Titolo della tesi: The catalytic and non-catalytic roles of chromatin modifiers in early mouse development
Chromatin modifications are essential for development and homeostasis by
contributing to cell-fate decisions, propagating gene expression programs, and acting
as a gene-environment interface. The functional significance of chromatin
modifications during embryonic development in vivo is typically interrogated by
knock-out of a specific chromatin-modifying enzyme. However, the implied causality
between disrupted marks and function is confounded, since many chromatinmodifiers have critical non-catalytic roles and/or structurally participate in many
regulatory complexes. Thus, it remains unclear to what extent molecular or
developmental phenotypes in knockout models arise as a consequence of loss of the
modification, or loss of the protein.
To address this, mouse lines harbouring catalytic-inactivating point-mutations for nine
different chromatin-modifying enzymes were generated, and compared to their knockout counterparts at E6.5 using single-embryo sequencing. Half the chromatin
modifiers were found to non-catalytic gene regulatory functions during early
gastrulation. Moreover, some of these non-catalytic functions are context-specific,
only occurring in a specific developmental tissue or affected by embryo sex. This
demonstrates that catalytic mutants are a crucial and complementary approach to
capture the functions of chromatin modifications per se, particularly when exploring
new systems.
The chromatin modifiers in this study are EZH2 (H3K27me3), RING1B
(H2A119ub), G9A (H3K9me2), SETDB1 (H3K9me3), DNMT1 (meCpG), P300
(H3K27ac), MLL2 (H3K4me3), SETD2 (H3K36me3) and DOT1L (H3K79me2). All
of the catalytic mutants are embryonic lethal in homozygosity. This confirms the vital
role of the associated chromatin modifications in development. Indeed, extensive
omics analysis captured the gene expression targets and network responses of each
modification, thereby reinforcing the centrality of chromatin landscapes in regulating
cell-type specific expression patterns that underpin developmental canalisation. By
intersecting the impact of perturbing multiple chromatin modifications within the
same system and genetic background, I further characterised the relative roles of each
mark. Specifically, I identified that H3K27me3 and H2A119ub have the strongest
pan-tissue effect on genome regulation, but SETDB1-mediated H3K9me3 has the
earliest lethality and impact on the transposable element, as well as other unexpected
relationships. The DNA methylome was also characterised to examine the reciprocal
interplay between chromatin marks and DNA methylation. This confirmed that
meCpG recruitment by Setd2 was facilitated by H3K36me3 within specific contexts,
and identified further functional relationships.
In summary, I applied a systems-level multi-omics analysis to contrast
catalytic mutant and knock-out of multiple chromatin modifiers using novel singleembryo data. I establish the ground-truth developmental functions of their catalytic
function on the regulation of gene expression and developmental phenotype, thereby
reassessing the role of chromatin landscapes in ontogeny