Exploring the dynamic epigenome in pluripotent stem cells using quantitative methods

A totipotent stem cell has the potential to give rise to the trillions of cells in the human body, all carrying the very same genetic information. Through differentiation events, gene expression changes guided by epigenetic mechanisms resulting in specialized phenotypes. The more we understand of the highly dynamic epigenome, the better we will understand key phenomena such as human development, tissue regeneration and disease initiation and progression. In this thesis, focus lays on the role of key players in the field of molecular mechanisms of epigenetics using both mouse and human embryonic stem cells.

In Paper I we investigated the non-catalytic function of ten-eleven translocation 1 (TET1) methylcytosine dioxygenase in mouse embryonic stem cells (ESCs). Upon knocking out TET1, endogenous retroviral expression increased, while neither cells expressing wild type TET1 or a non-catalytic mutant showed the same trend. Studying the epigenetic landscape, we found that TET1, independently of its catalytic activity, is important for establishment of the silencing histone marks H3K9me3 and H4K20me3 at endogenous retroviral elements. This suggests that TET1 serves as an interaction hub for chromatin modifying complexes to repress the interstitial heterochromatin that the ERVs reside in.

In Paper II we investigated the role of Polycomb Repressive Complex 2 (PRC2) as an epigenetic regulator for cell type specification in human ESCs. By abolishing the function of PRC2 via drugs or knocking out its catalytic subunit we observed spurious differentiation of naïve pluripotent stem cells toward cells belonging to the mesoderm and trophectoderm lineages, indicating that PRC2 has a pivotal role in shielding naïve pluripotent stem cells from differentiating toward trophectoderm differentiation.

In Paper III we used genetic code expansion in mouse ESCs to produce acute and defined fractions of labelled histone variant H3.3 to study its chromatin deposition kinetics and turnover rate using quantitative methods for immunocytochemistry, chromatin immunoprecipitation sequencing and protein quantification. We revealed that H3.3 accumulates rapidly in a subnuclear space together with DAXX, ATRX, Smarcad1 and HP1 prior to significant chromatin incorporation both at enhancers and interstitial heterochromatin Moreover, this technique allowed for studying novel interactors of H3.3 in a temporal manner directly after protein synthesis. Furthermore, we found that one of the interactors plays a key role as a chromatin remodeler allowing for H3.3 turnover in enhancers.

Using the same methods, in Paper IV we expanded the study from Paper III to also focus on the kinetics of canonical histones H2A and H3 compared to the variants H2A.Z, macroH2A, H3.3 and CENP-A. Our results show that the histones are subjected differently to pre-assembly degradation, have defined individual genomic incorporation rates and distinctive half-life in chromatin. Using quantitative ChIP-seq allowed for studying the incorporation to repetitive elements, which is of essence when studying histone variants. Furthermore, we laid some ground work towards finding the enigmatic histone chaperone of macroH2A.

Taken together, we show that TET1, PRC2 and histone variants play essential and unique roles in the maintenance of homeostasis in ESCs. Continuing to unravel their dynamics and roles will be instrumental for understanding epigenetically regulated diseases and lead to improved diagnostics and treatments.

List of scientific papers

I. Paul Stolz, Angelo Salazar Mantero, Andrey Tvardovskiy, Enes Ugur, Lucas E. Wange, Christopher B. Mulholland, Yuying Cheng, Michael Wierer, Wolfgang Enard, Robert Schneider, Till Bartke, Heinrich Leonhardt, Simon J Elsässer and Sebastian Bultmann. TET1 regulates gene expression and repression of endogenous retroviruses independent of DNA demethylation. Nucleic Acids Research. 2022, vol. 50, p. 8491–8511.
https://doi.org/10.1093/nar/gkac642

II. Banushree Kumar*, Carmen Navarro*, Nerges Winblad*, John P Schell, Cheng Zhao, Jere Weltner, Laura Baqué-Vidal, Angelo Salazar Mantero, Sophie Petropoulos, Fredrik Lanner, Simon J Elsässer. Polycomb Repressive Complex 2 shields naïve human pluripotent cells from trophectoderm differentiation. Nature Cell Biology. 2022, vol. 24, p. 845–857. *Authors contributed equally.
https://doi.org/10.1038/s41556-022-00916-w

III. Anna-Maria Katsori*, Angelo Salazar Mantero*, Yuying Cheng, Carmen Navarro, Jürgen Eirich, Rozina Caridha, Banushree Kumar, Georgios Mermelekas, Simon J Elsässer. Quantitative histone H3.3 dynamics resolved by rapid protein pulsing. *Authors contributed equally. [Manuscript]

IV. Angelo Salazar Mantero, Yuying Cheng, Marius Jung, Rozina Caridha, Janice Linne, Jürgen Eirich, Simon J Elsässer. Quantitative temporal catalogue of histone variant dynamics in mESCs. [Manuscript]