Żylicz group: Epigenetic and metabolic regulation of early development

Transcriptional, epigenetic and metabolic changes orchestrate early development (Figure 1). However, how these processes are coordinated to allow for appropriate development remains largely unknown. Of particular interest are the first lineage choices made after the egg is fertilised. The delineation of extraembryonic trophectoderm (TE) and primitive endoderm (PrE) supports the development of the pluripotent epiblast (Epi), which in turn will give rise to the embryo proper. After implantation cells of the embryonic and extraembryonic lineages embark on a complex and distinct programme of transcriptional and chromatin rewiring. This culminates with the emergence of striking chromatin mark asymmetries between lineages, with extraembryonic tissues showing certain similarities to cancer cells (e.g. DNA hypomethylation). How such global epigenetic states emerge remains largely unknown. Similarly, it is unclear what function they might be playing during early development. This is a fundamental biological question of how wide-spread chromatin modifications actually regulate gene expression, and to what extent they are simply a read-out of transcriptional or metabolic states of cells? Are chromatin marks important? And if so, in what context?

Epigenetic and metabolic changes are coordinated during mouse development. A. Upon fertilisation mouse embryos embark on a complex developmental programme. This entails the activation of zygotic genome at around 2-cell stage. Blastomeres become first committed to the extra-embryonic trophectoderm (TE) or the pluripotent inner cell mass (ICM) at morula stage. ICM cells become further allocated to the embryonic epiblast lineage or extra-embryonic primitive endoderm (PrE) during blastocyst expansion. At the embryonic day 5 (E5.0) the embryo implants into the uterus leading to dramatic cell-number expansion in preparation for gastrulation.
B. Chromatin and metabolic changes are coordinated. DNA methylation is rapidly demethylated during pre-implantation leading to globally hypomethylated DNA in the blastocyst, by which stage H3K9me2 levels also decrease. Upon implantation there is extensive de novo DNA methylation and deposition of H3K9me2. High levels of oxidative phosphorylation (OxPhos) is linked to low levels of DNA methylation and H3K9me2.

The Żylicz team studies the role of chromatin modifiers during early mouse development as well as molecular mechanisms regulating them. Of particular interests are metabolic shifts affecting enzymatic activity of such epigenetic writers and erasers. Mouse development is our main model system but we also implement a wide range of in vitro stem cell cultures. The team goes beyond traditional developmental biology techniques and uses a wide spectrum of ultra-sensitive transcriptomic, epigenomic and metabolomic assays combined with live imaging.

Research Overview

Function of chromatin modifiers during development and differentiation

Recent technological advances helped to uncover dramatic transcriptional and epigenetic re-wiring of early mouse embryos. Our findings revealed a distinct chromatin landscape associated with primed pluripotent epiblast in vivo. Indeed, upon implantation epiblast accumulates high levels of histone H3 lysine 9 dimethylation (H3K9me2) and rearranges Polycomb-associated histone H3 lysine 27 trimethylation (H3K27me3). These two processes allow for embryo’s growth and successful gastrulation respectively. H3K9me2 and H3K27me3 marks decorate thousands of genes along the genome and yet are functionally relevant for the regulation of only about 100 loci. Similarly, we found that loss of a vital histone deacetylase: HDAC3 also leads to the deregulation of a very limited set of genes. In the Żylicz team we seek to understand not only the dynamic changes to chromatin states during development and differentiation but also their function. Why do cells expend large amounts of energy to modify swathes of the chromatin if this regulates only a handful of genes? Are chromatin marks important e.g. as a feed-forward loop to reinforce and maintain transcriptional states? Or maybe they are mainly a by-product of global cell states? To address these points we use a pallet of in vivo mouse knock-out strains as well as tuneable degron stem cell lines.

Mechanisms underlying facultative heterochromatin formation

During implantation embryonic lineages accumulate chromatin modifications associated with transcriptional repression e.g. H3K9me2 and DNA methylation. In some cases this is associated with the accumulation of facultative heterochromatin (fHC). fHC concerns the developmentally regulated heterochromatinisation of different regions of the genome and in the case of the mammalian X chromosome and imprinted loci, of only one allele of a homologous pair. The formation of fHC participates in the timely repression of genes, by resisting strong trans-activators. The Żylicz team is interested in how chromatin and transcriptional repression are coordinated along the genome. In the specific context of X chromosome inactivation fHC formation is aided by HDAC3-dependent rapid histone deacetylation. We seek to expand our understanding of how fHC forms in vivo and in vitro by performing ultra-sensitive time resolved chromatin and transcriptional mapping and integrating this with functional analysis of chromatin modifiers.

Epigenetic and metabolic coupling during mouse development

Multiple emerging evidence suggest an important functional role of metabolic changes in shaping the epigenetic landscape of a cell. This has been extensively studied in the context of cancer and stem cells but not development. Nevertheless, early mouse development is accompanied by a dramatic sequence of both metabolic and chromatin alterations, which must somehow be coordinated. This can occur by changing the availability of “epi-metabolites”, which are directly involved in chemically modifying both DNA and histones (Figure 2). Indeed, α-ketoglutarate, S-adenosylmethionine and acetyl-CoA are produced and consumed through normal metabolic processes but are also necessary for catalytic activity of chromatin-modifying enzymes. Thus dynamic metabolic changes have direct and global consequences to the chromatin state. Alternatively, expression or localisation of chromatin modifiers could be affected by factors sensing metabolic states. Żylicz team seeks to unravel this interplay between metabolism and chromatin during stem cell differentiation and development. A key question here is how global changes to metabolism and chromatin states result in distinct and specific transcriptional outcomes.

How metabolism can directly regulate chromatin and transcription? Some small metabolites are necessary for catalytic activity of chromatin modifiers. The concentration of such “epi-metabolites” can vary hugely between different cell types. Altered availability of these molecules will modify the activity of specific chromatin writers (or erasers) thus affecting chromatin modification landscape. This in turn, can have an effect on transcriptional activity of RNA polymerase II (RNAPII).

To achieve these goals we are utilising ultra-sensitive metabolomic and epigenomic assays both in vitro and in vivo. Our experimental design goes beyond characterisation of cell states but focuses on the issues of functionality of chromatin modifiers and of their coupling with metabolism. Studying the links between metabolism and chromatin provides an opportunity to address the importance of environmental factors on successful development and adult life.