Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: Deciphering the regulatory logic of chromatin modifications

Precise spatiotemporal control of gene expression underlies the vast diversity of cellular and organismal phenotypes. It is mediated at cis-regulatory elements by the interplay of sequence-specific transcription factors and epigenetic mechanisms. Among these, chromatin modifications - specifically post-translational histone and DNA modifications - are a key layer of epigenetic regulation influencing a range of DNA-templated processes, most prominently transcription. However, how precisely they regulate transcriptional states remains a long-standing question. This knowledge gap reflects experimental limitations in defining their causal and context-dependent function. My thesis addresses these limitations by employing precision epigenome perturbations at scale to capture the transcriptional impact of de novo programmed chromatin modifications across hundreds of genes. To this end, I developed a high-throughput epigenome editing method, termed epigenetic targeted perturb-seq (epiTAP-seq), by coupling a modular epigenome editing toolkit with a single-cell transcriptional readout. By implementing epiTAP-seq in mouse embryonic stem cells, I systematically assessed the quantitative impact of 4 distinct chromatin modifications on transcription across 1,000 target genes. Programming H2AK119ub, H3K9me2, DNA methylation and H3K4me3 at endogenous gene promoters induced heterogeneous transcriptional responses that ranged from robust to partially-penetrant. By analyzing (epi)genomic features at target loci, I identified pre-existing expression levels, chromatin states, and DNA sequence motifs as key determinants of transcriptional outcomes upon epigenome editing. In parallel, I sought to characterize the context-dependency of chromatin function across diverse cell types. By leveraging the single-cell readout of epiTAP-seq, I applied this approach in multi-lineage gastruloids - in vitro models of early embryonic development consisting of cell lineages derived from the three germ layers. Proof-of-principle data demonstrated that epiTAP-seq captured distinct cell identities in gastruloids and identified cell type-specific transcriptional outcomes for a subset of target genes. In summary, I systematically interrogated the function of chromatin modifications across diverse (epi)genomic contexts and cell types at single-cell resolution. By uncovering functional relationships with epigenomic features, this thesis advances our understanding of how chromatin states shape transcriptional programmes.