Periodic Reporting for period 1 - PIONEER (FROM SINGLE MOLECULES TO CELL REPROGRAMMING: DECIPHERING AND RECODING DISORDERED PIONEER TRANSCRIPTION FACTORS)
Berichtszeitraum: 2023-01-01 bis 2025-06-30
Pioneer transcription factors (pTFs) have unique capabilities beyond classical TFs in that they can remodel closed chromatin and promote cell-fate changes. Their remarkable abilities have been used to steer cell fate decisions and to induce a pluripotent stem cell state through poorly understood pathways. Like most TFs, pTFs consist of structured DNA-binding domains (DBDs) flanked by long intrinsically disordered regions (IDRs). In attempts to explain their pioneering functions, intense focus has been on how the structured DBDs of pTFs interact with the nucleosome core particle. Yet, the critical interactions with nucleosomes beyond the core particle, the interplay between DBDs and IDRs, and the molecular mechanism of chromatin invading and opening, remain unclear.
The extensive disorder of pTFs places them outside the scope of current structural biology efforts and understanding their functions therefore requires a different approach. Single-molecule spectroscopy offers a powerful toolbox to monitor dynamic molecular systems and measure their conformational distributions. These methods enable quantitative modeling of distances and dynamics in biomolecules over timescales reaching over 15 orders of magnitude. Building on our recent breakthroughs in single molecule techniques for studying highly disordered proteins in chromatin regulation and our preliminary data on pTF IDRs, we are in a unique position to apply our expertise to the molecular mechanism of pTFs.
Using five established pTFs involved in four distinct cell reprogramming pathways, the PIONEER project is intended to: 1) map conformational states, 2) decipher kinetic mechanisms, 3) engineer new pTFs, and 4) observe chromatin remodelling, both in vitro and within the complex cellular environment. A molecular-level understanding of pTF functions may break the barrier to fully controlling cell fate, unleashing the enormous medical potential of cell-based therapy.
The extensive disorder of pTFs places them outside the scope of current structural biology efforts and understanding their functions therefore requires a different approach. Single-molecule spectroscopy offers a powerful toolbox to monitor dynamic molecular systems and measure their conformational distributions. These methods enable quantitative modeling of distances and dynamics in biomolecules over timescales reaching over 15 orders of magnitude. Building on our recent breakthroughs in single molecule techniques for studying highly disordered proteins in chromatin regulation and our preliminary data on pTF IDRs, we are in a unique position to apply our expertise to the molecular mechanism of pTFs.
Using five established pTFs involved in four distinct cell reprogramming pathways, the PIONEER project is intended to: 1) map conformational states, 2) decipher kinetic mechanisms, 3) engineer new pTFs, and 4) observe chromatin remodelling, both in vitro and within the complex cellular environment. A molecular-level understanding of pTF functions may break the barrier to fully controlling cell fate, unleashing the enormous medical potential of cell-based therapy.
The pTFs are enriched in intrinsically disordered regions (IDRs) that are crucial for their function but currently there exist little structural information on these regions. We set out to address this knowledge-gap and have mapped the conformational ensembles of the pTFs to varying degrees using a highly interdisciplinary approach combinding experiments and simulations. We have mapped the conformational ensembles of free pTFs and their complexes with DNA, nucleosomes, or protein binding partners (Objective 1). The ensemble descriptions we have produced are some of the most detailed structural models of a full-length human TFs to date. Our models have led us to discover: complex rearrangements in IDRs upon DNA binding that lead to variable exposure of transcription activation domains, novel interactions of IDRs with the nucleosome histone core, charge-based interdomain interactions that tune conformational ensembles, and disordered regions involved in modulating DNA binding specificity. We are currently building on these discoveries in multiple ways. Our integrative approaches include a suite of biophysical methods such as smFRET, NMR spectroscopy, optical tweezers. We are combining these results with molecular simulations and machine learning/artificial intelligence to design modified IDR sequences and novel protein binders, to modulate function (Objective 3).
Our work in PIONEER has already established a new and deeper understanding of the role of IDRs in pTF function. The first part of the project focused on constructing accurate models of pioneer transcription factor ensembles. This has enabled us to uncover the remarkable conformational complexity that characterizes the disordered activation domains and how they rearrange upon target DNA binding. These conformational rearrangements may be tightly linked to binding partner selection to activate transcription. We have also discovered that these regions play a direct role in recognizing binding sites in closed, nucleosome-rich chromatin, which facilitates high affinity binding. The disordered regions therefore clearly play critical roles that extend beyond the classical paradigm of transcriptional activation. Importantly, our ensemble models allow us to dissect sequence features that can be modified, with the help of simulations and artificial intelligence, in attempts to enhance cell reprogramming efficiency and enable full control of cell identity.