IAP Seminar (Direct visualisation of Xist RNA-mediated X chromosome inactivation using in situ cryogenic correlative light and electron microscopy)
Date : May 22, 2025 14:30 ~ 15:30
Speaker : Dr. Jeongyoon Choi (University of Oxford)
Professor : Prof. Je-Kyung Ryu
Location : 56-219
Direct visualisation of Xist RNA-mediated X chromosome inactivation using in situ
cryogenic correlative light and electron microscopy
Jeongyoon Choi
The 3D structure and organisation of chromatin in the cell nucleus play a critical role in
regulating the fundamental biological processes of gene regulation in the eukaryotic cell. A
broad range of experimental approaches has been developed to better understand chromatin
3D architecture and its relationship to function, including genome-wide sequencing techniques
(e.g., Hi-C, ATAC-seq, RNA-seq and ChIP-seq), imaging methods (e.g., super-resolution light
microscopy, live-cell single molecule imaging and electron microscopy) and biochemical
reconstitution with recombinant proteins and/or cell extracts. A recently developed approach
applies cryogenic electron tomography (cryo-ET) to define the ultrastructural organisation of
chromatin1-4
. Because cryo-ET is free from chemical fixation and contrasting agents, it offers
the promise of capturing the near-native chromatin structure in the cell.
Here, we have developed a correlative light and electron microscopy (CLEM) strategy that
allows us to focus on in situ chromatin structure at distinct biological states in the near-native
cell. We have applied this to a fundamental biological process, X chromosome inactivation in
female mammalian cells. In X chromosome inactivation, almost all genes on one of the two X
chromosomes in each cell are silenced from early embryonic development throughout the
lifetime. This process is initiated by the long non-coding RNA Xist, which recruits chromatin
silencing complexes to the X chromosome. As a result of this, the inactive X chromosome has
been shown to have more compacted chromatin organisation at the light microscopic level5.
In this talk, I will present in situ cryo-ET structure of inactive X-chromosome from the mouse
embryonic stem cell and discuss the chromatin organisation in the near-native cell.
1. Mahamid, J. et al. Visualizing the molecular sociology at the HeLa cell nuclear periphery.
Science 351, 969–972 (2016).
2. Cai, S., Song, Y., Chen, C., Shi, J. & Gan, L. Natural chromatin is heterogeneous and selfassociates in vitro. Mol. Biol. Cell 29, 1652–1663 (2018).
3. Cai, S., Böck, D., Pilhofer, M. & Gan, L. The in situ structures of mono-, di-, and trinucleosomes
in human heterochromatin. Mol. Biol. Cell 29, 2450–2457 (2018).
4. Hou, Z., Nightingale, F., Zhu, Y., MacGregor-Chatwin, C. & Zhang, P. Structure of native
chromatin fibres revealed by Cryo-ET in situ. Nat. Commun. 14, 6324 (2023).
5. Smeets, D. et al. Three-dimensional super-resolution microscopy of the inactive X
chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA
foci. Epigenetics Chromatin 7, 8 (2014).