Elucidating mechanisms mediating epigenetic remodelling in the human germline

In mammals, the formation of sperm and eggs is crucial for initiating a new life cycle. The first step in this developmental process is the specification of primordial germ cells (PGCs) in the early post-implantation embryo. As PGCs develop, they undergo extensive epigenetic remodelling, contributing to the "rejuvenation" of the germline and establishing the foundation for normal gametogenesis and embryonic development in the following life cycle. Our previous work and that of others demonstrate that shaping the epigenome in the early germline is a highly regulated process involving the reduction of heterochromatic marks such as DNA methylation and repressive histone modifications (Gruhn et al. 2023). Interestingly, some regulatory regions evade this general heterochromatin weakening, which likely has significant implications for germ cell function and development.

This leads us to ask: How is transcription and epigenetic remodelling regulated in the early human germline? Addressing this question in human embryos is not feasible for technical and ethical reasons, which has limited a mechanistic understanding of early human germline development in the past.

Recent advancements in generating human PGC-like cells (hPGCLCs) from embryonic stem cells provide an important model for investigating human PGC development mechanistically (Tang et al. 2022). While hPGCLCs do not yet fully replicate in vivo germ cell development, they provide a valuable entry point for studying human germline biology.

This project seeks to elucidate the mechanisms underlying epigenetic remodelling in the human germline using hPGCLCs as a model system, with the following objectives:

1. Determine DNA interactions of key transcription factors in hPGCLCs. By analyzing transcriptional and epigenetic profiles, we have identified several transcription factors likely involved in epigenetic remodelling. This project will focus on five of these factors and assess their genome-wide DNA binding profiles using next-generation sequencing (NGS)-associated approaches including CUT&RUN to explore their roles in transcription and the epigenome.
2. Functional analysis of candidate transcription factors. Using gain and loss of function approaches, including CRISPR activation and inducible degron systems, we will evaluate the role of these transcription factors in hPGCLC development. Nascent transcriptome and epigenome profiling (e.g., SLAM-seq, CUT&RUN, ChIP-seq) will be used to assess their functional impact.
3. Identification of regulatory networks through NGS data integration. Computational integration of published transcriptional, epigenetic, and transcription factor binding profiles with the NGS datasets from this project will allow us to decipher regulatory networks that govern transcription and epigenetic remodelling in early germline development.

This project leverages the latest embryonic stem cell models, innovative CRISPR and degron technologies, and advanced bioinformatic data integration to uncover critical mechanisms of human germline development. The insights gained will enhance our understanding of how the potential of forming a totipotent zygote is established in the germline and help to establish in vitro models for human gametogenesis. Furthermore, these findings may help elucidate the origins of germline-associated pathologies, such as infertility and Type II Germ Cell Tumours.

• Gruhn et al., 2023 “Epigenetic Resetting in the Human Germ Line Entails Histone Modification Remodelling.” Science Advances 9 (3): eade1257.
• Tang et al., 2022 “Sequential Enhancer State Remodelling Defines Human Germline Competence and Specification.” Nature Cell Biology 24 (4): 448–60.

Techniques

Generation of transgenic human embryonic stem cells, Transcription factor CUT&RUN, Histone modification CUT&Tag or ChIP-seq, Auxine-inducible degrons, inducible CRISPR activation, inducible CRISPR interference, FACS, Whole genome bisulfite sequencing, SLAM-seq.

Primary Supervisor: Dr Wolfram Gruhn

Secondary Supervisor: Dr Daniel Hebenstreit

University of Registration: University of Warwick

BBSRC Strategic Research Priority:

• Understanding the Rules of Life (Stem Cells)