Affiliations: 1) Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 2) Division of Computational Genomics and Systems Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany
Keywords: a. chromatin structure; e. enhancers
Developmental progression and cellular diversity are largely driven by transcription factors (TFs), yet characterizing their loss-of-function phenotypes remains challenging and often disconnected from their underlying molecular mechanisms. To overcome this, we have combined single cell regulatory genomics with loss-of-function mutants to simultaneously assess both cellular and molecular phenotypes.
We performed single cell chromatin accessibility profiling by sci-ATAC-seq for over 20,000 cells at eight overlapping time-points during Drosophila mesoderm development, ensuring that key developmental transitions are captured and can be directly traced, including stages of multipotency, specification and terminal differentiation. This dataset captures the establishment of regulatory landscapes in the nascent embryonic mesoderm at high resolution and its progression along developmental time. We demonstrate that this information can reconstruct multiple developmental trajectories of all major and rare cell-types, including the heart, and uncover the enhancers and TFs involved.
To systematically assess the TFs mutant phenotypes, we developed a streamlined strategy to robustly profile chromatin accessibility and digitally genotype mutant cells coming from embryo collections of mixed genotypes. Applying this approach to four TF mutants could uncover and quantify their characterized phenotypes de novo, and discover new ones, while simultaneously revealing their regulatory input and mode of action. We observe strikingly different outcomes in cell fate, as one mutant (Mef2) results in abnormal ‘new’ cell states, while other TF mutants appear to block cell populations much earlier along their developmental trajectory.
Our approach is a general framework to dissect the functional input of TFs in a systematic unbiased manner, uncovering both cellular and molecular phenotypes at a scale and resolution that was not feasible before.