Investigation of transcriptional regulation by C. elegans SET-2/SETD1 H3K4 methyltransferase through single-nucleus profiling

Diverse epigenetic regulation modulates the transcriptional output of a cell. Among these, trimethylation of lysine 4 on histone H3 (H3K4me3), catalyzed by the SETD1 family of histone methyltransferases, is a well-established hallmark of active promoters in eukaryotes. Although loss of SETD1 function results in transcriptional deregulation in many systems, the relationship between changes in gene expression and H3K4me3 marking remains unclear. A potential reason is that H3K4me3 may regulate transcription in different ways, depending on the cell type, chromatin environment and locus. In this thesis, I investigated the consequences of loss of SET-2, the C. elegans orthologue of mammalian SETD1, in early embryogenesis using single-cell profiling of nuclear transcription and chromatin accessibility. Owing to the invariant lineage of C. elegans, these data could be lineage-resolved, enabling the study of gene expression changes in individual cell types of early development. I first generated a lineage-resolved single-cell multiomic atlas of early embryogenesis in a null mutant of set-2 and then analysed the resulting alterations in gene expression and chromatin accessibility. I found extensive deregulation of diverse transcriptional programmes including shared and lineage-specific changes. Upon characterization of these patterns, I found a strong association between loss of SET-2 and reduced induction of gene expression. These changes were accompanied by a reduction in chromatin accessibility at their promoters. To place these observations in the context of H3K4me3 marking, I next examined the H3K4me3 landscape in early embryos. I found two distinct patterns of H3K4me3 deposition: a canonical form localised to active promoters, and broad domains extending across gene bodies. Genes marked by broad domains showed generally higher levels of expression and RNA polymerase II occupancy than those marked only at their promoters. I also found that these broad H3K4me3 domains observed in early embryos were likely de novo established owing to their absence from the developing germline and sperm. Notably, broad H3K4me3 domains in early embryos were strongly associated with genes showing poor transcriptional induction in set-2 mutants. Thus, SET-2 establishes broad H3K4me3 domains on the genes it activates, although whether this marking precedes or follows transcriptional initiation remains unclear. These findings provide a link between SET-2, broad H3K4me3 marking, and diverse transcriptional induction programmes in the embryo. I also optimized an approach to fluorescently label and isolate nuclei from a single post-embryonic lineage for single-cell multiomic profiling and generated preliminary datasets. My work expands on the understanding of how SET-2 and H3K4me3 regulates transcription during early development. It suggests an instructive role for H3K4me3 in transcriptional activation rather than being merely a consequential marker. Finally, my work also shows the power of lineage-resolved single-nucleus profiling for uncovering context-dependent functions of transcriptional regulators.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Hill, Matthew

Advisor dc:contributor.advisor

• Ahringer, Julie

• Single-nucleus profiling
• C. elegans
• Histone modifications
• Gene regulation

Licence

• https://www.repository.cam.ac.uk/bitstreams/8cc7ab68-7b44-4e06-9575-0408ff8a0932/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

Author Identifier

0000-0002-7074-4051

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/397399