ORCID
https://orcid.org/0000-0002-4669-930X
Date of Award
12-19-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Understanding the dynamics of cell identities has always been a central question in developmental biology. Direct reprogramming, referring to methods that directly convert terminally differentiated cells into a variety of other cell types bypassing intermediate pluripotent states, provides an ideal model to explore the regulatory mechanisms driving cell fate decisions. In direct reprogramming, transcription factors (TFs) initiate cell fate changes by engaging with intrinsic gene regulatory networks (GRNs). However, understanding the impact of early TF binding events on reprogramming outcomes remains challenging. Most of current TF profiling technologies lack the resolution to dissect binding events within rare starting populations and are typically destructive, which makes it impossible to connect past binding data to future cell fate decisions. Here, I applied single-cell Calling Cards (scCC), a technique that records TF binding and gene expression simultaneously at single-cell resolution, to elucidate the dynamics of TF-mediated reprogramming of mouse embryonic fibroblasts (MEFs) into induced endoderm progenitors (iEPs). Our results suggest that FoxA1 initially binds widely across the genome. This promiscuity is refined by lineage-specific co-factors like Cepba, Nr2c2, and Foxl1, which steer FoxA1 towards tissue-specific enhancers critical for successful reprogramming. Ineffective enhancer reconfiguration leads to reprogramming failures or dead-end states. I further demonstrate that reprogramming efficiency correlates with the stoichiometry of reprogramming factors and is enhanced by inhibiting TGF-β signaling, which reduces mesenchymal TF activity and promotes precise enhancer engagement. By capturing the interplay between early TF activity, chromatin accessibility, and gene expression, scCC provides profound insights into the transcriptional and epigenetic orchestration underlying reprogramming. My dissertation is organized as follows: Chapter 1 introduces the biological and technical background relevant to this study, focusing on dissecting the TF-mediated regulatory mechanism underlying direct reprogramming. Chapter 2 discusses collaborative efforts within the Morris lab to elucidate the identities of induced endoderm progenitors (iEPs) and extends the investigation to early stages of reprogramming across various paradigms. Chapter 3 details the application of undirected single-cell Calling Cards (scCC) in iEP reprogramming to assess the technology's compatibility with the system and explore how early enhancer activities contribute to trajectory bifurcation. Chapter 4 focuses on employing FoxA1- and Hnf4a-directed scCC in iEP reprogramming to study the regulatory mechanism of early TF binding on subsequent cell fate decisions, with a particular focus on co-binding dynamics. Chapter 5 summarizes the findings from the preceding chapters and proposes future research directions. Overall, my thesis elucidates key regulatory factors that influence the efficiency and fidelity of reprogramming, offering insights that could enhance strategies in regenerative medicine.
Language
English (en)
Chair and Committee
Samantha Morris
Committee Members
Benjamin Humphreys; Helen McNeill; Michael Meers; Robi Mitra
Recommended Citation
Yang, Xue, "Recording Transcription Factor FoxA1 and Hnf4a Binding at Single-Cell Resolution During iEP Direct Reprogramming" (2024). Arts & Sciences Electronic Theses and Dissertations. 3341.
https://openscholarship.wustl.edu/art_sci_etds/3341