Single-cell sequencing has been instrumental to analyse cellular processes, such as transcriptional regulation, in a cell type- and tissue-specific context. Interpreting a single measurement of a heterogeneous pool of cells can be challenging, as not every cellular process correlates with transcription, complicating a direct coupling to cell identities and other cellular processes. Therefore, new approaches focus on the co-measurement of multiple modalities from the same cell (single-cell multi-omics). This allows a direct comparative analysis and interpretation relative to transcriptional cell states. To this extent, we have developed multiple single-cell multi-omic techniques that allow the co-acquisition of PTHMs together with a variety of measurements: cell type enrichment [sort-ChIC], additional histone modifications [ChIX], past chromatin states [Dam&ChIC] and full-length transcriptome [T-ChIC]. Our lab aims to continue expanding the repertoire of single-cell multi-omics techniques that enable unprecedented functional insights into chromatin regulation.
Most of the protein complexes that deposit (writer complexes) and remove (eraser complexes) PTHMs across the whole genome have been identified. However, we do not understand how these proteins are recruited to specific genomic locations and thereby establish cell type-specific distributions of PTHMs. Previous studies were unable to identify the responsible targeting factors, as they only associate with a fraction of writer complexes at a time and likely form transient interactions. Thus, new experimental approaches are needed to identify the proteins that establish localised epigenetic changes.
To identify these factors, we will integrate CRISPR-based perturbations with our multi-omics readouts. We will systematically perturb potential targeting factors and determine the consequences on PTHM distributions. Performing these experiments at the single-cell level allows us to perturb many candidates at once in a pooled perturbation format and distinguish target-specific results at the computational level. Together, these results allow us to identify novel regulators responsible for guiding histone modifications to their genomic positions in a cell-type specific manner.
Differentiation of the epiblast into the three germ layers during gastrulation is one of the most exciting specification steps during the development of most organisms. In order to achieve this, a transcriptionally indistinguishable pool of epiblast cells undergoes germ layer specification and splits into three distinct identities with specific transcriptional programs and epigenomes: endoderm, mesoderm and ectoderm. This process of lineage specification is accompanied with quick cell divisions and dynamic cell migration, all together leading to the establishment of the body plan. This rise in complexity, coupled to dynamic changes make single-cell approaches instrumental for the study of this window of development.
Since gastrulation occurs after implantation in mammals, this process was previously difficult to study. Recently, new in vitro models called gastruloids were developed that recapitulate key aspects of this developmental phase and can therefore be used to elucidate the underlying mechanisms.
Since gastruloids are generated by aggregation of a few hundred mouse embryonic stem cells, they are highly compatible with pooled CRISPR perturbation approaches, leading to a mosaic structure with various cell clusters containing different perturbations. Additionally, they allow for stringent control of exterior signals. This enables us to study the impact of extracellular signals on the resulting intracellular processes, such as epigenetic programs.
