Live-cell multiplex super-resolution imaging of chromatin state transitions

Project Summary
Chromatin structure and transcription regulation are essential for cellular function, and their dynamics are highly
correlated both in development and in disease. However, despite decades of amazing work identifying the
molecular players involved in these processes, and mapping their interactions genome-wide, we are currently
unable to describe the function connecting 3D chromatin structure and transcription dynamics. This limitation
stems from the fact that chromatin structure and gene expression emerge from intrinsically stochastic transitions
at the single-cell level, and we are missing the critical temporal parameters associated with these transitions.
Therefore, new tools to measure both chromatin structure and transcription over time in single cells are critical
for understanding how the human genome is read and for predictively controlling the epigenome.
Here, we propose to develop a new set of live single-cell imaging technologies to simultaneously measure
changes in 3D chromatin structures and their associated dynamics of gene expression across a large range of
timescales: from dynamics of individual topologically associated domains and enhancer-promoter interactions,
to changes associated with stable epigenetic memory across cell cycles. For the shorter timescales (under a cell
cycle), our new imaging approach combines live super-resolution microscopy of fluorescently labeled loci with
end-point demultiplexing of loci identity using Optical Reconstruction of Chromatin Architecture (ORCA), in order
to track and trace 3-12 points within a functional chromatin unit. This new technique, which we call live-ORCA,
will allow us to measure for the first time the temporal dynamics of an entire topologically associated domain in
single cells. We will use live-ORCA in conjunction with time-lapse imaging of transcriptional bursting to study
the dynamics of promoter-enhancer activity throughout cell differentiation and under perturbations of the
chromatin network. For the longer timescale (across multiple cell cycles), our approach will combine time-lapse
microscopy of gene expression, monitoring the distance between two tagged genomic loci as a live reporter of
chromatin structure, and end-point chromatin tracing of the entire gene neighborhood using ORCA. We will
perform these measurements in two systems: at a highly controlled synthetic reporter where we can induce
either short-term silencing or long-term epigenetic memory, and at time points in differentiation when genes
commit epigenetically to a new transcriptional state. Moreover, in order to further investigate the mechanism of
epigenetic inheritance, we will develop a novel microfluidic device that allows us to track changes in chromatin
3D structures across individual cell lineages. Finally, to test our quantitative understanding, we will go back and
forth between these single-cell data and theoretical modelling of chromatin dynamics. This research plan will
greatly advance our understanding of chromatin dynamics and its functional role in transcription regulation, while
at the same time contributing a whole new set of novel imaging technologies and engineered cell lines that will
serve as a jumping board for the 4D Nucleome and broader scientific community.

Grant Number: 5U01DK127419-05
NIH Institute/Center: NIH
Principal Investigator: Lacramioara Bintu