Characterization of Dynamic Nanoscale Chromatin Reorganization During Induced Cell Reprogramming

Abstract

Mammalian cells sharing the same genome can exist in notably distinct phenotypes. Since the ground-breaking work of Shinya Yamanaka, reprogramming terminally differentiated cells into induced pluripotent stem cells and other cell types such as neurons and heart muscle cells is one of the most fascinating topics in basic research and provides hope for regenerative medicine and curing diseases like Alzheimer’s disease and cardiomyopathy. However, the underlying mechanism of cell reprogramming is still poorly understood. It has been shown that during cell reprogramming, cells need to switch between some densely packed and transcriptionally inactive chromosome regions and open regions that allow active transcription and this chromatin structural reorganization serves as the rate-limiting step of cell reprogramming. As conventional approaches rely on analyzing averaged fixed cell population, it is unclear whether this reorganization takes place in a definite sequential order or stochastically throughout a chromosome region. Answering this question is critical for unraveling reprogramming mechanism, but existing biochemical methods cannot provide the needed temporal correlation information at a single cell level. The proposed research aims at mapping out the temporal sequences of open/close chromosome conformational changes and associated change of transcription factor expression during the early stage of a well-studied fibroblast-to-neuron reprogramming process. Specifically, we will first use a new CRISPR-based multiplex chromosome labeling of live cells coupled with 3D single-particle tracking for long-term simultaneous tracking of local conformational changes of multiple genomic loci during the progression of reprogramming. The local chromatin environment around each genomic locus will then be quantitatively assessed by super-resolution fluorescence imaging of nanoscale chromatin structures at different epigenetic states marked by various histone marks. Further, we will perform theoretical polymer physics modeling in the context of random block copolymer theory for mechanistic analysis of the experimental data. By the end of the funding period, we will, for the first time, provide direct temporal information on the interplay between chromosome reorganization and underlying gene regulatory network during the early stage (within five days) of fibroblast-to-neuron reprogramming. The research will establish an experimental and theoretical framework for future studies to investigate the overall reprogramming and other cell phenotype conversion processes.

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