Single-molecule microscopy reveals details of heterochromatin reorganisation at the onset of mouse ES cell differentiation

Change log
Jartseva, Aleksandra 

In the last decade, spatial organisation of the genome has been recognised as one of the important factors linked to gene expression and cell identity. Although we have come a long way in describing how chromatin folds – from the level of single nucleosomes all the way to compartments and chromosomal territories – it is still unclear what determines this structure and why it changes upon differentiation. Mouse embryonic stem cells provide an excellent system to address these questions, since upon exit from naive pluripotency their genome undergoes a dramatic structural reorganisation. The development of new fluorophores and imaging technologies has enabled researchers to observe single biological molecules, in both fixed and live cells. These approaches have already proven themselves to be extremely valuable in the chromatin field, for example by revealing the existence and dynamics of chromatin “blobs” (e.g. Ricci et al 2015, Barth et al 2020). This project employs the single-molecule microscopy methods to study chromatin structure and dynamics in ES cells and at the onset of their differentiation. Research conducted in the Laue lab suggested that heterochromatin might play a key role in the global rearrangement of chromatin as ES cells differentiate. Thus, the first part of the thesis focused on studying the role of heterochromatin protein 1β (HP1β) in this process. HP1β was found to increase in expression as cells exit from the ground state, and to change its distribution. Interestingly, this was concomitant with a decrease in both histone H3K9 and H3K27 methylation. 2D and 3D single-particle tracking in live cells was then employed to study HP1β and chromatin dynamics. By applying novel biophysical analysis methods to the data, features of HP1β diffusion not consistent with the current models of heterochromatin compartmentalisation were found, as well as evidence for non-equilibrium processes. Transient slowdown in HP1β diffusion upon differentiation was observed, potentially reflecting a change in the nuclear environment. However, analysis of small-scale chromatin movement showed no change in dynamics between the cell states. In the second part of this work, PALM- and PAINT-based approaches to image chromatin-binding proteins and DNA in 3D throughout the entire nuclear volume of fixed cells were developed. The microscopy protocol was designed to be compatible with single-cell Hi-C, enabling modeling of the genome structures of the imaged cells (Stevens et al 2017). Correlating the images and the structures will for the first time make it possible to obtain spatial sequence-specific information about the genome fold alongside protein localisation on a single-cell level. I hope that this method will shed light on the molecular functions of chromatin-binding proteins in their native context and help understand the rules of genome organisation.

Laue, Ernest
Chromatin, Single-molecule microscopy, Embryonic stem cells
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
BBSRC (1943806)
Biotechnology and Biological Sciences Research Council (1943806)
UKRI BBSRC Doctoral Training Programme