Integrated genomics for the identification of novel factors controlling midbrain dopaminergic neuron differentiation
Objective: In this project, we are studying the gene regulatory program underlying midbrain dopaminergic (mDAN) differentiation and identity for the identification of novel factors controlling this process.
Background: Induced pluripotent stem cell (iPSC) technology allows us to reprogram human somatic cells into specific cell types. iPSC lines generated either from healthy or diseased individuals carry their genetic background, making them a robust tool for disease modelling, drug screening and cell-replacement therapies. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a progressive and selective loss of mDANs. iPSC-derived mDAN cell-replacement therapies have long been proposed as a possible treatment for PD. However, the lack of accurate control of cell state and high heterogeneity in the current protocols hinder clinical progress.
Methods: We have generated time-series data on chromatin accessibility and transcriptome changes during neural precursor differentiation towards mDANs using a human tyrosine hydroxylase (TH) reporter cell line. Footprinting and transcription factor (TF) motif matching was performed per time point using the chromatin accessibility data. Integration of the TF binding profiles with the corresponding transcriptome data was performed using our tool EPIC-DREM. The generated time-point-specific gene regulatory networks were used to identify putative key TFs controlling mDAN differentiation. To further prioritize the identified TFs, we performed low input ChIP-seq for histone H3 lysine 27 acetylation (H3K27ac) to identify TFs controlled by super-enhancers (SE) in mDANs.
Results: Several TFs were selected and tested by RNAi assays to determine their relevance during mDAN differentiation. Those results helped us to narrow down the candidates that presented the strongest effect for further functional characterization in our in vitro system.
Conclusions: Novel TFs were identified by our approach and showed to be essential for normal mDAN differentiation in vitro. Our next step is to deeper characterize the function of those TFs in mDAN differentiation.