential therapeutic target, little is known about neither the physiological role of PU.1 nor the biochemical and biophysical properties of the protein. We use single-molecule spectroscopy in combination with FRET (smFRET) to study the dynamics of IDRs. smFRET is a powerful method to study IDRs, where site-specific labeling enables probing of distances within a single molecule as well as interactions between two molecules. The single-molecule resolution avoids the ensemble-averaging and allows us to see transient molecular events which enable the studying of structurally heterogeneous systems. By labeling PU.1 and nucleosomes in various positions, we can analyze the behavior of PU.1’s domains and identify important regions for PU.1´s function. Our results show that PU.1 binds to short DNA harboring its recognition sequence with high affinity and that the binding alters the conformations of the disordered region. We have also identified two distinct PU.1 populations that exhibit different behaviors, with varying degrees of sensitivity to denaturing agents and salt concentrations. Preliminary data suggests that these two populations show different affinities towards free DNA. Furthermore, we have observed a potential dimer formation, with low nanomolar affinity, that affects the conformations of the IDR. By characterizing the physical principles behind PU.1’s function, we may unlock a useful tool in research of the hematopoietic system and take a step forward in understanding intrinsically disordered proteins, pioneer transcription factors and their role in cell reprogramming.

Doctoral Committee
Pétur Orri Heiðarsson, Associate Professor at the Faculty of Life and Environmental Sciences, University of Iceland 
Erik Holmstrom, Assistant Professor, University of Kansas 
Eiríkur Steingrímsson, Professor at the Faculty of Medicine, University of Iceland